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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.

RNA-binding proteins (RBPs) participate in several steps of post-transcriptional regulation of gene expression, such as splicing, messenger RNA transport, mRNA localization, and translation. Gene-expression regulation in trypanosomatids occurs primarily at the post-transcriptional level, and RBPs play important roles in the process. Here, we characterized the RBP TcSgn1, which contains one RNA recognition motif (RRM). TcSgn1 is a close ortholog of yeast Saccharomyces cerevisiae protein ScSgn1, which plays a role in translational regulation in the cytoplasm. We found that TcSgn1 in Trypanosoma cruzi is localized in the nucleus in exponentially growing epimastigotes. By performing immunoprecipitation assays of TcSgn1, we identified hundreds of mRNAs associated with the protein, a significant fraction of them coding for nucleic acids binding, transcription, and endocytosis proteins. In addition, we show that TcSgn1 is capable of interacting directly with the poly(A) tail of the mRNAs. The study of parasites under nutritional stress showed that TcSgn1 was localized in cytoplasmic granules in addition to localizing in the nucleus. Similar to ScSgn1, we observed that TcSgn1 also interacts with the PABP1 protein, suggesting that this protein may play a role in regulating gene expression in T. cruzi. Taken together, our results show that RNA-binding protein TcSgn1 is part of ribonucleoprotein complexes associated with nuclear functions, stress response, and RNA metabolism.

Post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycinbased tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3'UTRs of cyclin-dependent kinase inhibitor 1B (CDKN1B/p27Kip1) mRNAs in vivo. P27Kip1 plays an important role in mediating a cell's response to cisplatin (CP), a widely used chemotherapeutic cancer drug that induces DNA damage and cell cycle arrest. We found that p27Kip1 mRNA is stabilized upon CP treatment of HEK293 cells through elements in its 3'UTR. Applying tobTRIP, we further compared the associated proteins in CP and non-treated cells, and identified more than fifty interacting RBPs, many functionally related and evoking a coordinated response. Knock-downs of several of the identified RBPs in HEK293 cells confirmed their involvement in CP-induced p27 mRNA regulation; while knock-down of the KH-type splicing regulatory protein (KHSRP) further enhanced the sensitivity of MCF7 adenocarcinoma cancer cells to CP treatment. Our results highlight the benefit of specific in vivo mRNA-protein interactome capture to reveal post-transcriptional regulatory networks implicated in cellular drug response and adaptation.

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.

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-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

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.

Cellular and Molecular Life Sciences, 65 (5), ISSN:1420-682X, ISSN:1420-9071

Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs) and small non-coding RNAs (e.g., microRNAs) that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

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 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. ISSN:1362-4962 ISSN:0301-5610

Whilst the profiling of the transcriptome and proteome even of single-cells becomes feasible, the analysis of the translatome, which refers to all messenger RNAs (mRNAs) engaged with ribosomes for protein synthesis, is still an elaborate procedure requiring millions of cells. Herein, we report the generation and use of “smart materials”, namely molecularly imprinted polymers (MIPs) to facilitate the isolation of ribosomes and translated mRNAs from merely 1,000 cells. In particular, we show that a hydrogel-based ribosome imprinted polymer could recover ribosomes and associated mRNAs from human, simian and mice cellular extracts, but did not selectively enrich yeast ribosomes, thereby demonstrating selectivity. Furthermore, ribosome imprinted polymers enabled the sensitive measurement of an mRNA translational regulatory event, requiring 1,000-fold less cells than current methodologies. These results provide first evidence for the suitability of MIPs to selectively recover ribonucleoprotein complexes such as ribosomes, founding a novel means for sensitive detection of gene regulation.

Caspases are key components of apoptotic pathways. Regulation of caspases occurs at several levels, including transcription, proteolytic processing, inhibition of enzymatic function, and protein degradation. In contrast, little is known about the extent of post-transcriptional control of caspases. Here, we describe four conserved RNA-binding proteins (RBPs)—PUF-8, MEX-3, GLD-1, and CGH-1—that sequentially repress the CED-3 caspase in distinct regions of the Caenorhabditis elegans germline. We demonstrate that GLD-1 represses ced-3 mRNA translation via two binding sites in its 3′ untranslated region (UTR), thereby ensuring a dual control of unwanted cell death: at the level of p53/CEP-1 and at the executioner caspase level. Moreover, we identified seven RBPs that regulate human caspase-3 expression and/or activation, including human PUF-8, GLD-1, and CGH-1 homologs PUM1, QKI, and DDX6. Given the presence of unusually long executioner caspase 3′ UTRs in many metazoans, translational control of executioner caspases by RBPs might be a strategy used widely across the animal kingdom to control apoptosis.

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

During the past decade, there has been a rapidly increased appreciation of the role of translation as a key regulatory node in gene expression. Thereby, the development of methods to infer the translatome, which refers to the entirety of mRNAs associated with ribosomes for protein synthesis, has facilitated the discovery of new principles and mechanisms of translation and expanded our view of the underlying logic of protein synthesis. Here, we review the three main methodologies for translatome analysis, and we highlight some of the recent discoveries made using each technique. We first discuss polysomal profiling, a classical technique that involves the separation of mRNAs depending on the number of bound ribosomes using a sucrose gradient, and which has been combined with global analysis tools such as DNA microarrays or high-throughput RNA sequencing to identify the RNAs in polysomal fractions. We then introduce ribosomal profiling, a recently established technique that enables the mapping of ribosomes along mRNAs at near-nucleotide resolution on a global scale. We finally refer to ribosome affinity purification techniques that are based on the cell-type-specific expression of tagged ribosomal proteins, allowing the capture of translatomes from specialized cells in organisms. We discuss the advantages and disadvantages of these three main techniques in the pursuit of defining the translatome, and we speculate about future developments.

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.

The development of genome-wide analysis tools has prompted global investigation of the gene expression program, revealing highly coordinated control mechanisms that ensure proper spatiotemporal activity of a cell’s macromolecular components. With respect to the regulation of RNA transcripts, the concept of RNA regulons, which – by analogy with DNA regulons in bacteria – refers to the coordinated control of functionally related RNA molecules, has emerged as a unifying theory that describes the logic of regulatory RNA-protein interactions in eukaryotes. Hundreds of RNA-binding proteins and small non-coding RNAs, such as microRNAs, bind to distinct elements in target RNAs, thereby exerting specific and concerted control over posttranscriptional events. In this review, we discuss recent reports committed to systematically explore the RNA-protein interaction network and outline some of the principles and recurring features of RNA regulons: the coordination of functionally related mRNAs through RNA-binding proteins or non-coding RNAs, the modular structure of its components, and the dynamic rewiring of RNA-protein interactions upon exposure to internal or external stimuli. We also summarize evidence for robust combinatorial control of mRNAs, which could determine the ultimate fate of each mRNA molecule in a cell. Finally, the compilation and integration of global protein-RNA interaction data has yielded first insights into network structures and provided the hypothesis that RNA regulons may, in part, constitute noise ‘buffers’ to handle stochasticity in cellular transcription.

The La-motif (LAM) is an ancient and ubiquitous RNA-binding domain defining a superfamily of proteins, which comprises the genuine La proteins and La-related proteins (LARPs). In contrast to La, which binds and stabilizes pre-tRNAs and other RNA polymerase III transcripts, data on function and RNA targets of the LARPs have remained scarce. We have undertaken a global approach to elucidate the previously suggested role of the yeast LARP Slf1p in copper homeostasis. By applying RNA-binding protein immunopurification-microarray (RIP-Chip) analysis, we show that Slf1p and its paralog Sro9p copurify with overlapping sets of hundreds of functionally related mRNAs, including many transcripts coding for ribosomal proteins and histones. Interestingly, among these potential RNA targets were also mRNAs coding for proteins critical for protection of cells against elevated copper concentrations. Mutations introduced in the conserved aromatic patch of the LAM in Slf1p drastically impaired both association with its targets and Slf1-mediated protection of cells against toxic copper concentrations. Furthermore, we show that Slf1p stabilizes copper-related mRNA targets in a LAM-dependent manner. These results provide the first evidence for post-transcriptional regulation of factors/pathways implicated in copper homeostasis by a cytoplasmic RBP.

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Vascular endothelial growth factor A (VEGFA) plays a key role in the angiogenesis of human skin. Elevated levels of VEGFA are associated with several pathological conditions, including chronic inflammatory skin diseases and several types of skin cancer. In particular, squamous cell carcinoma (SCC) of the skin, the second most common skin cancer in the general population, is characterized by invasive growth, pronounced angiogenesis and elevated levels of VEGFA. The processing, turnover and production of VEGFA are extensively regulated at the post-transcriptional level, both by RNA-binding proteins and microRNAs (miRNAs). In the present study, we identified a new miRNA recognition element in a downstream conserved region of the VEGFA 3′-UTR. We confirmed the repressive effect of miR-361-5p on this element in vitro, identifying the first target for this miRNA. Importantly, we found that miR-361-5p levels are inversely correlated with VEGFA expression in SCC and in healthy skin, indicating that miR-361-5p could play a role in cancers. ISSN:1932-6203

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.

Fetal hemoglobin, HbF (α(2)γ(2)), is the main hemoglobin synthesized up to birth, but it subsequently declines and adult hemoglobin, HbA (α(2)β(2)), becomes predominant. Several studies have indicated that expression of the HbF subunit γ-globin might be regulated post-transcriptionally. This could be confered by ∼22-nucleotide long microRNAs that associate with argonaute proteins to specifically target γ-globin mRNAs and inhibit protein expression. Indeed, applying immunopurifications, we found that γ-globin mRNA was associated with argonaute 2 isolated from reticulocytes that contain low levels of HbF (

RNA-binding proteins (RBPs) play important roles in the posttranscriptional control of gene expression. However, our understanding of how RBPs interact with each other at different regulatory levels to coordinate the RNA metabolism of the cell is rather limited. Here, we construct the posttranscriptional regulatory network among 69 experimentally studied RBPs in yeast to show that more than one-third of the RBPs autoregulate their expression at the posttranscriptional level and demonstrate that autoregulatory RBPs show reduced protein noise with a tendency to encode for hubs in this network. We note that in- and outdegrees in the posttranscriptional RBP-RBP regulatory network exhibit gaussian and scale-free distributions, respectively. This network was also densely interconnected with extensive cross-talk between RBPs belonging to different posttranscriptional steps, regulating varying numbers of cellular RNA targets. We show that feed-forward loops and superposed feed-forward/feedback loops are the most significant three-node subgraphs in this network. Analysis of the corresponding protein-protein interaction (posttranslational) network revealed that it is more modular than the posttranscriptional regulatory network. There is significant overlap between the regulatory and protein-protein interaction networks, with RBPs that potentially control each other at the posttranscriptional level tending to physically interact and being part of the same ribonucleoprotein (RNP) complex. Our observations put forward a model wherein RBPs could be classified into those that can stably interact with a limited number of protein partners, forming stable RNP complexes, and others that form transient hubs, having the ability to interact with multiple RBPs forming many RNPs in the cell.

Background Glucose inhibition of gluconeogenic growth suppressor 2 protein (Gis2p) and zinc-finger protein 9 (ZNF9) are conserved yeast and human zinc-finger proteins. The function of yeast Gis2p is unknown, but human ZNF9 has been reported to bind nucleic acids, and mutations in the ZNF9 gene cause the neuromuscular disease myotonic dystrophy type 2. To explore the impact of these proteins on RNA regulation, we undertook a systematic analysis of the RNA targets and of the global implications for gene expression. Results Hundreds of mRNAs were associated with Gis2p, mainly coding for RNA processing factors, chromatin modifiers and GTPases. Target mRNAs contained stretches of G(A/U)(A/U) trinucleotide repeats located in coding sequences, which are sufficient for binding to both Gis2p and ZNF9, thus implying strong structural conservation. Predicted ZNF9 targets belong to the same functional categories as seen in yeast, indicating functional conservation, which is further supported by complementation of the large cell-size phenotype of gis2 mutants with ZNF9. We further applied a matched-sample proteome-transcriptome analysis suggesting that Gis2p differentially coordinates expression of RNA regulons, primarily by reducing mRNA and protein levels of genes required for ribosome assembly and by selectively up-regulating protein levels of myosins. Conclusions This integrated systematic exploration of RNA targets for homologous RNA-binding proteins indicates an unexpectedly high conservation of the RNA-binding properties and of potential targets, thus predicting conserved RNA regulons. We also predict regulation of muscle-specific genes by ZNF9, adding a potential link to the myotonic dystrophy related phenotypes seen in ZNF9 mouse models., Genome Biology, 12 (1), ISSN:1474-760X