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Kürzlich wurde mithilfe von Ribo-seq Experimenten die Translation hunderter Mikroproteine in menschlichen Herzen entdeckt. Diese blieben zuvor aufgrund ihrer geringen Größe (< 100 Aminosäuren) unentdeckt, und ihre physiologische Rolle ist noch weitgehend unbekannt. Ziel dieser Promotionsarbeit ist es, potentielle Funktionen dieser neuartigen Mikroproteine zu entschlüsseln. Dabei sollen insbesondere die Aufklärung ihrer evolutionären Konservierungssignatur, subzellulären Lokalisierung und ihres Proteininteraktoms helfen. Die Konservierungsanalyse ergab, dass fast 90% der Mikroproteine nur in Primaten konserviert ist. Weiterhin konnte ich die Produktion von Mikroproteine in vitro und in vivo nachweisen, die subzelluläre Lokalisierung von 92 Mikroproteinen definieren, und Interaktionspartner für 60 Mikroproteine identifizieren. Dutzende dieser Mikroproteine lokalisieren in Mitochondrien. Dazu gehörte ein im Herzen angereichertes Mikroprotein, das aufgrund der Interaktions- und Lokalisationsdaten einen neuartigen Modulator der mitochondrialen Proteintranslation darstellen könnte. Der Interaktom-Screen zeigte außerdem, dass evolutionär junge Mikroproteine ähnliche Interaktionsfähigkeiten wie konservierte Kandidaten haben. Schließlich wurden kurze Sequenzmotive identifiziert, die Mikroprotein-Protein-Wechselwirkungen vermitteln, wodurch junge Mikroproteine mit zellulären Prozessen – wie z.B. Endozytose und Spleißen – in Verbindung gebracht werden konnten. Zusammenfassend wurde die Produktion vieler kleiner Proteine im menschlichen Herzen bestätigt, von denen die meisten lediglich in Primaten konserviert sind. Zusätzlich verknüpften umfangreiche Lokalisierungs- und Interaktionsdaten mehrere Mikroproteine mit Prozessen wie Spleißen, Endozytose und mitochondrialer Translation. Weitere Untersuchungen dieses zuvor verborgenen Teils des Herzproteoms werden zu einem besseren Verständnis von evolutionär jungen Proteinen und kardiologischen Prozessen beitragen., Recently, the active translation of hundreds of previously unknown microproteins was detected using ribosome profiling on tissues of human hearts. They had remained undetected due to their small size (< 100 amino acids), and their physiological roles are still largely unknown. This dissertation aims to investigate these novel microproteins and validate their translation by independent methods. Particularly, elucidating their conservation signature, subcellular localization, and protein interactome shall aid in deciphering their potential biological role. Conservation analysis revealed that sequence conservation of almost 90% of microproteins was restricted to primates. I next confirmed microprotein production in vitro and in vivo by in vitro translation assays and mass spectrometry-based approaches, defined the subcellular localization of 92 microproteins, and identified significant interaction partners for 60 candidates. Dozens of these microproteins localized to the mitochondrion. These included a novel cardiac-enriched microprotein that may present a novel modulator of mitochondrial protein translation based on its interaction profile and subcellular localization. The interactome screen further revealed that evolutionarily young microproteins have similar interaction capacities to conserved candidates. Finally, it allowed identifying short linear motifs that may mediate microprotein-protein interactions and implicated several young microproteins in distinct cellular processes such as endocytosis and splicing. I conclude that many novel small proteins are produced in the human heart, most of which exhibit poor sequence conservation. I provide a substantial resource of microprotein localization and interaction data that links several to cellular processes such as splicing, endocytosis, and mitochondrial translation. Further investigation into this hidden part of the cardiac proteome will contribute to our understanding of recently evolved proteins and heart biology.

Heterosis, the phenomenon in which hybrids outperform their parents, has been utilized in maize (Zea mays L.) for over 100 years. To provide a more complete understanding of heterosis, we collected a comprehensive transcriptome and translatome dataset on seedling leaves for B73, Mo17, and their F1 hybrid, which provided a dynamic landscape of transcriptomic and translatomic variation in maize. Although additivity accounted for a large proportion of variation at two omics-levels, an elevated nonadditive effect was observed in the translatome, especially in the translated subgenome maize1 genes, and the genes that switched from additivity in the transcriptome to nonadditivity in the translatome were significantly enriched in the subgenome maize1. Many genes with allele-specific expression and translation show dramatic regulatory switches between the transcriptome and translatome, and partial genes with allele-specific translation underlying regulatory mechanism also exhibited subgenome bias. Interestingly, we found the translated isoforms show different expression patterns compared with transcriptome and more genes changed their dominant isoforms during the genetic flow from parents to the hybrid at the translatome level. The translated genes with switched dominant isoforms significantly biased to the subgenome maize2 while genes with conserved dominant isoforms significantly enriched in subgenome maize1. Together, the dynamic changed patterns in translatome across hybrid and parental lines show translational fractionation of the maize subgenomes, which may be associated with heterosis in maize and provides a potential theoretical basis for breeding.

The present study aimed to better understand the possibility of utilizing all-trans retinoic acids (ATRA) in acute myeloid leukemia (AML). We found that ATRA significantly suppressed global translation and protein synthesis in AML cells. The efficacy of ATRA in treating AML required its translational regulatory functions, as shown by the fact that the decrease in the universal eukaryotic initiation factor 4E (eIF4E) was essential to maintain the induction of cell growth arrest and differentiation by ATRA. By establishing a specific translational landscape, we suggested that transcripts with simple 5′UTR gained a translational advantage in AML cells during ATRA stress. Based on that, the genes translationally regulated by ATRA were mainly enriched in phosphatidylinositol-3-kinase/Akt (PI3K/AKT) signaling; we subsequently revealed that PI3K/AKT activation was required for ATRA to effectively induce AML cell differentiation. However, PI3K/AKT has been reported to promote the stemness of AML cells. As such, we further suggested that sequential treatment including ATRA and PI3K/AKT inhibitor induced robust apoptosis, extremely inhibited the clonality of AML cells, and suppressed the FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD)-driven transformation of CD34+ hematopoietic stem/progenitor cells. Future clinical studies are warranted to further support the clinical application of the sequential strategy for the effective treatment of AML.

Soybean (Glycine max (L.) Merr.) establishes symbiosis with rhizobacteria, developing the symbiotic nodule, where the biological nitrogen fixation (BNF) occurs. The redox control is key for guaranteeing the establishment and correct function of the BNF process. Plants have many antioxidative systems involved in ROS homeostasis and signaling, among them a network of thio- and glutaredoxins. Our group is particularly interested in studying the differential response of nodulated soybean plants to water-deficit stress. To shed light on this phenomenon, we set up an RNA-seq experiment (for total and polysome-associated mRNAs) with soybean roots comprising combined treatments including the hydric and the nodulation condition. Moreover, we performed the initial identification and description of the complete repertoire of thioredoxins (Trx) and glutaredoxins (Grx) in soybean. We found that water deficit altered the expression of a greater number of differentially expressed genes (DEGs) than the condition of plant nodulation. Among them, we identified 12 thioredoxin (Trx) and 12 glutaredoxin (Grx) DEGs, which represented a significant fraction of the detected GmTrx and GmGrx in our RNA-seq data. Moreover, we identified an enriched network in which a GmTrx and a GmGrx interacted with each other and associated through several types of interactions with nitrogen metabolism enzymes.

Ribosome profiling experiments demonstrate widespread translation of eukaryotic genomes outside of annotated protein-coding genes. However, it is unclear how much of this “noncanonical” translation contributes biologically relevant microproteins rather than insignificant translational noise. Here, we developed an integrative computational framework (iRibo) that leverages hundreds of ribosome profiling experiments to detect signatures of translation with high sensitivity and specificity. We deployed iRibo to construct a reference translatome in the model organism S. cerevisiae. We identified ∼19,000 noncanonical translated elements outside of the ∼5,400 canonical yeast protein-coding genes. Most (65%) of these non-canonical translated elements were located on transcripts annotated as non-coding, or entirely unannotated, while the remainder were located on the 5’ and 3’ ends of mRNA transcripts. Only 14 non-canonical translated elements were evolutionarily conserved. In stark contrast with canonical protein-coding genes, the great majority of the yeast noncanonical translatome appeared evolutionarily transient and showed no signatures of selection. Yet, we uncovered phenotypes for 53% of a representative subset of evolutionarily transient translated elements. The iRibo framework and reference translatome described here provide a foundation for further investigation of a largely unexplored, but biologically significant, evolutionarily transient translatome.

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Here we apply ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and to analyse the host response to infection. We calculated programmed ribosomal frameshift (PRF) efficiency at both sites on the viral genome. This revealed the nsp2 PRF site as the second known example where temporally regulated frameshifting occurs, with increasing -2 PRF efficiency likely facilitated by accumulation of the PRF-stimulatory viral protein, nsp1β. Surprisingly, we find that PRF efficiency at the canonical ORF1ab frameshift site also increases over time, in contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a highly expressed 125-codon ORF overlapping nsp12, which is likely translated from novel subgenomic RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV-2, suggesting a potential conserved mechanism for temporally regulating expression of the 3'-proximal region of ORF1b. We also identified a highly translated, short upstream ORF in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. These findings reveal hidden complexity in the gene expression programmes of these important nidoviruses.Viruses have tiny genomes. Rather than carry all the genetic information they need, they rely on the cells they infect. This makes the few genes they do have all the more important. Many viruses store their genes not in DNA, but in a related molecule called RNA. When the virus infects cells, it uses the cells’ ribosomes – the machines in the cells that make proteins – to build its own proteins. One of the central ideas in biology is that one molecule of RNA carries the instructions for just one type of protein. But many viruses break this rule. The ribosomes in cells read RNA instructions in blocks of three: three RNA letters correspond to one protein building block. But certain sequences in the RNA of viruses act as hidden signals that affect how ribosomes read these molecules. These signals make the ribosomes skip backward by one or two letters on the viral RNA, restarting part way through a three-letter block. Scientists call this a ‘frameshift’, and it is a bit like changing the positions of the spaces in a sentence. The virus causes these frameshifts using proteins or by folding its RNA into a knot-like structure. The frameshifts result in the production of different viral proteins over time. The porcine reproductive and respiratory syndrome virus (PRRSV) uses frameshifts to cause devastating disease in pigs. Besides the sequences in its RNA that allow the ribosomes to skip backwards, the viral enzyme that copies the RNA can also skip forward. This results in shortened copies of its genes, which also changes the proteins they produce. To find out exactly how PRRSV uses these frameshifting techniques, Cook et al. examined infected cells in the laboratory. They monitored the RNA made by the virus and looked closely at the way the cells read it using a technique called ribosome profiling. This revealed that frameshifting increases over the course of an infection. This is partly because the viral protein that causes frameshifts builds up as infection progresses, but it also happened with frameshifts caused by RNA knots. The reason for this is less clear. Cook et al. also discovered several new RNAs made later in infection, which could also change the proteins the virus makes. RNA viruses cause disease in humans as well as pigs. Examples include coronaviruses and HIV. Many of these also have frameshift sites in their genomes. A better understanding of how frameshifts change during infection may aid drug development. Future work could help researchers to understand which proteins viruses make at which stage of infection. This could lead to new treatments for viruses like PRRSV.

Global translational remodeling has emerged as a principal mechanism of biological adaptation. Oxygen deficiency (hypoxia) disables the basal protein synthesis machinery ('Jekyll') and activates a hypoxic translational architecture ('Hyde') to drive translatome remodeling. Independent from mRNA-level fluctuations, this newer paradigm modernizes a field traditionally dominated by the hypoxia-inducible factor (HIF) transcriptional program.

We investigated the gene-expression variation among humans by analyzing previously published mRNA-seq and ribosome footprint profiling (RP) of heart left-ventricles from healthy donors. We ranked the genes according to their coefficient of variation values and found that the top 5% most variable genes had special features compared to the rest of the genome, such as lower mRNA levels and shorter half-lives coupled to increased translation efficiency. We observed that these genes are mostly involved with immune response and have a pleiotropic effect on disease phenotypes, indicating that asymptomatic conditions contribute to the gene expression diversity of healthy individuals.

The phellem is a specialized boundary tissue providing the first line of defense against abiotic and biotic stresses in organs undergoing secondary growth. Phellem cells undergo several differentiation steps, which include cell wall suberization, cell expansion and programmed cell death. Yet, the molecular players acting particularly in phellem cell differentiation remain poorly described, particularly in the widely used model plant Arabidopsis thaliana. Using specific marker lines we followed the onset and progression of phellem differentiation in A. thaliana roots, and further targeted the translatome of new developed phellem cells using Translating Ribosome Affinity Purification followed by mRNA sequencing (TRAP-SEQ). We showed that phellem suberization is initiated early after phellogen (cork cambium) division. The specific translational landscape was organized in three main domains related to energy production, synthesis and transport of cell wall components, and response to stimulus. Novel players in phellem differentiation, related to suberin monomer transport and assembly, as well as novel transcription regulators were identified. This strategy provided an unprecedented resolution of the translatome of developing phellem cells, giving a detailed and specific view on the molecular mechanisms acting on cell differentiation in periderm tissues of the model plant Arabidopsis.

Developmental and physiological processes depend on the transcriptional and translational activity of heterogeneous cell populations. A main challenge in gene expression studies is dealing with this intrinsic complexity while keeping sequencing efficiency. Translating ribosome affinity purification (TRAP) methods have allowed cell-specific recovery of polyribosome-associated RNAs by genetic tagging of ribosomes in selected cell populations. Here we combined the TRAP approach with adapted enhancer trap methods (trap-TRAP) to systematically generate zebrafish transgenic lines suitable for tissue-specific translatome interrogation. Through the random integration of a GFP-tagged version of the large subunit ribosomal protein L10a (EGFP-Rpl10a), we have generated stable lines driving expression in a variety of tissues, including the retina, skeletal muscle, lateral line primordia, rhombomeres, or jaws. To increase the range of applications, a UAS:TRAP transgenic line compatible with available Gal4 lines was also generated and tested. The resulting collection of lines and applications constitutes a resource for the zebrafish community in developmental genetics, organ physiology and disease modelling., This work is supported by grants awarded to JM-M from the Fundacio´n Ramón Areces (program-2016); PY20_00006 from Junta de Andalucía; as well as Spanish Ministry of Science, Innovation and Universities (MICINN, AEI/FEDER) BFU2017-91324-EXP, BFU2017-86339P, RED2018-102553-T, PID2020-112566GB-I00, and MDM-2016-0687.

For plant viruses, the ability to load into the vascular phloem and spread systemically within a host is an essential step in establishing a successful infection. However, access to the vascular phloem is highly regulated, representing a significant obstacle to virus loading, movement, and subsequent unloading into distal uninfected tissues. Recent studies indicate that during virus infection, phloem tissues are a source of significant transcriptional and translational alterations, with the number of virus-induced differentially expressed genes being four- to sixfold greater in phloem tissues than in surrounding nonphloem tissues. In addition, viruses target phloem-specific components as a means to promote their own systemic movement and disrupt host defense processes. Combined, these studies provide evidence that the vascular phloem plays a significant role in the mediation and control of host responses during infection and as such is a site of considerable modulation by the infecting virus. This review outlines the phloem responses and directed reprograming mechanisms that viruses employ to promote their movement through the vasculature.

Pulsed Stable Isotope Labeling in Cell culture (SILAC) approaches allow measurement of protein dynamics, including protein translation and degradation. However, its use for quantifying acute changes has been limited due to low labeled peptide stoichiometry. Here, we describe the use of instrument logic to select peaks of interest via targeted mass differences (TMD) for overcoming this limitation. Comparing peptides artificially mixed at low heavy-to-light stoichiometry measured using standard data dependent acquisition with or without TMD revealed 2–3-fold increases in identification without significant loss in quantification precision for both MS2 and MS3 methods. Our benchmarked method approach increased throughput by reducing the necessary machine time. We anticipate that all pulsed SILAC measurements, combined with tandem mass tagging (TMT) or not, would greatly benefit from instrument logic based approaches.

Globalni trend staranja prebivalstva s seboj prinaša številne izzive na področju zagotavljanja zdravstveno-socialne varnosti. Med njih sodi tudi naraščanje incidence nevrodegenerativnih bolezni, ki trenutno veljajo za neozdravljive. Posebno skupino nevrodegenerativnih bolezni predstavljajo prionske bolezni oz. transmisivne spongiformne encefalopatije, ki jih povzročajo prioni – infektivni delci v celoti sestavljeni iz napačno zvite oblike endogenega prionskega proteina, ki se v večji meri izraža na površini celic centralnega živčnega sistema. Prva oblika prionske bolezni imenovana praskavec je bila odkrita pri drobnici, za najbolj zloglasno pa velja prionska bolezen govedi, poznana tudi kot bolezen “norih krav”. Pri ljudeh glede na etiologijo ločimo pridobljene, dedne in sporadične prionske bolezni. Vsem je skupna značilna histopatološka slika z vakuolizacijo sive možganovine, ki daje tkivu prepoznaven gobast videz. Tako kot pri številnih drugih nevrodegenerativnih boleznih so tudi pri prionskih boleznih patološke spremembe sprva omejene le na določen predel možganov, kar je razlog za njihovo fenotipsko raznolikost. Mehanizem tega pojava, ki ga imenujemo selektivna dovzetnost, ostaja neznan. Ključnega pomena za njegovo razumevanje je obravnava molekulskih sprememb na ravni posameznih možganskih predelov in celic. Pri tem igrajo pomembno vlogo metode, ki omogočajo pridobitev celično specifičnih podatkov o spremembah v genskem izražanju. Med njih sodita metodi RiboTag in TRAP (angleško translating ribosome affinity purification), ki si delita osnovni koncept in vivo epitopskega označevanja ribosomalnih proteinov s pomočjo mišje mutageneze. To omogoča izolacijo na ribosome vezanih mRNA molekul (translatom), ki v primerjavi s celotno mRNA bolje odražajo proteinsko sestavo celice. S sekvenciranjem nove generacije lahko te molekule natančno okarakteriziramo in s tem pridobimo vpogled v spremembe, ki se kot odgovor na različne dražljaje odvijajo v določeni celični populaciji. Tovrsten pristop je ustrezen predvsem pri obravnavi patoloških stanj, katerih mehanizem redko vključuje le spremembe posameznih celičnih komponent. Te so namreč del interaktoma – kompleksne mreže molekulskih interakcij, ki ponazarja medsebojno odvisnost celičnih procesov. Preučevanje interaktoma na podlagi teorije grafov omogoča boljše razumevanje bolezni, kar predstavlja osnovo za iskanje novih terapevtskih tarč in biomarkerjev. Pogosto je omejeno na mreže, v katerih vozlišča predstavljajo proteine, veje oz. robovi pa fizične povezave med njimi. Proteinske mreže so tako kot številne druge mreže, ki ponazarjajo kompleksne sisteme (npr. svetovni splet), skalno neodvisne. Pomembno strukturno značilnost skalno neodvisnih mrež predstavljajo skupnosti vozlišč, katerih pripadniki med seboj tvorijo več povezav kot s preostalimi mrežnimi elementi. V proteinskih mrežah se v skupnosti, ki jih imenujemo bolezenski moduli, pogosto združujejo proteini povezani z določenim patološkim stanjem. Vozlišča, ki sestavljajo bolezenske module, lahko identificiramo s pomočjo različnih algoritmov. Eden izmed takšnih je DIAMOnD, s katerim vozlišča v bolezenski modul dodajamo postopno, na podlagi signifikantnosti števila njihovih povezav z začetnim setom znanih bolezenskih proteinov. V magistrski nalogi smo mrežni pristop uporabili za preučevanje odgovora različnih možganskih celic na prionsko okužbo. Pri tem smo izhajali iz podatkov, ki so bili pridobljeni v predhodno izvedeni študiji na mišjem modelu praskavca, s pomočjo metode RiboTag. Podatki vsebujejo informacije o spremembah v genskem izražanju pri petih vrstah možganskih celic v dveh časovnih točkah: pred pojavom zaznavnih sprememb možganske aktivnosti in po pojavu bolezenskih znakov. Ker se pri napredovali bolezni možnosti za uspešno terapevtsko intervencijo manjše, smo se osredotočili le na spremembe v prvi časovni točki. Kot izhodišče analize smo uporabili seznam genov s pripadajočimi p-vrednostmi, ki določajo signifikantnost spremembe njihovega izražanja. Pogosto pri razvoju bolezni sodelujejo funkcionalno povezani geni, ki posamezno ne izkazujejo statistične pomembnosti, temveč imajo velik kumulativni učinek. Izbira diferencialno izraženih genov na podlagi arbitrarne mejne p-vrednosti je torej lahko nepopolna. Da bi bolje definirali ključne bolezenske spremembe v zgodnji fazi prionske okužbe, smo gene, katerih izražanje je signifikantno spremenjeno, uporabili kot izhodiščna vozlišča pri iskanju celično specifičnih bolezenskih modulov. Sprva smo v ta namen s pomočjo spletne podatkovne baze STRING izgradili mrežo proteinskih interakcij. Le 50.1 % teh interakcij je podprto z eksperimentalnimi dokazi, večji delež proteinov pa si povezave deli na podlagi sonavedb v znanstveni literaturi. Število povezav, ki si jih določen protein deli z drugimi vozlišči je tako odvisno od tega kako dobro je ta protein raziskan. Morebitna pristranskost do bolje raziskanih proteinov lahko vpliva na identifikacijo vozlišč bolezenskih modulov. Da bi ta vpliv zmanjšali, smo razvili modificirano različico algoritma DIAMOnD, pri katerem je vsakemu kandidatnemu bolezenskemu proteinu/genu pripisan rang glede na število njegovih povezav z izhodiščnimi vozlišči in glede na p-vrednost, ki določa signifikantnost spremembe njegovega izražanja. Gen z najvišjo vsoto obratne vrednosti posameznih rangov se vključi v bolezenski modul. Hkrati sestavlja tudi set izhodiščnih vozlišč v nadaljnjih ponovitvah algoritma. Z uporabo modificirane različice algoritma DIAMOnD smo odkrili bolezenske module treh vrst možganskih celic: astrocitov, GABAergičnih nevronov in glutamatergičnih nevronov. Uspešnost algoritma smo ocenili na podlagi primerjave z osnovnim algoritmom. S slednjim smo identificirali večje število genov, ki si z izhodiščnimi vozlišči delijo funkcionalne značilnosti. Število funkcionalno sorodnih genov je bilo signifikantno tudi v bolezenskih modulih zaznanih z modificiranim algoritmom, ki pa so vsebovali večji delež izhodiščnih vozlišč. Največ genov je bilo vključenih v astrocitni modul, med njimi številni, ki kodirajo komponente dihalne verige. Znižano izražanje teh genov najverjetneje vodi v oksidativni stres in zmanjšano proizvodnjo ATP. Posledično pride do zmanjšane sinteze proteinov in sprememb v procesiranju RNA molekul. Zaradi prionske okužbe lahko pride do porasta prostih kisikovih radikalov tudi v GABAergičnih nevronih. Poleg tega so v teh celicah prisotne spremembe v izražanju genov, ki sodelujejo pri uravnavanju cirkadialnega ritma. Po drugi strani se v populaciji ekscitatornih nevronov aktivirajo nekateri obrambni mehanizmi. Povečano izražanje genov, ki kodirajo citoskeletne proteine npr. pripomore k mehanski stabilizaciji celic in ohranjanju sinaptične plastičnosti. Na podlagi rezultatov mrežne analize lahko sklepamo, da se različne možganske celice na razvijajočo se prionsko bolezen odzovejo drugače. Pravilnost te ugotovitve je med drugim odvisna od učinkovitosti metode RiboTag. Ta omogoča izolacijo mRNA, vezane na ribosome, ki vsebujejo označen ribosomalni protein Rpl22. S tem se RiboTag razlikuje od metode TRAP, pri kateri je epitopski označevalec pripet na ribosomalni protein Rpl10a. Nedavni dokazi, ki pričajo o obstoju ribosomov s specializiranimi funkcijami, kažejo na morebitne razlike v učinkovitosti med metodama. Rpl22 in Rpl10 se lahko namreč prednostno vključujeta v ribosome, ki sodelujejo pri translaciji le določenih mRNA molekul. V magistrski nalogi smo želeli razviti sistem, ki bi omogočal ustrezno vrednotenje te hipoteze. V ta namen smo izdelali dva plazmidna vektorja z zapisom za označena Rpl22 in Rpl10a in ju uporabili za transfekcijo mišjih embrionalnih matičnih celic. Z uporabo sistema CRISPR/Cas9 smo omogočili vstavitev kodirajočih zaporedij za modificirana proteina v lokus Rosa26. Uspešnost tarčnega genskega preurejanja smo preverili z reakcijo PCR. Neurodegenerative diseases are a group of disorders affecting the nervous system that develop as a consequence of protein misfolding and aggregation. These pathological changes affect only specific brain areas and the mechanisms behind this regional preference are unknown. Elucidating the strategies employed by different brain cell types in confronting protein aggregates would lead to a better understanding of this phenomenon, known as selective vulnerability. To facilitate this task, several transgenic tools were developed to study cell-type-specific gene regulation in mice. Among the most notable are RiboTag and TRAP (translating ribosome affinity purification), which rely on epitope-tagging of polysomes (translating ribosomes) for the extraction of ribosome-bound mRNA. Combining RiboTag or TRAP with RNA sequencing provides a genome-wide snapshot of the changes occurring in specific cell populations under various conditions. This strategy is particularly suited for studying pathophysiological states which rarely develop as a consequence of a single gene or protein abnormality. Rather, the cellular events resulting in disease can be viewed as a perturbation of an intricate network of interactions between diverse molecular components. Applying the concepts of graph/network theory to molecular interaction networks can contribute to understanding of disease mechanisms, thereby facilitating drug-target prediction and biomarker discovery. In this thesis, we used a network-based approach to study how different brain cell types respond to a prion disease called scrapie, a fatal neurological disorder caused by misfolding of the endogenous prion protein. Prion disease-related communities within cell-type-specific molecular networks (disease modules) were identified using a modified DIAMOnD algorithm in which information on protein interaction patterns was combined with data from a RiboTag-based gene expression study. We show that the modified algorithm can outperform the original version. Moreover, we reveal that astrocytes, GABAergic neurons, and glutamatergic neurons each face ensuing prion disease with unique changes to gene expression. To verify these results in the context of the recently discovered phenomenon of ribosome specialization, we developed a tool for side-by-side comparison of RiboTag and TRAP technologies. Particularly, we established two murine cell lines using CRISPR/Cas9 which allow for an unbiased comparison of mRNA profiles obtained by both methods.

Cyanobacteria are promising industrial platforms owing to their ability to produce diverse natural secondary metabolites and nonnative value-added biochemicals from CO2 and light. To fully utilize their industrial potency, it is critical to understand their photosynthetic efficiency under various environmental conditions. In this study, we elucidated the inhibitory mechanisms of photosynthesis under high-light and low-temperature stress conditions in the model cyanobacterium Synechocystis sp. PCC 6803. Under each stress condition, the transcript abundance and translation efficiency were measured using transcriptome sequencing (RNA-seq) and ribosome profiling, and the genome-wide transcription unit architecture was constructed by data integration of transcription start sites and transcript 3′-end positions obtained from differential RNA-seq and sequencing of 3′-ends (Term-seq), respectively. Our results suggested that the mode of photosynthesis inhibition differed between the two stress conditions; high light stress induced photodamage responses, while low temperature stress impaired the translation efficiency of photosynthesis-associated genes. In particular, poor translation of photosystem I resulted from ribosome stalling at the untranslated regions, affecting the overall photosynthetic yield under low temperature stress. Our comprehensive multiomics analysis with transcription unit architecture provides foundational information on photosynthesis for future industrial strain development. IMPORTANCE Cyanobacteria are a compelling biochemical production platform for their ability to propagate using light and atmospheric CO2 via photosynthesis. However, the engineering of strains is hampered by limited understanding of photosynthesis under diverse environmental conditions such as high-light and low-temperature stresses. Herein, we decipher the transcriptomic and translatomic responses of the photosynthetic efficiency to stress conditions using the integrative analysis of multiomic data generated by RNA-seq and ribosome profiling, respectively. Through the generated massive data, along with the guide of the genome-wide transcription unit architecture constructed by transcription start sites and transcript 3′-end positions, we identified the factors affecting photosynthesis at transcription, posttranscription, and translation levels. Importantly, the high-light stress induces photodamage responses, and the low-temperature stress cripples the translation efficiency of photosynthesis-associated genes. The resulting insights provide pivotal information for future cyanobacterial cell factories powered by the engineering toward robust photosynthesis ability.

In eukaryotes, stalled and collided ribosomes are recognized by several conserved multicomponent systems, which either block protein synthesis in situ and resolve the collision locally, or trigger a general stress response. Yeast ribosome-binding GTPases RBG1 (DRG1 in mammals) and RBG2 (DRG2) form two distinct heterodimers with TMA46 (DFRP1) and GIR2 (DFRP2), respectively, both involved in mRNA translation. Accumulated evidence suggests that the dimers play partially redundant roles in elongation processivity and resolution of ribosome stalling and collision events, as well as in the regulation of GCN1-mediated signaling involved in ribosome-associated quality control (RQC). They also genetically interact with SLH1 (ASCC3) helicase, a key component of RQC trigger (RQT) complex disassembling collided ribosomes. Here, we present RNA-Seq and ribosome profiling (Ribo-Seq) data from S. cerevisiae strains with individual deletions of the TMA46 and GIR2 genes. Raw RNA-Seq and Ribo-Seq data as well as gene-level read counts are available in NCBI Gene Expression Omnibus (GEO) repository under GEO accession GSE185458 and GSE185286.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus which causes significant economic losses to the swine industry worldwide. Here, the viral translatome was characterised using ribosome profiling (RiboSeq), a high-throughput sequencing-based technique that allows the positions of translating ribosomes to be mapped to a genome with sub-codon precision. This was used in parallel with RNA sequencing (RNASeq) to analyse changes in viral gene expression over a timecourse of infection in MARC-145 cells. The PRRSV genome contains two programmed ribosomal frameshift (PRF) signals, at which conserved elements promote the backwards slippage of the ribosome by one or two nucleotides. PRRSV encodes a canonical −1 PRF site, at which frameshifting is directed by a downstream RNA pseudoknot structure, to facilitate expression of the ORF1b-encoded viral replicase. At a second PRF site, both −1 and −2 PRF are stimulated by a trans-acting complex of a viral (nsp1β) and cellular (PCBP) protein in a unique, non-canonical mechanism, generating alternative forms (nsp2N and nsp2TF) of a viral non-structural protein, nsp2. Frameshift efficiency at both sites was quantified and, at the non-canonical site, was found to increase with time, rendering this the second known example of temporally regulated PRF during infection. This novel aspect of viral gene expression regulation is likely facilitated by accumulation of the PRF-stimulatory viral protein, nsp1β, during the virus life cycle. Surprisingly, frameshift efficiency at the canonical ORF1ab PRF site was also found to increase over time, overturning the common assumption that RNA structure-directed sites operate at a fixed efficiency, with potential implications for the numerous other viruses which encode canonical PRF sites. Several highly translated additional ORFs were discovered in the PRRSV genome, the translation of which is potentially facilitated by multiple novel viral transcripts. For example, a 125-codon ORF overlapping nsp12 was discovered, which is expressed as highly as nsp12 itself in the late stages of replication and likely translated from novel subgenomic (sg) RNA transcripts overlapping the 3′ end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV-2, suggesting a potential conserved mechanism for temporal regulation of expression of the 3′-proximal region of ORF1b. In addition, a highly translated, short upstream ORF (uORF) was identified in the 5′ UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. This work is the first application of RiboSeq to arterivirus-infected cells and reveals new features which add to our understanding of the complexity of gene expression programmes in this important family of nidoviruses.

The emergence of ribosome profiling as a tool for measuring the translatome has provided researchers with valuable insights into the post-transcriptional regulation of gene expression. Despite the biological insights and technical improvements made since the technique was initially described by Ingolia et al. (Science 324(5924):218-223, 2009), ribosome profiling measurements and subsequent data analysis remain challenging. Here, we describe our lab's protocol for performing ribosome profiling in bacteria, yeast, and mammalian cells. This protocol has integrated elements from three published ribosome profiling methods. In addition, we describe a tool called RiboViz (Carja et al., BMC Bioinformatics 18:461, 2017) ( https://github.com/riboviz/riboviz ) for the analysis and visualization of ribosome profiling data. Given raw sequencing reads and transcriptome information (e.g., FASTA, GFF) for a species, RiboViz performs the necessary pre-processing and mapping of the raw sequencing reads. RiboViz also provides the user with various quality control visualizations.

Significance Proteins are the key drivers of neuronal synaptic function. The regulation of gene expression is important for the formation and modification of synapses throughout the lifespan. The complexity of dendrites and axons imposes unique challenges for protein supply at remote locations. The discovery of messenger RNAs (mRNAs) and ribosomes near synapses has shown that local protein synthesis represents an important solution to this challenge. Here we used RNA sequencing and ribosome sequencing to determine directly the population of mRNAs that is present and in the process of translation in neuronal cell bodies, dendrites, and axons. Thousands of transcripts were differentially translated between the cell body and synaptic regions with over 800 mRNAs exhibiting more translation in the dendritic–axonal compartment., To form synaptic connections and store information, neurons continuously remodel their proteomes. The impressive length of dendrites and axons imposes logistical challenges to maintain synaptic proteins at locations remote from the transcription source (the nucleus). The discovery of thousands of messenger RNAs (mRNAs) near synapses suggested that neurons overcome distance and gain autonomy by producing proteins locally. It is not generally known, however, if, how, and when localized mRNAs are translated into protein. To investigate the translational landscape in neuronal subregions, we performed simultaneous RNA sequencing (RNA-seq) and ribosome sequencing (Ribo-seq) from microdissected rodent brain slices to identify and quantify the transcriptome and translatome in cell bodies (somata) as well as dendrites and axons (neuropil). Thousands of transcripts were differentially translated between somatic and synaptic regions, with many scaffold and signaling molecules displaying increased translation levels in the neuropil. Most translational changes between compartments could be accounted for by differences in RNA abundance. Pervasive translational regulation was observed in both somata and neuropil influenced by specific mRNA features (e.g., untranslated region [UTR] length, RNA-binding protein [RBP] motifs, and upstream open reading frames [uORFs]). For over 800 mRNAs, the dominant source of translation was the neuropil. We constructed a searchable and interactive database for exploring mRNA transcripts and their translation levels in the somata and neuropil [MPI Brain Research, The mRNA translation landscape in the synaptic neuropil. https://public.brain.mpg.de/dashapps/localseq/. Accessed 5 October 2021]. Overall, our findings emphasize the substantial contribution of local translation to maintaining synaptic protein levels and indicate that on-site translational control is an important mechanism to control synaptic strength.

Understanding how roots modulate development under varied irrigation or rainfall is crucial for development of climate-resilient crops. We established a toolbox of tagged rice lines to profile translating mRNAs and chromatin accessibility within specific cell populations. We used these to study roots in a range of environments: plates in the lab, controlled greenhouse stress and recovery conditions, and outdoors in a paddy. Integration of chromatin and mRNA data resolves regulatory networks of the following: cycle genes in proliferating cells that attenuate DNA synthesis under submergence; genes involved in auxin signaling, the circadian clock, and small RNA regulation in ground tissue; and suberin biosynthesis, iron transporters, and nitrogen assimilation in endodermal/exodermal cells modulated with water availability. By applying a systems approach, we identify known and candidate driver transcription factors of water-deficit responses and xylem development plasticity. Collectively, this resource will facilitate genetic improvements in root systems for optimal climate resilience.

Widespread sleep deprivation is a continuing public health problem in the United States and worldwide affecting adolescents and adults. Acute sleep deprivation results in decrements in spatial memory and cognitive impairments. The hippocampus is vulnerable to acute sleep deprivation with changes in gene expression, cell signaling, and protein synthesis. Sleep deprivation also has long lasting effects on memory and performance that persist after recovery sleep, as seen in behavioral studies from invertebrates to humans. Although previous research has shown that acute sleep deprivation impacts gene expression, the extent to which sleep deprivation affects gene regulation remains unknown. Using an unbiased deep RNA sequencing approach, we investigated the effects of acute sleep deprivation on gene expression in the hippocampus. We identified 1,146 genes that were significantly dysregulated following sleep deprivation with 507 genes upregulated and 639 genes downregulated, including protein coding genes and long non-coding RNAs not previously identified as impacted by sleep deprivation. Notably, genes significantly upregulated after sleep deprivation were associated with RNA splicing and the nucleus. In contrast, downregulated genes were associated with cell adhesion, dendritic localization, the synapse, and postsynaptic membrane. Furthermore, we found through independent experiments analyzing a subset of genes that three hours of recovery sleep following acute sleep deprivation was sufficient to normalize mRNA abundance for most genes, although exceptions occurred for some genes that may affect RNA splicing or transcription. These results clearly demonstrate that sleep deprivation differentially regulates gene expression on multiple transcriptomic levels to impact hippocampal function. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00835-1.

Non-alcoholic fatty liver disease (NAFLD) is a common disease with a multitude of complications. Increasing evidence shows that the dietary supplement with betaine, a natural chemical molecule, can effectively reduce the fat accumulation in the liver. Translational regulation is considered to play a vital role in gene expression, but whether betaine functions through the regulation of gene translational level is still unclear. To this end, RNC-seq (ribosome-nascent chain complex bound mRNA sequencing) and RNA-seq co-analyses were performed to identify betaine target genes by using the liver samples from high-fat diet + betaine treated and high-fat diet treated mice. The results showed that betaine does play a lipid-lowering role by regulating the expression of gene translation levels; some NAFLD- and lipid metabolism- associated genes were differentially expressed at translational level, for example. And the mRNA translation ratio (TR) of gene significantly increased after betaine treatment. Besides, it is found that the regulation of some genes at transcriptional level is opposite to that at translational level, which indicates that transcriptional regulation and translational regulation may be independent from each other. Finally, we identified several candidate genes, especially Gpc1 , which may mediate the lipid-lowering effect of betaine in the liver. To sum up, this study depicted the molecular portrait of mice liver with or without betaine treatment from the angel of translatome and transcriptome, giving insights into the molecular mechanism of betaine-mediated lipid-lowering effect and also providing new clues for understanding and prevention of NAFLD.

In human primary dendritic cells (DC) rapamycin—an autophagy inducer and protein synthesis inhibitor—overcomes the autophagy block induced by Mycobacterium tuberculosis (Mtb) and promotes a Th1 response via IL-12 secretion. Here, the immunostimulatory activity of rapamycin in Mtb-infected DC was further investigated by analyzing both transcriptome and translatome gene profiles. Hundreds of differentially expressed genes (DEGs) were identified by transcriptome and translatome analyses of Mtb-infected DC, and some of these genes were found further modulated by rapamycin. The majority of transcriptome-associated DEGs overlapped with those present in the translatome, suggesting that transcriptionally stimulated mRNAs are also actively translated. In silico analysis of DEGs revealed significant changes in intracellular cascades related to cytokine production, cytokine-induced signaling and immune response to pathogens. In particular, rapamycin treatment of Mtb-infected DC caused an enrichment of IFN-β, IFN-λ and IFN-stimulated gene transcripts in the polysome-associated RNA fraction. In addition, rapamycin led to an increase of IL-12, IL-23, IL-1β, IL-6, and TNF-α but to a reduction of IL-10. Interestingly, upon silencing or pharmacological inhibition of GSK-3β, the rapamycin-driven modulation of the pro- and anti-inflammatory cytokine balance was lost, indicating that, in Mtb-infected DC, GSK-3β acts as molecular switch for the regulation of the cytokine milieu. In conclusion, our study sheds light on the molecular mechanism by which autophagy induction contributes to DC activation during Mtb infection and points to rapamycin and GSK-3β modulators as promising compounds for host-directed therapy in the control of Mtb infection.

Although transcriptome changes have long been recognized as a mechanism to induce tentative substitution of expressed genes in diverse biological processes in plants, the regulation of translation—the final step of the central dogma of molecular biology—emerged as an alternative and prominent layer in defining the output of genes. Despite these demands, the genome-wide analysis of protein synthesis has posed technical challenges, resulting in the plant translatome being poorly understood. The development of ribosome profiling promises to address the hidden aspects of translation, and its application to plants is revolutionizing our knowledge of the translatome. This review outlines the array of recent findings provided by ribosome profiling and illustrates the power of the versatile technique in green organisms.

Despite the critical position of translation in the multilevel gene expression regulation program, high-resolution and genome-wide view of the landscape of RNA translation in solid tumors is still limited. Methods: With a ribosome profiling procedure optimized for solid tissue samples, we profiled the translatomes of liver tumors and their adjacent noncancerous normal liver tissues from 10 patients with hepatocellular carcinoma (HCC). A set of bioinformatics tools was then applied to these data for the mining of novel insights into the translation shifts in HCC. Results: This is the first translatome data resource for dissecting dysregulated translation in HCC at the sub-codon resolution. Based on our data, quantitative comparisons of mRNA translation rates yielded the genes and processes that were subjected to patient specific or universal dysregulations of translation efficiencies in tumors. For example, multiple proteins involved in extracellular matrix organization exhibited significant translational upregulation in tumors. We then experimentally validated the tumor-promoting functions of two such genes as examples: AGRN and VWA1. In addition, the data was also used for de novo annotation of the translatomes in tumors and normal tissues, including multiple types of novel non-canonical small ORFs, which would be a resource for further functional studies. Conclusions: The present study generates the first survey of the HCC translatome with ribosome profiling, which is an insightful data resource for dissecting the translatome shift in liver cancer, at sub-codon resolution.

riboCIRC is a translatome data-oriented circRNA database specifically designed for hosting, exploring, analyzing, and visualizing translatable circRNAs from multi-species. The database provides a comprehensive repository of computationally predicted ribosome-associated circRNAs, a manually curated collection of experimentally verified translated circRNAs, an evaluation of cross-species conservation of translatable circRNAs, a systematic de novo annotation of putative circRNA-encoded peptides, including sequence, structure, and function, and a genome browser to visualize the context-specific occupant footprints of circRNAs. It represents a valuable resource for the circRNA research community and is publicly available at http://www.ribocirc.com.

Jasmonoyl-L-isoleucine (JA-Ile) is a powerful oxylipin responsible for the genome-wide transcriptional reprogramming in plants that results in major physiological shifts from growth to defense. The double T-DNA insertion Arabidopsis mutant, cyp94b1cyp94b3 (b1b3), defective in cytochrome p450s, CYP94B1 and CYP94B3, which are responsible for oxidizing JA-Ile, accumulates several fold higher levels of JA-Ile yet displays dampened JA-Ile–dependent wound responses—the opposite of what is expected. Transcriptomic and proteomic analyses showed that while the transcriptional response to wounding was largely unchanged in b1b3 compared to wild type (WT), many proteins were found to be significantly reduced in the mutant, which was verified by immunoblot analyses of marker proteins. To understand this protein phenotype and their hypothesized contribution to the b1b3 phenotypes, wounded rosette leaf samples from both WT and b1b3 were subject to a translating ribosome affinity purification RNA sequencing analysis. More than 1,600 genes whose transcripts do not change in abundance by wounding changed their association with the ribosomes after wounding in WT leaves. Consistent with previous observations, the total pool of mRNA transcripts was similar between WT and b1b3; however, the ribosome-associated pool of transcripts was changed significantly. Most notably, fewer transcripts were associated with the ribosome pool in b1b3 than in WT, potentially explaining the reduction of many proteins in the mutant. Among those genes with fewer ribosome-associated transcripts in b1b3 were genes relating to stress response, specialized metabolism, protein metabolism, ribosomal subunits, and transcription factors, consistent with the biochemical phenotypes of the mutant. These results show previously unrecognized regulations at the translational level that are affected by misregulation of JA homeostasis during the wound response in plants.

The global COVID-19 pandemic caused by SARS-CoV-2 has resulted in over 2.2 million deaths. Disease outcomes range from asymptomatic to severe with, so far, minimal genotypic change to the virus so understanding the host response is paramount. Transcriptomics has become incredibly important in understanding host-pathogen interactions, however, post-transcriptional regulation plays an important role in infection and immunity through translation and mRNA stability, allowing tight control over potent host responses by both the host and the invading virus. Here, we apply ribosome profiling to assess post-transcriptional regulation of host genes during SARS-CoV-2 infection of a human lung epithelial cell line (Calu-3). We have identified numerous transcription factors (JUN, ZBTB20, ATF3, HIVEP2 and EGR1) as well as select antiviral cytokine genes, namely IFNB1, IFNL1,2 and 3, IL-6 and CCL5, that are restricted at the post-transcriptional level by SARS-CoV-2 infection and discuss the impact this would have on the host response to infection. This early phase restriction of antiviral transcripts in the lungs may allow high viral load and consequent immune dysregulation typically seen in SARS-CoV-2 infection.

A Correction to this paper has been published: https://doi.org/10.1038/s41597-021-00858-2.

One gene could be transcribed to different RNA isoforms, and then produce various forms of protein sequences. This mechanism largely diversifies the cellular pool and allows natural selection to select from a wider range of substrates. Most of the deleterious changes should be either purged or only be observed in patients with deficiencies or diseases. In the cancer field, the “intra-gene” changes between tumor and normal tissues such as the alternative splicing, stop codon read-through, or protein domestication could not be captured by differential expression analyses. In this work, we collected public transcriptome and translatome data from ten patients with liver cancer, and performed genome-wide comparison on the stop codon read-through and protein domestication events. Both events could diversify the proteome without changing the genome sequence. Surprisingly, we found that compared to normal tissues, the tumor tissues globally have significantly higher occurrence of stop codon read-through events. Similarly, the translation signals of non-coding repetitive elements (protein domestication) are elevated in tumor samples. These read-through and domestication events show limited overlapping across the ten patients, suggesting the randomness of the occurrence. It also indicates that these tumor-specific read-through and domestication events should be deleterious, and should be purged by natural selection if they are not collected timely. Our work manifests the role of protein extension and domestication in liver cancer oncogenesis, and adds new aspects to the cancer field.

The reprogramming of gene expression is one of the key responses to environmental stimuli, whereas changes in mRNA do not necessarily bring forth corresponding changes of the protein, which seems partially due to the stress-induced selective translation. To address this issue, we systematically compared the transcriptome and translatome using self-produced and publicly available datasets to decipher how and to what extent the coordination and discordance between transcription and translation came to be in response to wounding (self-produced), dark to light transition, heat, hypoxia, Pi starvation and the pathogen-associated molecular pattern (elf18) in Arabidopsis. We found that changes in total mRNAs (transcriptome) and ribosome-protected fragments (translatome) are highly correlated upon dark to light transition or heat stress. However, this close correlation was generally lost under other four stresses analyzed in this study, especially during immune response, which suggests that transcription and translation are differentially coordinated under distinct stress conditions. Moreover, Gene Ontology (GO) enrichment analysis showed that typical stress responsive genes were upregulated at both transcriptional and translational levels, while non-stress-specific responsive genes were changed solely at either level or downregulated at both levels. Taking wounding responsive genes for example, typical stress responsive genes are generally involved in functional categories related to dealing with the deleterious effects caused by the imposed wounding stress, such as response to wounding, response to water deprivation and response to jasmonic acid, whereas non-stress-specific responsive genes are often enriched in functional categories like S-glycoside biosynthetic process, photosynthesis and DNA-templated transcription. Collectively, our results revealed the differential as well as targeted coordination between transcriptome and translatome in response to diverse stresses, thus suggesting a potential model wherein preferential ribosome loading onto the stress-upregulated mRNA pool could be a pacing factor for selective translation.

Ribosomes execute the transcriptional program in every cell. Critical to sustain nearly all cellular activities, ribosome biogenesis requires the translation of ~200 factors of which 80 are ribosomal proteins (RPs). As ribosome synthesis depends on RP mRNA translation, a priority within the translatome architecture should exist to ensure the preservation of ribosome biogenesis capacity, particularly under adverse growth conditions. Here, we show that under critical metabolic constraints characterized by mTOR inhibition, LARP1 complexed with the 40S subunit protects from ribophagy the mRNAs regulon for ribosome biogenesis and protein synthesis, acutely preparing the translatome to promptly resume ribosomes production after growth conditions return permissive. Characterizing the LARP1-protected translatome revealed a set of 5′TOP transcript isoforms other than RPs involved in energy production and in mitochondrial function, among other processes, indicating that the mTOR-LARP1-5′TOP axis acts at the translational level as a primary guardian of the cellular anabolic capacity.

SUMMARYA vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode uncharacterized proteins, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated lncRNAs and peptide-producing lncRNAs.Here we present AHARIBO, a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs and correspondingde novosynthesized polypeptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds lncRNAs.

Over the past decade, ample transcriptome data have been generated at different stages during seed germination; however, far less is known about protein synthesis during this important physiological process. Generally, the correlation between transcript levels and protein abundance is low, which strongly limits the use of transcriptome data to accurately estimate protein expression. Polysomal profiling has emerged as a tool to identify mRNAs that are actively translated. The association of the mRNA to the polysome, also referred to as translatome, provides a proxy for mRNA translation. In this study, the correlation between the changes in total mRNA, polysome-associated mRNA, and protein levels across seed germination was investigated. The direct correlation between polysomal mRNA and protein abundance at a single time-point during seed germination is low. However, once the polysomal mRNA of a time-point is compared to the proteome of the next time-point, the correlation is much higher. 35% of the investigated proteome has delayed changes at the protein level. Genes have been classified based on their delayed protein changes, and specific motifs in these genes have been identified. Moreover, mRNA and protein stability and mRNA length have been found as important predictors for changes in protein abundance. In conclusion, polysome association and/or dissociation predicts future changes in protein abundance in germinating seeds.

Jasmonoyl-L-isoleucine (JA-Ile) is a powerful oxylipin responsible for the genome-wide transcriptional reprogramming in plants that results in major physiological shifts from growth to defense. The double T-DNA insertion Arabidopsis mutant, cyp94b1cyp94b3 (b1b3), defective in cytochrome p450s, CYP94B1 and CYP94B3, which are responsible for oxidizing JA-Ile, accumulates several fold higher levels of JA-Ile yet displays dampened JA-Ile–dependent wound responses—the opposite of what is expected. Transcriptomic and proteomic analyses showed that while the transcriptional response to wounding was largely unchanged in b1b3 compared to wild type (WT), many proteins were found to be significantly reduced in the mutant, which was verified by immunoblot analyses of marker proteins. To understand this protein phenotype and their hypothesized contribution to the b1b3 phenotypes, wounded rosette leaf samples from both WT and b1b3 were subject to a translating ribosome affinity purification RNA sequencing analysis. More than 1,600 genes whose transcripts do not change in abundance by wounding changed their association with the ribosomes after wounding in WT leaves. Consistent with previous observations, the total pool of mRNA transcripts was similar between WT and b1b3; however, the ribosome-associated pool of transcripts was changed significantly. Most notably, fewer transcripts were associated with the ribosome pool in b1b3 than in WT, potentially explaining the reduction of many proteins in the mutant. Among those genes with fewer ribosome-associated transcripts in b1b3 were genes relating to stress response, specialized metabolism, protein metabolism, ribosomal subunits, and transcription factors, consistent with the biochemical phenotypes of the mutant. These results show previously unrecognized regulations at the translational level that are affected by misregulation of JA homeostasis during the wound response in plants.

Background Copper (Cu) is an essential metal mediating a variety of vital biological reactions with its redox property. Its dyshomeostasis has been associated with accelerated cognitive decline and neurodegenerative disorders, such as Alzheimer’s disease (AD). However, underlying neurotoxic mechanisms elicited by dysregulated Cu remain largely elusive. We and others previously demonstrated that exposure to Cu in drinking water significantly exacerbated pathological hallmarks of AD and pro-inflammatory activation of microglia, coupled with impaired phagocytic capacity, in mouse models of AD. Methods In the present study, we extended our investigation to evaluate whether chronic Cu exposure to wild-type (WT) and J20 mouse model of AD perturbs homeostatic dynamics of microglia and contributes to accelerated transformation of microglia towards degenerative phenotypes that are closely associated with neurodegeneration. We further looked for evidence of alterations in the microglial morphology and spatial memory of the Cu-exposed mice to assess the extent of the Cu toxicity. Results We find that chronic Cu exposure to pre-pathological J20 mice upregulates the translation of degenerative genes and represses homeostatic genes within microglia even in the absence amyloid-beta plaques. We also observe similar expression signatures in Cu-exposed WT mice, suggesting that excess Cu exposure alone could lead to perturbed microglial homeostatic phenotypes and contribute to accelerated cognitive decline. Conclusion Our findings highlight the risk of chronic Cu exposure on cognitive decline and altered microglia activation towards degenerative phenotypes. These changes may represent one of the key mechanisms linking Cu exposure or its dyshomeostasis to an increased risk for AD.

The translatome, a profile of the translational status of genetic information within cells, provides a new perspective on gene expression. Although many plant genomes have been sequenced, comprehensive translatomic annotations are not available for plants due to a lack of efficient translatome profiling techniques. Here, we developed a new technique termed 3′ ribosome-profiling sequencing (3′Ribo-seq) for reliable, robust translatomic profiling. 3′Ribo-seq combines polysome profiling and 3′ selection with a barcoding and pooling strategy. Systematic translatome profiling of different tissues of Arabidopsis, rice, and maize using conventional ribosome profiling (Ribo-seq) and 3′Ribo-seq revealed many novel translational genomic loci, thereby complementing functional genome annotation in plants. Using the low-cost, efficient 3′Ribo-seq technique and genome-wide association mapping of translatome expression (eGWAS), we performed a population-level dissection of the translatomes of 159 diverse maize inbred lines and identified 1,777 translational expression quantitative trait loci (eQTLs). Notably, local eQTLs are significantly enriched in the 3′ untranslated regions of genes. Detailed eQTL analysis suggested that sequence variation around the polyadenylation (polyA) signal motif plays a key role in translatomic variation. Our study provides a comprehensive translatome annotation of plant functional genomes and introduces 3′Ribo-seq, which paves the way for deep translatomic analysis at the population level., This study reports a new method, 3′Ribo-Seq, for translatome profiling and systematically annotates the genomes of Arabidopsis, rice, and maize using both conventional Ribo-seq and 3′Ribo-Seq, thereby uncovering many new functional loci in plants. A genome-wide association study of translatomic expression at the population level reveals a key role for genic 3′ untranslated regions in translatomic variation.

Key message Arabidopsis pollen transcriptome analysis revealed new intergenic transcripts of unknown function, many of which are long non-coding RNAs, that may function in pollen-specific processes, including the heat stress response. Abstract The male gametophyte is the most heat sensitive of all plant tissues. In recent years, long noncoding RNAs (lncRNAs) have emerged as important components of cellular regulatory networks involved in most biological processes, including response to stress. While examining RNAseq datasets of developing and germinating Arabidopsis thaliana pollen exposed to heat stress (HS), we identified 66 novel and 246 recently annotated intergenic expressed loci (XLOCs) of unknown function, with the majority encoding lncRNAs. Comparison with HS in cauline leaves and other RNAseq experiments indicated that 74% of the 312 XLOCs are pollen-specific, and at least 42% are HS-responsive. Phylogenetic analysis revealed that 96% of the genes evolved recently in Brassicaceae. We found that 50 genes are putative targets of microRNAs and that 30% of the XLOCs contain small open reading frames (ORFs) with homology to protein sequences. Finally, RNAseq of ribosome-protected RNA fragments together with predictions of periodic footprint of the ribosome P-sites indicated that 23 of these ORFs are likely to be translated. Our findings indicate that many of the 312 unknown genes might be functional and play a significant role in pollen biology, including the HS response.

Our understanding of the transition from physiological to pathological cardiac hypertrophy remains elusive and largely based on reductionist hypotheses. Here, we profiled the translatomes of 15 mouse hearts to provide a molecular blueprint of altered gene networks in early cardiac remodeling. Using co-expression analysis, we reveal how sub-networks are orchestrated into functional modules associated with pathological phenotypes. We show how transcriptome networks are only partially reproducible at the translatome level. We find unappreciated hub genes and genes in the transcriptional network that were rewired in the translational network, and associated with semantically different subsets of enriched functional terms, providing novel insights into the complexity of the organization of in vivo cardiac regulatory networks.

Motivation Ribosome profiling (Ribo-seq) is a powerful approach based on deep sequencing of cDNA libraries generated from ribosome-protected RNA fragments to explore the translatome of a cell, and is especially useful for the detection of small proteins (50–100 amino acids) that are recalcitrant to many standard biochemical and in silico approaches. While pipelines are available to analyze Ribo-seq data, none are designed explicitly for the automatic processing and analysis of data from bacteria, nor are they focused on the discovery of unannotated open reading frames (ORFs). Results We present HRIBO (High-throughput annotation by Ribo-seq), a workflow to enable reproducible and high-throughput analysis of bacterial Ribo-seq data. The workflow performs all required pre-processing and quality control steps. Importantly, HRIBO outputs annotation-independent ORF predictions based on two complementary bacteria-focused tools, and integrates them with additional feature information and expression values. This facilitates the rapid and high-confidence discovery of novel ORFs and their prioritization for functional characterization. Availability and implementation HRIBO is a free and open source project available under the GPL-3 license at: https://github.com/RickGelhausen/HRIBO.

Summary Understanding the impact of elevated CO2 (eCO2) in global agriculture is important given climate change projections. Breeding climate‐resilient crops depends on genetic variation within naturally varying populations. The effect of genetic variation in response to eCO2 is poorly understood, especially in crop species. We describe the different ways in which Solanum lycopersicum and its wild relative S. pennellii respond to eCO2, from cell anatomy, to the transcriptome, and metabolome. We further validate the importance of translational regulation as a potential mechanism for plants to adaptively respond to rising levels of atmospheric CO2., Significance Statement In a domesticated and a wild Solanum species, translational and transcriptional regulation feedback onto each other and show interspecific differences in response to elevated CO2.

A novel coronavirus was recently discovered and termed SARS-CoV-2. Human infection can cause coronavirus disease 2019 (COVID-19), for which, at this point, over 80,000 cases resulting in over 2,500 deaths have been reported in over 40 countries. SARS-CoV-2 shows some similarities to other coronaviruses. However, treatment options and a cellular understanding of SARS-CoV-2 infection are lacking. Here we identify the host cell pathways modulated by SARS-CoV-19 infection and reveal that drugs targeting pathways prevent viral replication in human cells. We established a human cell culture model for infection with SARS-CoV-2 clinical isolate. Employing this system, we determined the SARS-CoV-2 infection profile by translatome and proteome proteomics at different times after infection.These analyses revealed that SARS-CoV-2 reshapes central cellular pathways, such as translation, splicing, carbon metabolism and nucleic acid metabolism. Small molecule inhibitors targeting these pathways were tested in cellular infection assays and prevented viral replication. Our results reveal the cellular infection profile of SARS-CoV-2 and led to the identification of drugs inhibiting viral replication. We anticipate our results to guide efforts to develop therapy options for COVID-19.Authors Denisa Bojkova, Kevin Klann, and Benjamin Koch contributed equally to this workData associated with the preprint has been made available on the authors' website.

International audience; Seeds characteristics such as germination ability, dormancy, and storability/longevity are important traits in agriculture, and various genes have been identified that are involved in its regulation at the transcriptional and post-transcriptional level. A particularity of mature dry seeds is a special mechanism that allows them to accumulate more than 10,000 mRNAs during seed maturation and use them as templates to synthesize proteins during germination. Some of these stored mRNAs are also referred to as long-lived mRNAs because they remain translatable even after seeds have been exposed to long-term stressful conditions. Mature seeds can germinate even in the presence of transcriptional inhibitors, and this ability is acquired in mid-seed development. The type of mRNA that accumulates in seeds is affected by the plant hormone abscisic acid and environmental factors, and most of them accumulate in seeds in the form of monosomes. Release of seed dormancy during after-ripening involves the selective oxidation of stored mRNAs and this prevents translation of proteins that function in the suppression of germination after imbibition. Non-selective oxidation and degradation of stored mRNAs occurs during long-term storage of seeds so that the quality of stored RNAs is linked to the degree of seed deterioration. After seed imbibition, a population of stored mRNAs are selectively loaded into polysomes and the mRNAs, involved in processes such as redox, glycolysis, and protein synthesis, are actively translated for germination

Meiotic maturation of oocyte relies on pre-synthesised maternal mRNA, the translation of which is highly coordinated in space and time. Here, we provide a detailed polysome profiling protocol that demonstrates a combination of the sucrose gradient ultracentrifugation in small SW55Ti tubes with the qRT-PCR-based quantification of 18S and 28S rRNAs in fractionated polysome profile. This newly optimised method, named Scarce Sample Polysome Profiling (SSP-profiling), is suitable for both scarce and conventional sample sizes and is compatible with downstream RNA-seq to identify polysome associated transcripts. Utilising SSP-profiling we have assayed the translatome of mouse oocytes at the onset of nuclear envelope breakdown (NEBD)&mdash, a developmental point, the study of which is important for furthering our understanding of the molecular mechanisms leading to oocyte aneuploidy. Our analyses identified 1847 transcripts with moderate to strong polysome occupancy, including abundantly represented mRNAs encoding mitochondrial and ribosomal proteins, proteasomal components, glycolytic and amino acids synthetic enzymes, proteins involved in cytoskeleton organization plus RNA-binding and translation initiation factors. In addition to transcripts encoding known players of meiotic progression, we also identified several mRNAs encoding proteins of unknown function. Polysome profiles generated using SSP-profiling were more than comparable to those developed using existing conventional approaches, being demonstrably superior in their resolution, reproducibility, versatility, speed of derivation and downstream protocol applicability.

Blood vessels are lined by endothelial cells engaged in distinct organ-specific functions but little is known about their characteristic gene expression profiles. RNA-Sequencing of the brain, lung, and heart endothelial translatome identified specific pathways, transporters and cell-surface markers expressed in the endothelium of each organ, which can be visualized at http://www.rehmanlab.org/ribo. We found that endothelial cells express genes typically found in the surrounding tissues such as synaptic vesicle genes in the brain endothelium and cardiac contractile genes in the heart endothelium. Complementary analysis of endothelial single cell RNA-Seq data identified the molecular signatures shared across the endothelial translatome and single cell transcriptomes. The tissue-specific heterogeneity of the endothelium is maintained during systemic in vivo inflammatory injury as evidenced by the distinct responses to inflammatory stimulation. Our study defines endothelial heterogeneity and plasticity and provides a molecular framework to understand organ-specific vascular disease mechanisms and therapeutic targeting of individual vascular beds., eLife digest Blood vessels supply nutrients, oxygen and other key molecules to all of the organs in the body. Cells lining the blood vessels, called endothelial cells, regulate which molecules pass from the blood to the organs they supply. For example, brain endothelial cells prevent toxic molecules from getting into the brain, and lung endothelial cells allow immune cells into the lungs to fight off bacteria or viruses. Determining which genes are switched on in the endothelial cells of major organs might allow scientists to determine what endothelial cells do in the brain, heart, and lung, and how they differ; or help scientists deliver drugs to a particular organ. If endothelial cells from different organs switch on different groups of genes, each of these groups of genes can be thought of as a ‘genetic signature’ that identifies endothelial cells from a specific organ. Now, Jambusaria et al. show that brain, heart, and lung endothelial cells have distinct genetic signatures. The experiments used mice that had been genetically modified to have tags on their endothelial cells. These tags made it possible to isolate RNA – a molecule similar to DNA that contains the information about which genes are active – from endothelial cells without separating the cells from their tissue of origin. Next, RNA from endothelial cells in the heart, brain and lung was sequenced and analyzed. The results show that each endothelial cell type has a distinct genetic signature under normal conditions and infection-like conditions. Unexpectedly, the experiments also showed that genes that were thought to only be switched on in the cells of specific tissues are also on in the endothelial cells lining the blood vessels of the tissue. For example, genes switched on in brain cells are also active in brain endothelial cells, and genes allowing heart muscle cells to pump are also on in the endothelial cells of the heart blood vessels. The endothelial cell genetic signatures identified by Jambusaria et al. can be used as “postal codes” to target drugs to a specific organ via the endothelial cells that feed it. It might also be possible to use these genetic signatures to build organ-specific blood vessels from stem cells in the laboratory. Future work will try to answer why endothelial cells serving the heart and brain use genes from these organs.

Highlights • Ribosome profiling in synaptosomes. • Transcriptome and translatome profiling from synaptic fractions. • Powerful tool to study local translation at the synapse., Background The aim of this study was to develop a method to study genome-wide local translation in biochemically isolated synaptic fractions (synaptoneurosomes). This methodology is of particular interest for neurons, due to the cardinal role of local translational control in neuronal sub-compartments, such as dendrites, for plasticity, learning, memory, and for disorders of the nervous system. New method We combined established methods for purifying synaptoneurosomes with translational profiling (ribosome profiling), a method that employs unbiased next generation sequencing to simultaneously assess transcription and translation in a single sample. Results The two existing methods are compatible to use in combination and yield high quality sequencing data, which are specific to synaptic compartments. This new protocol provides an easy to implement workflow, which combines biochemical isolation of synaptoneurosomes of varying levels of purity (crude or Percoll gradient purified) with the use of a commercial kit to generate sequencing libraries. Comparison with existing methods Compared to previous studies of the synaptic translatome, our method shows less contamination with non-neuronal cell types or non-synaptic compartments, increasing the specificity of the data obtained. Conclusions Combining the isolation of functional synaptic units with ribosome profiling offers a powerful tool to study local translation in synaptic compartments both in health and disease.

Local translation can support memory consolidation by supplying new proteins to synapses undergoing plasticity. Translation in adult forebrain dendrites is an established mechanism of synaptic plasticity and is regulated by learning, yet there is no evidence for learning-regulated protein synthesis in adult forebrain axons, which have traditionally been believed to be incapable of translation. Here, we show that axons in the adult rat amygdala contain translation machinery, and use translating ribosome affinity purification (TRAP) with RNASeq to identify mRNAs in cortical axons projecting to the amygdala, over 1200 of which were regulated during consolidation of associative memory. Mitochondrial and translation-related genes were upregulated, whereas synaptic, cytoskeletal, and myelin-related genes were downregulated; the opposite effects were observed in the cortex. Our results demonstrate that axonal translation occurs in the adult forebrain and is altered after learning, supporting the likelihood that local translation is more a rule than an exception in neuronal processes.

Since the introduction of the ribosome profiling technique in 2009 its popularity has greatly increased. It is widely used for the comprehensive assessment of gene expression and for studying the mechanisms of regulation at the translational level. As the number of ribosome profiling datasets being produced continues to grow, so too does the need for reliable software that can provide answers to the biological questions it can address. This review describes the computational methods and tools that have been developed to analyze ribosome profiling data at the different stages of the process. It starts with initial routine processing of raw data and follows with more specific tasks such as the identification of translated open reading frames, differential gene expression analysis, or evaluation of local or global codon decoding rates. The review pinpoints challenges associated with each step and explains the ways in which they are currently addressed. In addition it provides a comprehensive, albeit incomplete, list of publicly available software applicable to each step, which may be a beneficial starting point to those unexposed to ribosome profiling analysis. The outline of current challenges in ribosome profiling data analysis may inspire computational biologists to search for novel, potentially superior, solutions that will improve and expand the bioinformatician's toolbox for ribosome profiling data analysis. This article is characterized under: Translation > Ribosome Structure/Function RNA Evolution and Genomics > Computational Analyses of RNA Translation > Translation Mechanisms Translation > Translation Regulation.

There are 27 small molecule protein-protein interaction (PPI) modulators in Phase I, II, and III clinical trials targeting cancer, viruses, autoimmune disorders, and as immune suppression agents. Targeting PPIs as an antibiotic drug discovery strategy remains in relative infancy by comparison. However, a number of molecules are in development which target PPI within the replisome, divisome, transcriptome, and translatome are showing significant promise at the medicinal chemistry stage of drug development. Hence, the success of future PPI agents as antibiotics will build upon the techniques and design strategies of these molecules.

The turtle carapace is composed of severely deformed fused dorsal vertebrae, ribs, and bone plates. In particular, the lateral growth in the superficial layer of turtle ribs in the dorsal trunk causes an encapsulation of the scapula and pelvis. The recent study suggested that the carapacial ridge (CR) is a new model of epithelial–mesenchymal transition which is essential for the arrangement of the ribs. Therefore, it is necessary to explore the regulatory mechanism of carapacial ridge development to analyze the formation of the turtle shell. However, the current understanding of the regulatory network underlying turtle carapacial ridge development is poor due to the lack of both systematic gene screening at different carapacial ridge development stages and gene function verification studies. In this study, we obtained genome-wide gene transcription and gene translation profiles using RNA sequencing and ribosome nascent-chain complex mRNA sequencing from carapacial ridge tissues of Chinese soft-shell turtle at different development stages. A correlation analysis of the transcriptome and translatome revealed that there were 129, 670, and 135 codifferentially expressed genes, including homodirection and opposite-direction differentially expressed genes, among three comparison groups, respectively. The pathway enrichment analysis of codifferentially expressed genes from the Kyoto Encyclopedia of Genes and Genomes showed dynamic changes in signaling pathways involved in carapacial ridge development. Especially, the results revealed that the Wnt signaling pathway and MAPK signaling pathway may play important roles in turtle carapacial ridge development. In addition, Wnt and Fgf were expressed during the carapacial ridge development. Furthermore, we discovered that Wnt5a regulated carapacial ridge development through the Wnt5a/JNK pathway. Therefore, our studies uncover that the morphogenesis of the turtle carapace might function through the co-operation between conserved WNT and FGF signaling pathways. Consequently, our findings revealed the dynamic signaling pathways acting on the carapacial ridge development of Chinese soft-shell turtle and provided new insights into uncover the molecular mechanism underlying turtle shell morphogenesis.