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Aging is associated with substantial physiological changes and constitutes a major risk factor for neurological disorders including dementia. Alterations in gene expression upon aging have been extensively studied; however, an in-depth characterization of post-transcriptional regulatory events remains elusive. Here, we profiled the age-related changes of the transcriptome and translatome in the female mouse hippocampus by RNA sequencing of total RNA and polysome preparations at four ages (3-, 6-, 12-, 20-month-old); and we implemented a variety of bioinformatics approaches to unravel alterations in transcript abundance, alternative splicing, and polyadenylation site selection. We observed mostly well-coordinated transcriptome and translatome expression signatures across age including upregulation of transcripts related to immune system processes and neuroinflammation, though transcripts encoding ribonucleoproteins or associated with mitochondrial functions, calcium signaling and the cell-cycle displayed substantial discordant profiles, suggesting translational control associated with age-related deficits in hippocampal-dependent behavior. By contrast, alternative splicing was less preserved, increased with age and was associated with distinct functionally-related transcripts encoding proteins acting at synapses/dendrites, RNA-binding proteins; thereby predicting regulatory roles for RBM3 and CIRBP. Only minor changes in polyadenylation site selection were identified, indicating pivotal 3′-end selection in young adults compared to older groups. Overall, our study provides a comprehensive resource of age-associated post-transcriptional regulatory events in the mouse hippocampus, enabling further examination of the molecular features underlying age-associated neurological diseases.

The regulation of mRNA translation plays an essential role in neurons, contributing to important brain functions, such as brain plasticity and memory formation. Translation is conducted by ribosomes, which at their core consist of ribosomal proteins (RPs) and ribosomal RNAs. While translation can be regulated at diverse levels through global or mRNA-specific means, recent evidence suggests that ribosomes with distinct configurations are involved in the translation of different subsets of mRNAs. However, whether and how such proclaimed ribosome heterogeneity could be connected to neuronal functions remains largely unresolved. Here, we postulate that the existence of heterologous ribosomes within neurons, especially at discrete synapses, subserve brain plasticity. This hypothesis is supported by recent studies in rodents showing that heterogeneous RP expression occurs in dendrites, the compartment of neurons where synapses are made. We further propose that sleep, which is fundamental for brain plasticity and memory formation, has a particular role in the formation of heterologous ribosomes, specialised in the translation of mRNAs specific for synaptic plasticity. This aspect of our hypothesis is supported by recent studies showing increased translation and changes in RP expression during sleep after learning. Thus, certain RPs are regulated by sleep, and could support different sleep functions, in particular brain plasticity. Future experiments investigating cell-specific heterogeneity in RPs across the sleep-wake cycle and in response to different behaviour would help address this question.

As our understanding of the complex network of regulatory pathways for gene expression continues to grow, avenues of investigation for how these new findings can be utilised in therapeutics are emerging. The recent growth of interest in the RNA binding protein (RBP) interactome has revealed it to be rich in targets linked to, and causative of diseases. While this is, in and of itself, very interesting, evidence is also beginning to arise for how the RBP interactome can act to modulate the response of diseases to existing therapeutic treatments, especially in cancers. Here we highlight this topic, providing examples of work that exemplifies such modulation of chemotherapeutic sensitivity.

DICER1 syndrome is a cancer pre-disposition disorder caused by mutations that disrupt the function of DICER1 in miRNA processing. Studying the molecular, cellular and oncogenic effects of these mutations can reveal novel mechanisms that control cell homeostasis and tumor biology. Here, we conduct the first analysis of pathogenic DICER1 syndrome allele from the DICER1 3′UTR. We find that the DICER1 syndrome allele, rs1252940486, abolishes interaction with the PUMILIO RNA binding protein with the DICER1 3′UTR, resulting in the degradation of the DICER1 mRNA by AUF1. This single mutational event leads to diminished DICER1 mRNA and protein levels, and widespread reprogramming of miRNA networks. The in-depth characterization of the rs1252940486 DICER1 allele, reveals important post-transcriptional regulatory events that control DICER1 levels.

Post-transcriptional gene regulation is driven by RNA-binding proteins (RBPs). Recent global approaches suggest widespread autoregulation of RBPs through binding to their own mRNA; however, little is known about the regulatory impact and quantitative models remain elusive. By integration of several independent kinetic parameters and abundance data, we modelled autoregulatory feedback loops for six canonical and non-canonical RBPs from the yeast

Cardiac pathologies are characterized by intense remodeling of the extracellular matrix (ECM) that eventually leads to heart failure. Cardiomyocytes respond to the ensuing biomechanical stress by re-expressing fetal contractile proteins via transcriptional and post-transcriptional processes, like alternative splicing (AS). Here, we demonstrate that the heterogeneous nuclear ribonucleoprotein C (hnRNPC) is upregulated and relocates to the sarcomeric Z-disk upon ECM pathological remodeling. We show that this is an active site of localized translation, where the ribonucleoprotein associates to the translation machinery. Alterations in hnRNPC expression and localization can be mechanically determined and affect the AS of numerous mRNAs involved in mechanotransduction and cardiovascular diseases, like Hippo pathway effector YAP1. We propose that cardiac ECM remodeling serves as a switch in RNA metabolism by impacting an associated regulatory protein of the spliceosome apparatus. These findings offer new insights on the mechanism of mRNAs homeostasis mechanoregulation in pathological conditions.

Summary RNA-binding proteins (RBPs) are key post-transcriptional regulators that play a substantial role during stress adaptation. Recent proteome-wide surveys have uncovered a large number of new and “unconventional” RBPs such as metabolic enzymes, yet little is known about the reconfiguration of the RNA-binding proteome (RBPome) and RNA-enzyme interactions in response to cellular stress. Here, we applied RNA-interactome capture to monitor the dynamics of the mRBPome upon mild oxidative stress in the yeast Saccharomyces cerevisiae. Among the 257 proteins that significantly changed RNA associations, we observed the coordinated remodeling of RNA-binding enzymes — particularly of the central carbon metabolism — that complemented known metabolic responses. Furthermore, we recognized the propensity for paralogous specific alterations of enzyme-RNA interactions. Our results suggest coordinated cross talk between RNA-enzyme interactions and intermediary metabolism to maintain the physiological and molecular balance upon oxidative stress, perhaps through specialization of paralogous during evolution., Graphical abstract, Highlights • Oxidative stress induces the rearrangement of 257 proteins on polyadenylated RNAs • Coordinated response of RNA-enzyme interactions and metabolism • Yeast RNA-binding enzymes are paralog specific • Integration of three different mass spectrometry analysis tools, Molecular network; Microbial metabolism; Proteomics

RNA-binding proteins (RBPs) are key post-transcriptional regulators that play a substantial role during stress adaptation. Recent proteome-wide surveys have uncovered a large number of new and ‘unconventional’ RBPs such as metabolic enzymes, yet little is known about the reconfiguration of the RNA-binding proteome (RBPome) and RNA-enzyme interactions in response to cellular stress. Here we applied RNA-interactome capture to monitor the dynamics of the RBPome upon mild oxidative stress in the yeast Saccharomyces cerevisiae. Among the 257 proteins that significantly changed RNA associations, we observed the coordinated remodeling of RNA-binding enzymes - particularly of the central carbon metabolism - that complemented known metabolic responses. Furthermore, we recognized the propensity for paralogous specific alterations of enzyme-RNA interactions. Our results suggest coordinated crosstalk between RNA-enzyme interactions and intermediary metabolism to maintain the physiological and molecular balance upon oxidative stress, perhaps through specialization of paralogous during evolution.

The RNA-binding proteins play essential roles in the post-transcriptional regulation of gene expression. While hundreds of RNA-binding proteins can be predicted computationally, the recent introduction of proteome-wide approaches has dramatically expanded the repertoire of proteins interacting with RNA. Besides canonical RNA-binding proteins that contain characteristic RNA-binding domains, many proteins that lack such domains but have other well-characterized cellular functions were identified; including metabolic enzymes, heat shock proteins, kinases, as well as transcription factors and chromatin-associated proteins. In the context of these recently published RNA-protein interactome datasets obtained from yeast, nematodes, flies, plants and mammalian cells, we discuss examples for seemingly evolutionary conserved 'unconventional' RNA-binding proteins that act in central carbon metabolism, stress response or regulation of transcription.

Summary In vivo characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Saccharomyces cerevisiae. Proteins are crosslinked to nucleic acids in vivo by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT]25 beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress. For complete details on the use and execution of this protocol, please refer to Matia-González et al. (2021, 2015)., Graphical abstract, Highlights • Detection of proteins that interact with polyadenylated RNAs in vivo • UV irradiation of cells to crosslink RNA-protein interactions • Polyadenylated RNAs are captured with oligo[dT] beads from yeast cell lysate • Suitable for monitoring dynamic changes of the mRNA-binding proteome, In vivo characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Saccharomyces cerevisiae. Proteins are crosslinked to nucleic acids in vivo by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT]25 beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress.

In the early years of Molecular Biology, the role of RNA was largely considered to be the transmission of the genetic information stored in the DNA into polypeptides. Three major types of RNA engaged in these processes were known: messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). However, within the last half century our knowledge about RNA classes, abundance and diversity of functions has increased dramatically. The discovery that the ribosome is essentially a ribozyme 1, 2 especially brought the central role of RNA for cellular life into the spotlight. The turn of the millennium was also the time of another far‐reaching realization, that of the pervasiveness of small RNA‐dependent regulation of gene expression 3. Within the decade that followed, RNAs were found in essentially all regulatory layers of gene expression, from the epigenetic layer at the top of the gene expression cascade 4, 5 to the most distal translation layer 6. Much of this rapid transition was enabled by sequencing technologies that were developed in the wake of the human genome project. Coupled with ingenious protocols for isolating RNAs of various sizes and molecular properties (e.g. 7) and computational methods to annotate various classes of small RNAs 8, the set of regulatory RNAs has expanded and diversified rapidly. We now know that < 2% of the human genome contains protein‐coding genes, whereas 80% is transcribed into noncoding RNAs (ncRNAs), classified based on their size into small and long noncoding RNAs (lncRNAs). Small ncRNAs include the highly investigated microRNAs (miRNAs) that were found to be increased in various cancer types where they exhibit pro‐oncogenic activity 9. LncRNAs are long (> 200 nucleotides) transcripts that functionally contribute to the control of cell differentiation and maintenance of cell identity. Notably, as most (~ 90%) of disease‐associated single nucleotide polymorphisms are located in gene regulatory or intergenic regions, long‐intergenic ncRNAs (lincRNAs) in particular may have pivotal impact for the development of personalized medicine therapies in the future10. Whether small or long, ncRNAs often bear domains enabling direct and specific interactions with other RNAs, forming RNA‐RNA hybrids that participate in the control of gene expression and biogenesis of RNAs 11.

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

Post-transcriptional control of mRNAs by RNA-binding proteins (RBPs) has a prominent role in the regulation of gene expression. RBPs interact with mRNAs to control their biogenesis, splicing, transport, localization, translation, and stability. Defects in such regulation can lead to a wide range of human diseases from neurological disorders to cancer. Many RBPs are conserved between Caenorhabditis elegans and humans, and several are known to regulate apoptosis in the adult C. elegans germ line. How these RBPs control apoptosis is, however, largely unknown. Here, we identify mina-1(C41G7.3) in a RNA interference-based screen as a novel regulator of apoptosis, which is exclusively expressed in the adult germ line. The absence of MINA-1 causes a dramatic increase in germ cell apoptosis, a reduction in brood size, and an impaired P granules organization and structure. In vivo crosslinking immunoprecipitation experiments revealed that MINA-1 binds a set of mRNAs coding for RBPs associated with germ cell development. Additionally, a system-wide analysis of a mina-1 deletion mutant compared to wild type, including quantitative proteome and transcriptome data, hints to a post-transcriptional regulatory RBP network driven by MINA-1 during germ cell development in C. elegans. In particular, we found that the germline-specific Argonaute WAGO-4 protein levels are increased in mina-1 mutant background. Phenotypic analysis of double mutant mina-1;wago-4 revealed that contemporary loss of MINA-1 and WAGO-4 strongly rescues the phenotypes observed in mina-1 mutant background. To strengthen this functional interaction, we found that upregulation of WAGO-4 in mina-1 mutant animals causes hypersensitivity to exogenous RNAi. Our comprehensive experimental approach allowed us to describe a phenocritical interaction between two RBPs controlling germ cell apoptosis and exogenous RNAi. These findings broaden our understanding of how RBPs can orchestrate different cellular events such as differentiation and death in C. elegans.

Identifying the interaction partners of noncoding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA crosslinking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-crosslinking and Argonaute 2 immunopurification followed by streptavidin affinity purification of probe-linked RNAs provided selectivity in the capture of targets, which were identified by deep sequencing. miR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. miR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long noncoding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation, levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a 'sponge' for these miRNAs.

© 2015 Nature Publishing Group a division of Macmillan Publishers Limited. All Rights Reserved. RNA binding proteins (RBPs) are essential for post transcriptional regulation of gene expression. Recent high throughput screens have dramatically increased the number of experimentally identified RBPs; however comprehensive identification of RBPs within living organisms is elusive. Here we describe the repertoire of 765 and 594 proteins that reproducibly interact with polyadenylated mRNAs in Saccharomyces cerevisiae and Caenorhabditis elegans respectively. Furthermore we report the differential association of mRNA binding proteins (mRPBs) upon induction of apoptosis in C. elegans L4 stage larvae. Strikingly most proteins composing mRBPomes including components of early metabolic pathways and the proteasome are evolutionarily conserved between yeast and C. elegans. We speculate on the basis of our evidence that glycolytic enzymes bind distinct glycolytic mRNAs that enzyme mRNA interactions relate to an ancient mechanism for post transcriptional coordination of metabolic pathways that perhaps was established during the transition from the early 'RNA world' to the 'protein world'.

We describe a tandem RNA isolation procedure (TRIP) that enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues.

Some of the classical paradigms of gene regulation have been challenged by global-scale analysis of eukaryotic transcriptional and post-transcriptional gene regulation (PTGR), made possible by the development of genomics and proteomics tools. Post-transcriptional events in particular are increasingly being recognized as important sources of gene regulation. The hundreds of regulatory RNA-binding proteins that exist in eukaryotes may regulate dozens to hundreds of functionally related RNA targets. Likewise, the expression of considerable fractions of many eukaryotic genomes is affected by hundreds of non-coding RNAs, e.g., microRNAs. These findings suggest an enormous regulatory potential for PTGR that may affect virtually every message in a cell. All gene regulatory systems are composed of simple network circuits that coordinate the transfer of regulatory signals to a target gene/message.

The RNA-binding protein Khd1p (KH-domain protein 1) is required for efficient localization of ASH1 mRNA to the bud-tip, probably acting as a translational repressor during mRNA transport in yeast. Here, we have systematically examined Khd1p mRNA targets and colocalization with known bud-tip-localized mRNAs in vivo. Affinity purification and DNA microarray analysis of Khd1p-associated mRNAs revealed hundreds of potential mRNAs targets, many of them encoding membrane-associated proteins. The putative targets include the messages for MID2, MTL1, WSC2, SRL1, EGT2, CLB2, ASH1, and Khd1p colocalizes with these mRNAs at the bud-tip. The combination of bioinformatics, RNA localization, and in vitro RNA-binding assays revealed that Khd1p binds to CNN repeats in coding regions of mRNA targets. Among the proteins encoded by previously known bud-tip-localized mRNAs, only Mtl1p levels were decreased in khd1Δ mutant cells, whereas Ash1p and Srl1p were reduced in cells overexpressing KHD1. Hence, Khd1p differentially affects gene expression possibly due to combinatorial arrangement with additional factors reflecting the redundant structure of post-transcriptional regulatory systems.

Standard risk stratification methods are incapable of identifying many individuals subsequently experiencing major adverse cardiovascular events (MACE). Coronary artery calcification (CAC) is a powerful predictor of MACE however the routine use of this technique is limited by radiation exposure and

Cytoplasmic mRNA localization provides a means of generating cell asymmetry and segregating protein activity. Previous studies have identified two mRNAs that localize to the bud tips of the yeast Saccharomyces cerevisiae . To identify additional localized mRNAs, we immunoprecipitated the RNA transport components She2p, She3p, and Myo4p and performed DNA microarray analysis of their associated RNAs. A secondary screen, using a GFP-tagged RNA reporter assay, identified 22 mRNAs that are localized to bud tips. These messages encode a wide variety of proteins, including several involved in stress responses and cell wall maintenance. Many of these proteins are asymmetrically localized to buds. However, asymmetric localization also occurs in the absence of RNA transport, suggesting the existence of redundant protein localization mechanisms. In contrast to findings in metazoans, the untranslated regions are dispensable for mRNA localization in yeast. This study reveals an unanticipated widespread use of RNA transport in budding yeast.

RNA-binding proteins (RBPs) play pivotal roles in post-transcriptional regulation, acting as master controllers of the RNA’s life. The development of a variety of global analysis tools during the last decade has tremendously enriched our understanding of the function of RBPs and their implications in environmental and cellular cues. In this chapter, we discuss the properties and functions of the RBPs in fungi, and we outline the genome-wide approaches that have been developed during the last decade to systematically identify their RNA targets. We further sketch recent approaches aimed to search for novel RBPs, revealing that many more proteins such as metabolic enzymes could have additional functions in RNA regulation. Based on a rich dataset obtained in budding yeast, we further refer to attempts to integrate RNA targets for more than 60 RBPs into molecular networks to reveal structures and motifs. Finally, we elaborate on the future tasks and challenges on our way to complete our understanding of this highly connected and interwoven protein-RNA interaction network and its importance for cell physiology.

The posttranscriptional modification of messenger RNA precursors (pre-mRNAs) by base deamination can profoundly alter the physiological function of the encoded proteins. The recent identification of tRNA-specific adenosine deaminases (ADATs) has led to the suggestion that these enzymes, as well as the cytidine and adenosine deaminases acting on pre-mRNAs (CDARs and ADARs), belong to a superfamily of RNA-dependent deaminases. This superfamily might have evolved from an ancient cytidine deaminase. This article reviews the reactions catalysed by these enzymes and discusses their evolutionary relationships.

Several transfer RNAs (tRNAs) contain inosine (I) at the first position of their anticodon (position 34); this modification is thought to enlarge the codon recognition capacity during protein synthesis. The tRNA-specific adenosine deaminase of Saccharomyces cerevisiae that forms I 34 in tRNAs is described. The heterodimeric enzyme consists of two sequence-related subunits (Tad2p/ADAT2 and Tad3p/ADAT3), both of which contain cytidine deaminase (CDA) motifs. Each subunit is encoded by an essential gene ( TAD2 and TAD3 ), indicating that I 34 is an indispensable base modification in elongating tRNAs. These results provide an evolutionary link between the CDA superfamily and RNA-dependent adenosine deaminases (ADARs/ADATs).

The double-stranded RNA-specific adenosine deaminases ADAR1 and ADAR2 convert adenosine (A) residues to inosine (I) in messenger RNA precursors (pre-mRNA). Their main physiological substrates are pre-mRNAs encoding subunits of ionotropic glutamate receptors or serotonin receptors in the brain. ADAR1 and ADAR2 have similar sequence features, including double-stranded RNA binding domains (dsRBDs) and a deaminase domain. The tRNA-specific adenosine deaminases Tad1p and Tad2p/Tad3p modify A 37 in tRNA-Ala1 of eukaryotes and the first nucleotide of the anticodon (A 34) of several bacterial and eukaryotic tRNAs, respectively. Tad1p is related to ADAR1 and ADAR2 throughout its sequence but lacks dsRBDs. Tad1p could be the ancestor of ADAR1 and ADAR2. The deaminase domains of ADAR1, ADAR2 and Tad1p are very similar and resemble the active site domains of cytosine/cytidine deaminases.

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Post-transcriptional gene regulation is largely mediated by RNA-binding proteins (RBPs) that modulate mRNA expression at multiple levels, from RNA processing to translation, localization, and degradation. Thereby, the genome-wide identification of mRNAs regulated by RBPs is crucial to uncover post--transcriptional gene regulatory networks. In this chapter, we provide a detailed protocol for one of the techniques that has been developed to systematically examine RNA targets for RBPs. This technique involves the purification of endogenously formed RBP-mRNA complexes with specific antibodies from cellular extracts, followed by the identification of associated RNAs using DNA microarrays. Such RNA-binding protein immunopurification-microarray profiling, also called RIP-Chip, has also been applied to identify mRNAs that are transported to distinct subcellular compartments by RNP-motor complexes. The application and further development of this method could provide global insights into the subcellular architecture of the RBP-RNA network, and how it is restructured upon changing environmental conditions, during development, and possibly in disease.

Post-transcriptional gene regulation is largely mediated by RNA-binding proteins (RBPs) that modulate mRNA expression at multiple levels, from RNA processing to translation, localization, and degradation. Thereby, the genome-wide identification of mRNAs regulated by RBPs is crucial to uncover post--transcriptional gene regulatory networks. In this chapter, we provide a detailed protocol for one of the techniques that has been developed to systematically examine RNA targets for RBPs. This technique involves the purification of endogenously formed RBP-mRNA complexes with specific antibodies from cellular extracts, followed by the identification of associated RNAs using DNA microarrays. Such RNA-binding protein immunopurification-microarray profiling, also called RIP-Chip, has also been applied to identify mRNAs that are transported to distinct subcellular compartments by RNP-motor complexes. The application and further development of this method could provide global insights into the subcellular architecture of the RBP-RNA network, and how it is restructured upon changing environmental conditions, during development, and possibly in disease.

mRNAs encoding mitochondrial proteins are enriched in the vicinity of mitochondria, presumably to facilitate protein transport. A possible mechanism for enrichment may involve interaction of the translocase of the mitochondrial outer membrane (TOM) complex with the precursor protein while it is translated, thereby leading to association of polysomal mRNAs with mitochondria. To test this hypothesis, we isolated mitochondrial fractions from yeast cells lacking the major import receptor, Tom20, and compared their mRNA repertoire to that of wild-type cells by DNA microarrays. Most mRNAs encoding mitochondrial proteins were less associated with mitochondria, yet the extent of decrease varied among genes. Analysis of several mRNAs revealed that optimal association of Tom20 target mRNAs requires both translating ribosomes and features within the encoded mitochondrial targeting signal. Recently, Puf3p was implicated in the association of mRNAs with mitochondria through interaction with untranslated regions. We therefore constructed a tom20 Delta puf3 Delta double-knockout strain, which demonstrated growth defects under conditions where fully functional mitochondria are required. Mislocalization effects for few tested mRNAs appeared stronger in the double knockout than in the tom20 Delta strain. Taken together, our data reveal a large-scale mRNA association mode that involves interaction of Tom20p with the translated mitochondrial targeting sequence and may be assisted by Puf3p.

We describe ribosome affinity purification (RAP), a method that allows rapid purification of ribosomes and associated messages from the yeast Saccharomyces cerevisiae. The method relies on the expression of protein A tagged versions of the ribosomal protein Rpl16, which is used to efficiently recover endogenously formed ribosomes and polysomes from cellular extracts with IgG-coupled spherical microbeads. This approach can be applied to profile reactions of the translatome, which refers to all messages associated with ribosomes, with those of the transcriptome using DNA microarrays. In addition, ribosomal proteins, their modifications, and/or other associated proteins can be mapped with mass spectrometry. Finally, application of this method in other organisms provides a valuable tool to decipher cell-type specific gene expression patterns.

While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

Homeostatic regulation of ionic currents is of paramount importance during periods of synaptic growth or remodeling. Our previous work has identified the translational repressor Pumilio (Pum) as a regulator of sodium current (INa) and excitability inDrosophilamotoneurons. In this current study, we show that Pum is able to bind directly the mRNA encoding theDrosophilavoltage-gated sodium channelparalytic(para). We identify a putative binding site for Pum in the 3′ end of theparaopen reading frame (ORF). Characterization of the mechanism of action of Pum, using whole-cell patch clamp and real-time reverse transcription-PCR, reveals that the full-length protein is required for translational repression ofparamRNA. Additionally, the cofactor Nanos is essential for Pum-dependentpararepression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation ofINain motoneurons requires both Nanos and Brat, whereas regulation in other neuronal types seemingly requires only Nanos but not Brat. We also show that Pum is able to reduce the level ofnanosmRNA and as such identify a potential negative-feedback mechanism to protect neurons from overactivity of Pum. Finally, we show coupling betweenINa(para) andIK(Shal) such that Pum-mediated change inpararesults in a compensatory change inShal. The identification ofparaas a direct target of Pum represents the first ion channel to be translationally regulated by this repressor and the location of the binding motif is the first example in an ORF rather than in the canonical 3′-untranslated region of target transcripts.

Introduction Conventional risk stratification fails to identify many individuals presenting with major adverse cardiovascular events (MACE). Coronary artery calcification (CAC) is a powerful independent predictor of MACE however its use is limited by radiation and expense. MicroRNAs are non-coding RNAs that regulate transcription and their differential expression is acknowledged to be a hallmark of a number of diseases. We aim to determine if a peripheral blood-based microRNA profile is predictive of the presence and extent of CAC in humans. Methods Study patients met the following inclusion criteria: attendance for elective cardiac computed tomography; age 18–65 years; no history of coronary artery disease, cardiomyopathy or tachyarrhythmia; normal renal function; no history of diabetes mellitus; no autoimmune disease; no infection; no malignancy. Peripheral venesection was performed and an Agatston score was derived using default software. RNA was extracted using the LeukoLOCK Total RNA Isolation System and stored at –80 o C until Toray’s microarray analysis was undertaken. Results 24 participants were recruited (mean age 54 years; 67% male) and divided into the following categories: [CAC score 0] n = 6; [CAC score 1–10] n = 6; [CAC score 11–100] n = 6; [CAC score > 100] n = 6. Groups were matched according to their baseline characteristics; Table 1. The Student’s t-test was performed between groups. MiR-1181 was significantly down-regulated in all case groups compared to controls: [CAC 1–10] effect size (ES) = 1.76, p = 0.012; [CAC 11–100] ES = 1.74, p = 0.013; [CAC > 100] ES = 3.86, p 100 compared to controls (ES = 3, p Conclusions Human blood-based miRNA-1181 appears to predict the presence and extent of CAC. Likewise miRNAs miR-138-2-3p, miR-6816-3p and miR-8059 are differentially expressed in patients with high CAC scores. We plan to validate these findings using quantitative real-time PCR and to test these results in a prospective cohort. Ultimately miRNAs could act as a biomarker for MACE and identifying their associated molecular pathways may explain the mechanisms underpinning CAC.

Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3′-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins.

Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes. Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically. We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs. Distinct groups of 40–220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. We identified distinct sequence motifs in the 3′-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p. Three-hybrid assays confirmed the role of these motifs in specific RNA–protein interactions in vivo. The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program., Messenger RNAs that are associated with each of the five Puf RNA-binding proteins can be grouped according to function and localization, suggesting that tagging of transcripts by specific RNA-binding proteins coordinates the control of their localization, translation, and decay

To date, two structurally related RNA-editing enzymes with adenosine deaminase activity have been identified in mammalian tissue: ADAR1 and ADAR2 [Bass B. I. et al. (1997) RNA 3, 947–949]. In rodents, ADAR2 undergoes alternative RNA splicing, giving rise to two splice variants that differ by the presence or absence of a 10-amino-acid insert in the carboxy-terminal catalytic domain. However, the physiological significance of the splicing and its regional and developmental regulation are as yet unknown. The present study examined spatial and temporal patterns of ADAR2 gene transcripts within specific neuronal populations of rat brain. The two rodent ADAR2 isoforms were expressed at comparable levels at all ages examined. rADAR2 messenger RNA expression was first detectable in the thalamic nuclei formation at embryonic day E19. The rADAR2b insert and rADAR2a splice probes produced images similar to that of the rADAR2 pan probe. At birth, rADAR2a messenger RNA splice variants were abundantly expressed in the thalamic nuclei. No signal for any probe was detectable in other brain regions, including neocortex, hippocampus, striatum and cerebellum at this stage of development. During the first week of postnatal life, rADAR2 messenger RNA expression (detected with the pan probe) increased gradually in several brain regions, with low expression detected at postnatal day P7 in the olfactory bulb, inferior colliculus, and within the pyramidal and granule cell layers of the hippocampus. Hybridization patterns of the rADAR2a variant probe reached peak expression at about the second week of life, while peak expression of the rADAR2b probe was reached at about the third week of life. At the end of the first week of life (P7), expression of both splice variants was strongest in the thalamic nuclei. By P14, rADAR2 messenger RNA expression was more consolidated in the deeper structures, including the thalamic nuclei and the granule cell layer of the cerebellum. By P21, maximal levels of rADARb expression were observed in the thalamic nuclei, inferior colliculus, cerebellum and pontine nuclei. In the adult, rADAR2 messenger RNA expression was of highest intensity in the thalamic nuclei, with high levels of expression in the olfactory bulb, inferior colliculus, cerebellum and pontine nuclei. At the level of the hippocampus, positive labelling was restricted to the CA3 region of the Ammon's horn and the dentate gyrus, with weak signals in the CA1 subfield. rADAR2 pan expression was at near background levels throughout the neocortex and caudate putamen. In summary, our study shows that ADAR2 messenger RNA expression is regulated in a cell-specific manner throughout development. At early ages, ADAR2 messenger RNA is expressed only within (and restricted to) the thalamic nuclei. By the third postnatal week, expression of the editase enzyme is more widely distributed throughout the olfactory bulb, CA3 and dentate gyrus of the hippocampus, thalamus, inferior colliculus and the molecular cell layer of the cerebellum. ADAR2 is thought to act at specific nucleotide positions in primary transcripts encoding glutamate receptor subunits, thereby altering gating and ionic permeability properties of AMPA- and kainate-activated channels. ADAR2 also acts at pre-messenger RNA encoding the serotonin 5HT-2C receptor to alter G-protein coupling. Thus, RNA editing may be an important mechanism for fine-tuning of the physiological and pharmacological properties of transmitter receptors of the central nervous system.

ADAR1 and ADAR2 are members of a family of enzymes that catalyze the conversion of adenosine to inosine in double-stranded RNA. Unlike the other types of RNA editing that involve multiprotein editing complexes, the site-specific deamination of an adenosine to inosine is catalyzed by single enzymes. ADAR1 and ADAR2 have been purified and the genes cloned from various sources. Each gene encodes multiple splice variants. As it is crucial to have an adequate supply of pure protein to investigate this type of RNA editing, we describe in this article methods for both the purification and the overexpression of either full-length or partial ADAR1 and ADAR2 isoforms.

RNAs encoding subunits of glutamate-gated ion channel receptors are posttranscriptionally modified by RNA editing and alternative splicing. The change in amino acid sequence caused by RNA editing can affect both the kinetics and the permeability of the ion channel receptors to cations. Here, we report the purification of a 90-kDa double-stranded RNA-specific adenosine deaminase from HeLa cell nuclear extract that specifically edits the glutamine codon at position 586 in the pre-mRNA of the glutamate receptor B subunit. Site-specific deamination of an adenosine to an inosine converts the glutamine codon to that of arginine. Recently, a gene encoding a double-stranded-specific editase (RED1) was cloned from a rat brain cDNA library. Antibodies generated against the deaminase domain of its human homolog specifically recognized and inhibited the activity of the 90-kDa enzyme, indicating that we have purified hRED1 the human homolog of rat RED1. This enzyme is distinct from double-stranded RNA-specific adenosine deaminase which we and others have previously purified and cloned.

Abstract 3220 Background: Fetal hemoglobin, HbF (α2γ2), is the main hemoglobin synthesized until birth; it subsequently declines and switches to adult hemoglobin, HbA (α2β2). Certain mutations within the globin gene locus can cause persistent HbF synthesis after birth, ameliorating symptoms in hemoglobinopathies. Analysis from e.g. Hb Corfu and δβ-thalassemia indicates that γ-globin gene expression is post-transcriptionally regulated (Efremov DG., Br J Haematol, Oct 1994; Chakalova L., Blood, 2005). The post-transcriptional regulation of HbF might involve regulatory non-coding RNAs. MicroRNAs (miRNAs) are a small non-coding family of 21 nucleotide RNAs that regulate gene expression post-transcriptionally by targeting mRNAs. After transcription and maturation the miRNA is incorporated into the miRNA-induced silencing complex (miRISC). The miRISC, which contains argonaute proteins (AGO), binds and silences the target mRNAs. Previous studies have reported a role of miRNAs in the developmental switch from HbF to HbA synthesis (Noh SJ, J Transl Med, 2009; Bianchi N., BMB Rep, 2009), however so far no direct interaction between miRNAs and the γ-globin mRNA has been reported. Moreover a function of miRISC in mature red blood cells (RBC) has not been shown yet. Therefore, we aimed (I) to characterize the miRISC composition in RBCs, (II) to identify miRNAs that are directly involved in the regulation of HbF and (III) to study their function during erythropoiesis. Methods: To characterize miRISC in RBCs, we performed co-immunoprecipitations of the miRISC using antibodies directed against human AGO. The immunoprecipitated complexes were analyzed by Western-blot, mass spectrometry and qPCR. To identify miRNAs potentially involved in the regulation of HbF miRNA expression patterns in RBCs from adult and umbilical cord blood were analyzed by qPCR. To confirm the role of selected miRNAs, γ-globin protein levels were measured by ELISA in HEL cell cultures transfected with miRNA precursors or inhibitors as well as in primary erythroid cultures overexpressing miRNAs after lentiviral transduction. The direct interaction between miRNAs and the γ-globin mRNA was analyzed by luciferase reporter assay. Results: AGO1, 2, 3 and 4 and Importin 8 were detected in RBCs both from adult and cord blood. In adult RBC samples (n=3), expressing Conclusions: This study identifies miR-96 as direct inhibitor of the HbF expression during erythropoiesis. Specifically we observed that in cells with low HbF content, γ-globin mRNAs are bound to miRISC and are therefore directly inhibited by miRNAs. We demonstrated that miR-96 is binding to a seedless target site within the γ-globin open reading frame. Finally we showed that miR-96 overexpression reduces the expression of HbF in erythroleukemia cells and during late stages of cord blood derived erythropoiesis. Disclosures: No relevant conflicts of interest to declare.

We describe a new broadly applicable methodology for screening in parallel interactions of RNA binding proteins (RBPs) with large numbers of microRNA (miRNA) precursors and for determining their affinities in native form in the presence of cellular factors. The assays aim at identifying pre miRNAs that are potentially affected by the selected RBP during their biogenesis. The assays are carried out in microtiter plates and use chemiluminescent readouts. Detection of bound RBPs is achieved by protein or tag specific antibodies allowing crude cell lysates to be used as a source of RBP. We selected 70 pre miRNAs with phylogenetically conserved loop regions and 25 precursors of other well characterized miRNAs for chemical synthesis in 3' biotinylated form. An equivalent set in unmodified form served as inhibitors in affinity determinations. By testing three RBPs known to regulate miRNA biogenesis on this set of pre miRNAs we demonstrate that Lin28 and hnRNP A1 from cell lysates or as recombinant protein domains recognize preferentially precursors of the let 7 family and that KSRP binds strongly to pre miR 1 2.