Search

RNA-binding proteins (RBPs) regulate essentially every event in the lifetime of an RNA molecule, from its production to its destruction. Whereas much has been learned about RNA sequence specificity and general functions of individual RBPs, the ways in which numerous RBPs instruct a much smaller number of effector molecules, that is, the core engines of RNA processing, as to where, when and how to act remain largely speculative. Here, we survey the known modes of communication between RBPs and their effectors with a particular focus on converging RBP-effector interactions and their roles in reducing the complexity of RNA networks. We discern the emerging unifying principles and discuss their utility in our understanding of RBP function, regulation of biological processes and contribution to human disease.

Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs—mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)—that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation.

Mechanistic target of rapamycin (mTOR) is a protein kinase that integrates multiple inputs to regulate anabolic cellular processes. For example , mTOR complex I (mTORC1) has key functions in growth control, autophagy and metabolism. However, much less is known about the signaling components that act downstream of mTORC1 to regulate cellular morphogenesis. Here we show that the RNA-binding protein Unkempt, a key regulator of cellular morphogenesis, is a novel substrate of mTORC1. We show that Unkempt phosphorylation is regulated by nutrient levels and growth factors via mTORC1. To analyze Unkempt phosphorylation, we immunoprecipitated Unkempt from cells in the presence or absence of the mTORC1 inhibitor rapamycin and used mass spectrometry to identify mTORC1-dependent phosphorylated residues. This analysis showed that mTORC1-dependent phosphorylation is concentrated in a serine-rich intrinsically disordered region in the C-terminal half of Unkempt. We also found that Unkempt physically interacts with and is directly phosphorylated by mTORC1 through binding to the regulatory-associated protein of mTOR, Raptor. Furthermore, analysis in the developing brain of mice lacking TSC1 expression showed that phosphorylation of Unkempt is mTORC1-dependent in vivo. Finally, mutation analysis of key serine/threonine residues in the serine-rich region indicates that phosphorylation inhibits the ability of Unkempt to induce a bipolar morphology. Phosphorylation within this serine-rich region thus profoundly affects the ability of Unkempt to regulate cellular morphogenesis. Taken together, our findings reveal a novel molecular link between mTORC1 signaling and cellular morphogenesis.

During hematopoiesis, stem and progenitor cells become progressively restricted in their differentiation potential. This process is driven by lineage-specific transcription factors and is accompanied by dynamic changes in chromatin structure. The chromatin assembly factor complex CAF-1 is a key regulator of cellular plasticity in various cell lineages in different organisms. However, whether CAF-1 sustains lineage identity during normal homeostasis is unclear. To address this question, we investigated the role of CAF-1 in myeloid progenitor cells. CAF-1 suppression in myeloid progenitors triggered their rapid commitment but incomplete differentiation toward granulocyte, megakaryocyte, and erythrocyte lineages, resulting in a mixed cellular state. Through comparison with a canonical paradigm of directed terminal myeloid differentiation, we define changes in chromatin accessibility that underlie a unique transcriptome of the aberrantly matured CAF-1 deficient cells. We further identify C/EBPα and ELF1 as key transcription factors whose control of myeloid lineage commitment is kept in check by CAF-1. These findings shed new light on molecular underpinnings of hematopoiesis and suggest that manipulation of chromatin accessibility through modulating CAF-1 levels may provide a powerful strategy for controlled differentiation of blood cells.

In 1959, while analysing the bacterial flagellar proteins, Ambler and Rees observed an unknown species of amino acid that they eventually identified as methylated lysine. Over half a century later, protein methylation is known to have a regulatory role in many essential cellular processes that range from gene transcription to signal transduction. However, the road to this now burgeoning research field was obstacle-ridden, not least because of the inconspicuous nature of the methyl mark itself. Here, we chronicle the milestone achievements and discuss the future of protein methylation research.

We present Barcoded Oligonucleotides Ligated On RNA Amplified for Multiplexed and parallel In-Situ analysis (BOLORAMIS), a reverse-transcription (RT)-free method for spatially-resolved, targeted, in-situ RNA identification of single or multiple targets. For this proof of concept, we have profiled 154 distinct coding and small non-coding transcripts ranging in sizes 18 nucleotides in length and upwards, from over 200, 000 individual human induced pluripotent stem cells (iPSC) and demonstrated compatibility with multiplexed detection, enabled by fluorescent in-situ sequencing. We use BOLORAMIS data to identify differences in spatial localization and cell-to-cell expression heterogeneity. Our results demonstrate BOLORAMIS to be a generalizable toolset for targeted, in-situ detection of coding and small non-coding RNA for single or multiplexed applications.

Cellular morphology is an essential determinant of cellular function in all kingdoms of life, yet little is known about how cell shape is controlled. Here we describe a molecular program that controls the early morphology of neurons through a metazoan-specific zinc finger protein, Unkempt. Depletion of Unkempt in mouse embryos disrupts the shape of migrating neurons, while ectopic expression confers neuronal-like morphology to cells of different nonneuronal lineages. We found that Unkempt is a sequence-specific RNA-binding protein and identified its precise binding sites within coding regions of mRNAs linked to protein metabolism and trafficking. RNA binding is required for Unkempt-induced remodeling of cellular shape and is directly coupled to a reduced production of the encoded proteins. These findings link post-transcriptional regulation of gene expression with cellular shape and have general implications for the development and disease of multicellular organisms.

Lysine-specific demethylase 1 (LSD1) has been reported to repress and activate transcription by mediating histone H3K4me1/2 and H3K9me1/2 demethylation, respectively. The molecular mechanism that underlies this dual substrate specificity has remained unknown. Here we report that an isoform of LSD1, LSD1+8a, does not have the intrinsic capability to demethylate H3K4me2. Instead, LSD1+8a mediates H3K9me2 demethylation in collaboration with supervillin (SVIL), a new LSD1+8a interacting protein. LSD1+8a knockdown increases H3K9me2, but not H3K4me2, levels at its target promoters and compromises neuronal differentiation. Importantly, SVIL co-localizes to LSD1+8a-bound promoters, and its knockdown mimics the impact of LSD1+8a loss, supporting SVIL as a cofactor for LSD1+8a in neuronal cells. These findings provide insight into mechanisms by which LSD1 mediates H3K9me demethylation and highlight alternative splicing as a means by which LSD1 acquires selective substrate specificities (H3K9 versus H3K4) to differentially control specific gene expression programs in neurons.

Unkempt is an evolutionarily conserved RNA-binding protein that regulates translation of its target genes and is required for the establishment of the early bipolar neuronal morphology. Here we determined the X-ray crystal structure of mouse Unkempt and show that its six CCCH zinc fingers (ZnFs) form two compact clusters, ZnF1-3 and ZnF4-6, that recognize distinct trinucleotide RNA substrates. Both ZnF clusters adopt a similar overall topology and use distinct recognition principles to target specific RNA sequences. Structure-guided point mutations reduce the RNA binding affinity of Unkempt both in vitro and in vivo, ablate Unkempt's translational control and impair the ability of Unkempt to induce a bipolar cellular morphology. Our study unravels a new mode of RNA sequence recognition by clusters of CCCH ZnFs that is critical for post-transcriptional control of neuronal morphology.

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

Although several lines of evidence support a role for serine proteases in apoptosis, little is known about the mechanisms involved. In the present study, we have examined the apoptosis-inducing potential and dissected the death-signalling pathways of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-tosyl-L-lysine chloromethyl ketone (TLCK), inhibitors of chymotrypsin- and trypsin-like proteases, respectively. Our results designate two distinct roles for serine proteases. Firstly, we show that both inhibitors induce biochemical and morphological characteristics of apoptosis, including proteolysis of poly(ADP-ribose) polymerase 1 (PARP-1) and inhibitor of caspase-activated DNase (ICAD), as well as mitochondrial dysfunction, and that their action is abrogated by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp.fluoromethylketone (z-VAD.fmk). These results suggest that inhibition of anti-apoptotic serine proteases governs the onset of the caspase-dependant apoptotic cascade. Secondly, we also demonstrate the involvement of a serine protease in the terminal stage of apoptosis. We showed that chymotrypsin-like protease activity is required for internucleosomal DNA fragmentation in apoptotic cells. Hence, DNA fragmentation is abrogated in TPCK-pre-treated WEHI 231 cells undergoing apoptosis triggered either by anti-IgM or TLCK. These results indicate that internucleosomal DNA cleavage in apoptotic cells is mediated by a chymotrypsin-like protease.

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human‐induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems‐level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types., National Institutes of Health (U.S.) (Grant P50 HG005550), Merkin, Richard N., National Science Foundation (U.S.). Emergent Behaviors of Integrated Cellular Systems (Grant 0939511), Synthetic Biology Engineering Research Center (Grant 0540879), Swiss National Science Foundation, Ernst Schering Research Foundation

Fragile X syndrome, a common form of inherited intellectual disability, is caused by loss of the fragile X mental retardation protein FMRP. FMRP is present predominantly in the cytoplasm, where it regulates translation of proteins that are important for synaptic function. We identify FMRP as a chromatin-binding protein that functions in the DNA damage response (DDR). Specifically, we show that FMRP binds chromatin through its tandem Tudor (Agenet) domain in vitro and associates with chromatin in vivo. We also demonstrate that FMRP participates in the DDR in a chromatin-binding-dependent manner. The DDR machinery is known to play important roles in developmental processes such as gametogenesis. We show that FMRP occupies meiotic chromosomes and regulates the dynamics of the DDR machinery during mouse spermatogenesis. These findings suggest that nuclear FMRP regulates genomic stability at the chromatin interface and may impact gametogenesis and some developmental aspects of fragile X syndrome., Howard Hughes Medical Institute

SummaryHyperactivation of the PI 3-kinase/AKT pathway is a driving force of many cancers. Here we identify the AKT-inactivating phosphatase PHLPP1 as a prostate tumor suppressor. We show that Phlpp1-loss causes neoplasia and, on partial Pten-loss, carcinoma in mouse prostate. This genetic setting initially triggers a growth suppressive response via p53 and the Phlpp2 ortholog, and reveals spontaneous Trp53 inactivation as a condition for full-blown disease. Surprisingly, the codeletion of PTEN and PHLPP1 in patient samples is highly restricted to metastatic disease and tightly correlated to deletion of TP53 and PHLPP2. These data establish a conceptual framework for progression of PTEN mutant prostate cancer to life-threatening disease.

BackgroundThe B cell antigen receptor (BCR) is a signaling complex that mediates the differentiation of stage-specific cell fate decisions in B lymphocytes. While several studies have shown differences in signal transduction components as being key to contrasting phenotypic outcomes, little is known about the differential BCR-triggered gene transcription downstream of the signaling cascades.ResultsHere we define the transcriptional changes that underlie BCR-induced apoptosis and proliferation of immature and mature B cells, respectively. Comparative genome-wide expression profiling identified 24 genes that discriminated between the early responses of the two cell types to BCR stimulation. Using mice with a conditionalMyc-deletion, we validated the microarray data by demonstrating thatMycis critical to promoting BCR-triggered B-cell proliferation. We further investigated theMyc-dependent molecular mechanisms and found thatMycpromotes a BCR-dependent clonal expansion of mature B cells by inducing proliferation and inhibiting differentiation.ConclusionThis work provides the first comprehensive analysis of the early transcriptional events that lead to either deletion or clonal expansion of B cells upon antigen recognition, and demonstrates thatMycfunctions as the hub of a transcriptional network that control B-cell fate in the periphery.

B cell receptor (BCR) signaling contributes to the pathogenesis of B cell malignancies, and most B cell lymphomas depend on BCR signals for survival. Identification of genes that restrain BCR-mediated proliferation is therefore an important goal toward improving the therapy of B cell lymphoma. Here, we identify Ptger4 as a negative feedback regulator of proliferation in response to BCR signals and show that its encoded EP4 receptor is a principal molecule conveying the growth-suppressive effect of prostaglandin E2 (PGE2). Stable knockdown of Ptger4 in B cell lymphoma markedly accelerated tumor spread in mice, whereas Ptger4 overexpression yielded significant protection. Mechanistically, we show that the intrinsic activity of Ptger4 and PGE2–EP4 signaling target a similar set of activating genes, and find Ptger4 to be significantly down-regulated in human B cell lymphoma. We postulate that Ptger4 functions in B cells as a candidate tumor suppressor whose activity is regulated by PGE2 in the microenvironment. These findings suggest that targeting EP4 receptor for prostaglandin may present a novel strategy for treatment of B cell malignancies.

New platelet glycoprotein IIb/IIIa (GP IIb/IIIa, integrin alpha(IIb)beta3) antagonists were prepared on a 2H-1,4-benzoxazine-3(4H)-one scaffold. Their anti-aggregatory activities in human platelet rich plasma and their affinity towards alpha(IIb)beta3 and alpha(V)beta3 integrins were assessed. Various substitution positions and side chain variations were studied. In contrast to the generally accepted model, compounds containing ethyl esters as aspartate mimetics were in general more active than the corresponding free acids. We suggest an explanation for the observed behaviour of these new compounds.

Hyper-activation of the PI 3-Kinase/ AKT pathway is common in many cancer types. Tumourigenesis through this pathway is prevented by concerted action of multiple tumour suppressor genes. Most notably, PTEN reverts PI 3-Kinase activity whereas excessive pathway activation triggers the p53-mediated senescence arrest. However, it remains ill defined if and at what stage this response acts in human prostate cancer. Here we identify the AKT-inactivating phosphatase PHLPP as a tumour suppressor and demonstrate how the p53-response can antagonise co-deletion of PTEN and PHLPP to form a barrier against prostate cancer progression. We show that Phlpp-loss causes neoplasia and upon partial Pten-loss, carcinoma in mouse prostate. In this setting, Phlpp-deficiency triggers growth arrest via mTorC1-dependent activation of p53 and we find that co-deletion of Pten and Phlpp selects for spontaneous inactivation of p53 in prostate. Validating this conditional gene inactivation scheme in a comprehensive genomic patient data set we find that co-deletion of PTEN and PHLPP is almost exclusively observed in metastatic prostate cancer and tightly correlated to deletion of TP53. Furthermore, PTEN/ PHLPP expression can be used to predict disease outcome in these patients, comparable to the standard histology based method, but adding actionable information on pathway status. Finally, we show that both known PHLPP isoforms compensate for PTEN-suppression in a novel pathway feedback explaining their co-deletion with PTEN in the metastatic samples. Surprisingly, we find that the feedback surge of these genes is sensitive to some pharmacological inhibitors of the PI 3-Kinase pathway. Collectively, our findings emphasise the need for careful evaluation of PI 3-Kinase target therapy effects in prostate cancer and highlight the value of genetically engineered mouse models in this process. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2405. doi:10.1158/1538-7445.AM2011-2405

Circular RNAs (circRNA) are generated during a non-canonical splicing event called backsplicing. It was long thought that these transcripts are only noise produced by aberrant splicing events and were therefore quickly discarded. In recent years however, with the advancement of technology, several circRNAs have been identified in humans and other organisms. These results have led researchers to question the actual role of circRNAs in cell biology. So far, several circular transcripts have been implied to play a part in different human diseases, including neurological disorders, cancer, and cardiovascular diseases. Their potential roles have also led circRNAs to be researched in terms of clinical applications, including their use as biomarkers and therapeutics. Thus far, only a minority of circRNAs identified with RNA sequencing have been sufficiently investigated. There are several methods currently in use to investigate circRNAs, including fluorescence in situ hybridization (FISH). With the numerous drawbacks of RNA-FISH, the need has arisen for a novel method for circRNA investigation in living cells. We developed a novel method that allows fluorescent labeling of circRNAs, but not linear RNAs, with known fluorogenic RNA aptamers (Pepper). These aptamers were halved and added to each side of the linear transcripts and only formed a functional RNA aptamer when the backsplice junction (BSJ) was formed. With the addition of the HBC fluorescence dye, which is able to incorporate into the RNA aptamer structure, the circRNA transcripts were visualized under fluorescence microscope. The novel method overcomes the drawbacks of FISH and allows the investigation of circRNAs in living cells. We demonstrated that the split RNA aptamer does not interfere with circRNA circularization and the formation of a BSJ region. We also demonstrated that a split RNA aptamer is able to form a whole and is also functional when adding HBC dye. In addition, the method has the potential to be used to track circRNA in living cells with sufficient optimization. All in all, the development of a novel method potentially enables a more efficient investigation of circRNAs in living cells. Krožne RNK uvrščamo v družino nekodirajočih RNK, ki nastanejo v procesu povratnega spajanja. Pri procesu povratnega spajanja prihaja do povezovanja akceptorskega 5' in donorskega 3' konca prepisa, kar omogoči nastanek kovalentno zaprte strukture v obliki kroga. Krožne RNK so bile prvič opažene pred več kot 40 leti, vendar jim raziskovalci niso namenjali veliko pozornosti, saj so mislili, da so krožni prepisi le posledica nepravilnega spajanja eksonov pri procesu dozorevanja prekurzorske mRNA (pre-mRNA). Poleg tega so bile številne krožne RNK zaradi odsotnosti poliadenilacijskega repa na 3' koncu spregledane pri uporabi RNK sekvenciranja. V zadnjih letih je kot posledica razvoja različnih tehnologij in bioinformatskih orodij prišlo do odkritja ogromnega števila krožnih RNK v različnih organizmih, vključno s človekom. Zaradi tega se je pojavilo vprašanje, ali so krožne RNK resnično le posledica napak v spajanju eksonov ali imajo pomembnejšo vlogo v celični biologiji. Intenzivno raziskovanje teh kovalentno zaprtih prepisov je pokazalo, da so ti veliko bolj stabilni v primerjavi z linearnimi RNK. Poleg tega se v določenih primerih pojavljajo celo pogosteje kot njihovi linearni dvojniki in je njihovo izražanje tkivno ter razvojno specifično, hkrati pa se pojavljajo tudi pri različnih živalskih vrstah, kar nakazuje na njihovo evolucijsko ohranjenost. Številne raziskave namigujejo na prisotnost in vlogo krožnih RNK pri različnih boleznih, vključno z nevrološkimi in kardiovaskularnimi boleznimi ter rakom. Eksperimentalne analize dokazujejo, da so določene krožne RNK vključene v številne poti v celici, vključno z različnimi signalnimi potmi, kot sta PI3K/AKT in mTOR signalna pot, katerih deregulacija je dokazano vključena v nastanek in razvoj raka. Krožne RNK delimo na več tipov glede na njihovo strukturo: eksonske, intronske in ekson-intronske krožne RNK. 80 odstotkov vseh krožnih RNK predstavljajo eksonske RNK, medtem ko se ostala dva tipa pojavljata v manjšem odstotku. V skladu z vrsto krožne RNK se pojavlja tudi več možnih modelov biogeneze krožnih prepisov. Krožne RNK najpogosteje nastanejo v procesu direktnega povratnega spajanja, kjer med izrezovanjem pre-mRNA pride do takojšnega nastanka krožnih RNK. Zelo pogosto se pojavlja tudi t. i. lariatni model, kjer pri obdelavi pre-mRNA pride najprej do nastanka lariatnega intermediata z ostankom preskočenih eksonov, ki se nato povratno spoji in tvori zrelo krožno RNK. Do nastanka intronske RNK lahko pride tudi, ko se normalno izrezani introni ne razgradijo in tvorijo krožno strukturo. Za nastanek krožnih RNK sta nujno potrebna RNK polimeraza II in izrezovalno-povezovalni kompleks. Poleg tega je regulacija odvisna tudi od cis in trans regulatornih elementov. Med cis elemente regulacije povratnega spajanja pogosto vključujemo ponavljajoče se elemente, kot so na primer ALU ponovitve v primatih. Med trans elemente pa vključujemo proteine, ki se vežejo na molekule RNK (RNK vezavni proteini). Ti proteini ob vezavi na prepis RNK omogočijo stabilizacijo povratnega spajanja in s tem nastanek kroga. Krožne RNK imajo lahko različne funkcije v celici, vključno s titracijo mikro RNK in vezavo z RNK vezavnimi proteini. Poleg tega lahko krožne RNK uravnavajo izražanje svojih starševskih genov preko interakcij z RNK polimerazo II v jedru ali preko vpliva na DNK hipometilacijo v CpG otočkih promotorja. Kljub temu da krožne RNK uvrščamo med nekodirajoče RNK, pa je bilo ugotovljeno, da imajo številni krožni prepisi tudi sposobnost prevajanja v proteine in peptide. Vloge krožnih RNK se intenzivno raziskujejo v različnih patofizioloških stanjih, saj je bilo pogosto opaženo njihovo prekomerno ali prenizko izražanje pri različnih boleznih. Na tej točki je potrebno pojasniti, da razlika v izražanju še ne pomeni nujno tudi korelacije z razvojem bolezenskega stanja, ampak so lahko krožne RNK tudi posledica bolezni. Zaradi tega je nujno potrebna natančna opredelitev vloge krožnih RNK v različnih stanjih. Krožne RNK se prav tako intenzivno raziskujejo zaradi njihove potencialne uporabe v kliniki kot diagnostični ali/in prognostični biooznačevalci ter kot terapevtiki. Lastnosti, zaradi katerih bi lahko bili krožni prepisi dobri pokazatelji različnih bolezenskih stanj, med drugim vključujejo njihovo visoko stabilnost, specifično izražanje v tkivih in določenih razvojnih stanjih ter pojavljanje v različnih (lahko dostopnih) telesnih tekočinah, kot sta kri in urin. Poleg tega so krožne RNK zaradi svoje vloge v različnih signalnih poteh zelo zanimive tudi kot terapevtske tarče ali celo kot terapevtske molekule. Trenutno se uporabljajo številne tehnike za preučevanje krožnih prepisov, vključno z visokozmogljivim sekvenciranjem krožnih RNK, prenosom northern, verižno reakcijo s polimerazo in fluorescenčno in situ hibridizacijo. Vse naštete metode pa se srečujejo z omejitvami pri preučevanju krožnih prepisov. Na primer fluorescenčna in situ hibridizacija (FISH) ne omogoča opazovanja živih celic in s tem preučevanja celotnega življenjskega cikla krožnih RNK. Poleg tega je težavno tudi načrtovanje sond, ki se morajo vezati na specifično spojitveno sekvenco. Težava se pojavi tudi pri razlikovanju med krožnimi in linearnimi RNK, saj imajo pogosto podobno sekvenco. Opazovanje krožnih prepisov v fiksiranih celicah je statično, kar pomeni, da je opazovanje možno le v določeni časovni točki v celici. Za boljše razumevanje funkcije krožnih RNK je tako pomembno, da so opazovane dinamično, torej v živih celicah, in širšem časovnem obdobju. Razviti so že številni načini, kako opazovati molekule RNK v živih celicah, ki pa se najpogosteje uporabljajo pri opazovanju mRNK. mRNK namreč sodi med ene izmed najbolj raziskanih molekul RNK, zato je rezultate dinamičnega opazovanja v živi celici enostavneje interpretirati v primerjavi z ostalimi oblikami molekul RNK. Načini, kako opazovati molekule RNK, vključujejo izkoriščanje naravnih interakcij, ki se lahko tvorijo med proteini in RNK. V tem primeru se RNK vezavni proteini, spojeni s fluorescenčno komponento, vežejo na tarčno RNK in na ta način omogočijo označevanje. Med te sisteme vključujemo sistema MS2 in PP7, ki temeljita na bakteriofagnih plaščnih proteinih, ki se vežejo na, s tarčnim proteinom označeno, preučevano molekulo RNK. Alternativa temu pristopu je označevanje RNK molekul s specifičnimi fluorescenčnimi RNK aptameri. Aptameri sicer niso sposobni samostojnega fluoresciranja, temveč ob dodatku specifičnega barvila omogočajo njegovo vgradnjo v svojo tridimenzionalno strukturo. Ta interakcija nato omogoči emisijo fluorescenčne svetlobe. Razviti so bili že številni takšni aptameri, vključno s t. i. »Vegetables« in »Peppers«, ki se uporabljajo predvsem za označevanje mRNK, vendar bi lahko njihovo uporabo prilagodili tudi za preučevanje drugih prepisov, tudi krožnih RNK. »Peppers« RNK aptameri so sposobni izražanja fluorescence, ko pride do vgradnje specifičnega barvila, imenovanega HBC, v njihovo strukturo. Ta vgradnja je možna zaradi ugodnih energijskih interakcij med aptamerom in barvilom. V primerjavi z »Vegetables« imajo »Peppers« boljše spektroskopske in biokemične lastnosti ter hkrati povzročijo zelo malo motenj v normalni biologiji označenih RNK. Zaradi teh lastnosti so skoraj popolni kandidati za preučevanje RNK, vključno s krožnimi RNK v živih celicah. V sklopu magistrske naloge smo razvijali novo metodo, ki bo ob ustrezni optimizaciji in validaciji omogočila sledenje krožnih RNK v živih celicah. Metoda temelji na fluorescenčnem označevanju različnih krožnih RNK s pomočjo znanih fluorogenih aptamerov RNK (»Peppers«). Ideja metode je v razpolovitvi fluorogenega aptamera na vsako stran linearnega transkripta, ki potem samo v primeru povratnega spajanja spet tvori celoto tj. funkcionalen RNK aptamer. Na ta način smo lahko selektivno označili ektopično izražene ali endogene krožne RNK, ne pa linearnih RNK. Označene krožne RNK smo lahko po dodatku fluorescentnega barvila opazovali v živih celicah pod fluorescenčnim mikroskopom. Za validacijo in razvoj nove metode smo izvedli številne eksperimente, s katerimi smo želeli potrditi koncept nove metode. Pripravili smo ekspresijski vektor, v katerega smo klonirali razcepljen 4Pepper aptamer RNK. V vmesni predel smo nato klonirali zapise za različne krožne RNK, vključno s FOXO3, HIPK3 in EIF3J. Tako pripravljene konstrukte smo nato vstavili v različne celične linije (HeLa, HEK 293 T in NIH/3T3) z uporabo različnih transfekcijskih reagentov (Dharmafect, Lipofectamine 3000, PEI in MIRUS LT-1). Transfecirane celice smo nato po dodatku HBC barvila opazovali pod fluorescenčnim mikroskopom. Za validacijo izražene fluorescence smo vključili tudi negativne kontrole, ki so vključevale krožno RNK brez oznake, nespremenjene celice s transfekcijskim reagentom in krožne RNK brez introna, potrebnega za cirkularizacijo. Fluorescenčni signal smo kvantificirali glede na število celic, ki so fluorescirale. Dobljene rezultate smo nato primerjali z že objavljenimi izsledki FISH eksperimentov v literaturi. Poleg tega smo prisotnost sekvence na stičišču koncev, ki nastane le ob nastanku kroga, preučevali z uporabo verižne reakcije s polimerazo. Verjetno najpomembnejša in najbolj uporabna lastnost nove metode fluorescenčnega sledenja krožnih RNK je ta, da je metoda neodvisna od sekvence na stičišču koncev RNK, s čimer ta sekvenca ni več edina unikatna sekvenca v krožnih transkriptih. Hkrati pa nova metoda omogoča tudi in vivo sledenje krožnih RNK brez predhodne fiksacije celice. Zaradi teh lastnosti metoda preseže pomanjkljivosti metode RNK-FISH kot sta fiksacija celic in možnost neuspešne detekcije zaradi omejitve na kratko sekvenco na stičišču koncev RNK. Z eksperimentalnim delom smo uspeli pokazati, da dodatek aptamera RNK sekvenci krožnih RNK ne moti njihove tvorbe. Poleg tega lahko razcepljen aptamer RNK tvori celoto hkrati s tvorbo spojitvene sekvence krožnih RNK in z dodatkom ustreznega fluorescenčnega barvila omogoča opazovanje krožnih RNK z uporabo fluorescenčnega mikroskopa. Za dokaz uporabe nove metode za sledenje krožnih RNK v živih celicah pa bi jih morali opazovati v določenem časovnem obdobju, kar je naslednji korak v razvoju nove metode. Sledenje krožnih RNK v živih celicah je pomembno za preučevanje njihovega mesta delovanja preko opazovanja razporeditve krožnih prepisov v jedru in citoplazmi ter preučevanje njihovega nastanka in funkcije v celici. Z ustrezno optimizacijo lahko ta metoda omogoča uspešno preučevanje biologije krožnih RNK.