We explore global and specific aspects of post-transcriptional gene regulation mediated by RNA-binding proteins and non-coding RNAs.
Gene expression must be tightly controlled to ensure coordinated synthesis of the cells’ macromolecular components. Besides transcriptional control, it has become evident that also the later post-transcriptional steps – namely the processing, transport, turnover and translation of mRNAs – are pivotal for the diversification and spatiotemporal control of gene expression. Hundreds of RNA-binding proteins and non-coding RNAs mediate post-transcriptional control with widespread implications in cell physiology and disease. Nevertheless, the targets and functions for most RNA-binding proteins and non-coding RNAs are still not known.
We have been combining genome-wide analysis with classical biochemical and genetic tools to identify the RNA targets of RNA-binding proteins and to investigate post-transcriptional gene regulation on a global scale. Importantly, these studies revealed that RNA-binding proteins bind to and coordinate groups of mRNAs that code for proteins, which are localized to the same subcellular compartment, act in the same pathway or are components of macromolecular complexes, forming so-called RNA regulons. Moreover, these set of RNAs often bear conserved sequence/ structural elements that likely represent binding sites for RNA-binding proteins. These findings suggested the presence of a highly-organized and elaborate post-transcriptional regulatory system that may affect virtually every mRNA in a cell.
We are further exploring the post-transcriptional regulatory landscape. On the one hand, we study specific RNA-binding proteins that coordinate the localization, decay or translation of mRNAs in the cytoplasm. On the other hand, we characterize the translatome – which refers to all mRNAs that are associated with ribosomes for protein synthesis (Halbeisen et al. 2009) – and we monitor its reaction upon stress or drug treatment of cells and in pathological conditions. We primarily use budding yeast as model to establish new techniques and to elucidate principles of post-transcriptional control, and we work with mammalian cells to unravel implications in disease.
Different proteins assemble on a given mRNA to form a ribonucleoprotein complex (mRNP), the composition of which changes dynamically, depending on the cellular context. The combinatorial control of associated regulatory, scaffolding and accessory proteins ultimately determines fate of the mRNA ("mRNP code").
Dr. Daniela Fiori Gradia - Visiting Scientist
RNA binding proteins and non-coding RNA metabolism
Dr. Helen King - Postdoctoral Fellow
Translatome analysis in mammalian cells
Dr. Ana Maria Matia - Postdoctoral Fellow
RNA-protein interactome in yeast and C. elegans
Maikel Wouters - Research Fellow
Combinatorial control of cancer-related mRNAs by RBPs and ncRNAs
Ian Wright - Technical Assitant (50%)
Anne Meiwald - Internship
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.
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.
Fetal hemoglobin, HbF (a2c2), is the main hemoglobin synthesized up to birth, but it subsequently declines and adult hemoglobin, HbA (a2b2), becomes predominant. Several studies have indicated that expression of the HbF subunit c-globin might be regulated post-transcriptionally. This could be confered by ,22-nucleotide long microRNAs that associate with argonaute proteins to specifically target c-globin mRNAs and inhibit protein expression. Indeed, applying immunopurifications, we found that c-globin mRNA was associated with argonaute 2 isolated from reticulocytes that contain low levels of HbF (,1%), whereas association was significantly lower in reticulocytes with high levels of HbF (90%). Comparing microRNA expression in reticulocytes from cord blood and adult blood, we identified several miRNAs that were preferentially expressed in adults, among them miRNA-96. The overexpression of microRNA-96 in human ex vivo erythropoiesis decreased c-globin expression by 50%, whereas the knock-down of endogenous microRNA-96 increased c-globin expression by 20%. Moreover, luciferase reporter assays showed that microRNA-96 negatively regulates expression of c-globin in HEK293 cells, which depends on a seedless but highly complementary target site located within the coding sequence of c-globin. Based on these results we conclude that microRNA-96 directly suppresses c-globin expression and thus contributes to HbF regulation.
RNA-bindingproteins (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–proteininteraction (posttranslational) network revealed that it is more modular than the posttranscriptional regulatory network. There is significant overlap between the regulatory and protein–proteininteraction 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.
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.
Hundreds of RNA-binding proteins (RBPs) control diverse aspects of post-transcriptional gene regulation. To identify novel and unconventional RBPs, we probed high-density protein microarrays with fluorescently labeled RNA and selected 200 proteins that reproducibly interacted with different types of RNA from budding yeast Saccharomyces cerevisiae. Surprisingly, more than half of these proteins represent previously known enzymes, many of them acting in metabolism, providing opportunities to directly connect intermediary metabolism with posttranscriptional gene regulation. We mapped the RNA targets for 13 proteins identified in this screen and found that they were associated with distinct groups of mRNAs, some of them coding for functionally related proteins. We also found that overexpression of the enzyme Map1 negatively affects the expression of experimentally defined mRNA targets. Our results suggest that many proteins may associate with mRNAs and possibly control their fates, providing dense connections between different layers of cellular regulation.
Trf4p and Trf5p are non-canonical poly(A polymerases and are part of the heteromeric protein complexes TRAMP4 and TRAMP5 that promote the degradation of aberrant and short-lived RNA substrates by interacting with the nuclear exosome. To assess the level of functional redundancy between the paralogous Trf4 and Trf5 proteins and to investigate the role of the Trf4-dependent polyadenylation in vivo, we used DNA microarrays to compare gene expression of the wild-type yeast strain of S. cerevisiae with either that of trf4D or trf5D mutant strains or the trf4D mutant expressing the polyadenylationdefective Trf4(DADA) protein. We found little overlap between the sets of transcripts with altered expression in the trf4D or the trf5D mutants, suggesting that Trf4p and Trf5p target distinct groups of RNAs for degradation. Surprisingly, most RNAs the expression of which was altered by the trf4 deletion were restored to wild-type levels by overexpression of TRF4(DADA), showing that the polyadenylation activity of Trf4p is dispensable in vivo. Apart from previously reported Trf4p and Trf5p target RNAs, this analysis along with in vivo cross-linking and RNA immunopurification-chip experiments revealed that both the TRAMP4 and the TRAMP5 complexes stimulate the degradation of spliced-out introns via a mechanism that is independent of the polyadenylation activity of Trf4p. In addition, we show that disruption of trf4 causes severe shortening of telomeres suggesting that TRF4 functions in the maintenance of telomere length. Finally, our study demonstrates that TRF4, the exosome, and TRF5 participate in antisense RNA–mediated regulation of genes involved in phosphate metabolism. In conclusion, our results suggest that paralogous TRAMP complexes have distinct RNA selectivities with functional implications in RNA surveillance as well as other RNA–related processes. This indicates widespread and integrative functions of TRAMP complexes for the coordination of different gene expression regulatory processes.
Cells rapidly alter gene expression in response to environmental stimuli such as nutrients, hormones, and drugs. During the imposed ‘‘remodeling’’ of gene expression, changes in the levels of particular mRNAs do not necessarily correlate with those of the encoded proteins, which could in part rely on the differential recruitment of mRNAs to translating ribosomes. To systematically address this issue, we have established an approach to rapidly access the translational status of each mRNA in the yeast Saccharomyces cerevisiae by affinity purification of endogenously formed ribosomes and the analysis of associated mRNAs with DNA microarrays. Using this method, we compared changes in total mRNA levels (transcriptome) with ribosome associations (translatome) after the application of different conditions of cellular stress. Severe stresses, induced by amino acid depletion or osmotic shock, stimulated highly correlated responses affecting about 15% of both total RNA levels and translatome. Many of the regulated messages code for functionally related proteins, thus reflecting logical responses to the particular stress. In contrast, mild stress provoked by addition of Calcofluor-white and menadione altered the translatome of approximately 1% of messages with only marginal effects on total mRNA, suggesting largely uncorrelated responses of transcriptome and translatome. Among these putative translationally regulated messages were most components of the mitochondrial ATPase. Increased polysome associations of corresponding messages and higher mitochondrial ATPase activities upon treatment confirmed the relevance for regulation of this macromolecular complex. Our results suggest the presence of highly sensitive translational regulatory networks that coordinate functionally related messages. These networks are preferentially activated for rapid adaptation of cells to minor environmental perturbations.
RNA-binding proteins (RBPs) have roles in the regulation of many post-transcriptional steps in gene expression, but relatively few RBPs have been systematically studied. We searched for the RNA targets of 40 proteins in the yeast Saccharomyces cerevisiae: a selective sample of the approximately 600 annotated and predicted RBPs, as well as several proteins not annotated as RBPs. At least 33 of these 40 proteins, including three of the four proteins that were not previously known or predicted to be RBPs, were reproducibly associated with specific sets of a few to several hundred RNAs. Remarkably, many of the RBPs we studied bound mRNAs whose protein products share identifiable functional or cytotopic features. We identified specific sequences or predicted structures significantly enriched in target mRNAs of 16 RBPs. These potential RNA-recognition elements were diverse in sequence, structure, and location: some were found predominantly in 3'-untranslated regions, others in 5'-untranslated regions, some in coding sequences, and many in two or more of these features. Although this study only examined a small fraction of the universe of yeast RBPs, 70% of the mRNA transcriptome had significant associations with at least one of these RBPs, and on average, each distinct yeast mRNA interacted with three of the RBPs, suggesting the potential for a rich, multidimensional network of regulation. These results strongly suggest that combinatorial binding of RBPs to specific recognition elements in mRNAs is a pervasive mechanism for multi-dimensional regulation of their post-transcriptional fate.
Genome-wide identification of mRNAs regulated by RNA-binding proteins is crucial to uncover post-transcriptional gene regulatory systems. The conserved PUF family RNA-binding proteins repress gene expression post-transcriptionally by binding to sequence elements in 3'-UTRs of mRNAs. Despite their well-studied implications for development and neurogenesis in metazoa, the mammalian PUF family members are only poorly characterized and mRNA targets are largely unknown. We have systematically identified the mRNAs associated with the two human PUF proteins, PUM1 and PUM2, by the recovery of endogenously formed ribonucleoprotein complexes and the analysis of associated RNAs with DNA microarrays. A largely overlapping set comprised of hundreds of mRNAs were reproducibly associated with the paralogous PUM proteins, many of them encoding functionally related proteins. A characteristic PUF-binding motif was highly enriched among PUM bound messages and validated with RNA pull-down experiments. Moreover, PUF motifs as well as surrounding sequences exhibit higher conservation in PUM bound messages as opposed to transcripts that were not found to be associated, suggesting that PUM function may be modulated by other factors that bind conserved elements. Strikingly, we found that PUF motifs are enriched around predicted miRNA binding sites and that high-confidence miRNA binding sites are significantly enriched in the 3'-UTRs of experimentally determined PUM1 and PUM2 targets, strongly suggesting an interaction of human PUM proteins with the miRNA regulatory system. Our work suggests extensive connections between the RBP and miRNA post-transcriptional regulatory systems and provides a framework for deciphering the molecular mechanism by which PUF proteins regulate their target mRNAs.
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 (I Na) and excitability in Drosophila motoneurons. In this current study, we show that Pum is able to bind directly the mRNA encoding the Drosophila voltage-gated sodium channel paralytic (para). We identify a putative binding site for Pum in the 3′ end of the para open 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 of para mRNA. Additionally, the cofactor Nanos is essential for Pum-dependent para repression, whereas the requirement for Brain Tumor (Brat) is cell type specific. Thus, Pum-dependent regulation of I Na in 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 of nanos mRNA and as such identify a potential negative-feedback mechanism to protect neurons from overactivity of Pum. Finally, we show coupling between I Na (para) and I K (Shal) such that Pum-mediated change in para results in a compensatory change in Shal. The identification of para as 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.
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.
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