scholarly journals ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing

PLoS Genetics ◽  
2021 ◽  
Vol 17 (4) ◽  
pp. e1009439
Author(s):  
Seungjae Lee ◽  
Lu Wei ◽  
Binglong Zhang ◽  
Raeann Goering ◽  
Sonali Majumdar ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Direct Action ◽  
Ectopic Expression ◽  
Exon Skipping ◽  
Mrna Processing ◽  
Cleavage Sites ◽  
Polyadenylation Sites ◽  
Polyadenylation Signals

ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member,DrosophilaElav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3’ UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets ofDrosophilaare known, ectopic expression of each of the three family members (Elav, Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants ofelav/fne, but notelavalone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation ofDrosophilaELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3’ UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification inDrosophilaand mammalian neurons.

scholarly journals Overlapping activities of ELAV/Hu RNA binding proteins specify multiple neural alternative splicing programs

2020 ◽  
Author(s):  
Seungjae Lee ◽  
Binglong Zhang ◽  
Lu Wei ◽  
Raeann Goering ◽  
Sonali Majumdar ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Exon Skipping ◽  
Mrna Processing ◽  
Cassette Exon ◽  
Cleavage Sites ◽  
Motif Analysis ◽  
Hu Proteins ◽  
Polyadenylation Sites

AbstractELAV/Hu factors are conserved RNA binding proteins that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, still little is known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3’ UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. In ectopic contexts, all three members (Elav, Fne and Rbp9) induce similar and broad changes to cassette exon and alternative last exon (ALE) splicing. Reciprocally, double mutants of elav/fne, but not elav alone, have opposite effects on both types of mRNA processing events in the larval CNS. Accordingly, while fne mutants are normal, fne loss strongly enhances elav mutants with respect to neuronal differentiation. While manipulation of Drosophila ELAV/Hu factors induces both exon skipping and inclusion, motif analysis indicates their major direct effects are to suppress cassette exon usage. Moreover, we find direct analogies in their roles in global promotion of distal ALE splicing and terminal 3’ UTR extension, since both involve local suppression of proximal polyadenylation signals via ELAV/Hu binding sites downstream of cleavage sites. Finally, we provide evidence for analogous co-implementation of distal ALE and APA lengthening programs in mammalian neurons, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu proteins orchestrate multiple conserved programs of neuronal mRNA processing by suppressing alternative exons and polyadenylation sites.

scholarly journals APA-Scan: Detection and Visualization of 3’-UTR APA with RNA-seq and 3’-end-seq Data

2020 ◽  
Author(s):  
Naima Ahmed Fahmi ◽  
Jae-Woong Chang ◽  
Heba Nassereddeen ◽  
Khandakar Tanvir Ahmed ◽  
Deliang Fan ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Alternative Polyadenylation ◽  
Transcriptional Level ◽  
Cleavage Sites ◽  
Read Coverage ◽  
Rna Seq ◽  
Regulate Gene Expression ◽  
Polyadenylation Sites ◽  
Polyadenylation Signals

AbstractThe eukaryotic genome is capable of producing multiple isoforms from a gene by alternative polyadenylation (APA) during pre-mRNA processing. APA in the 3’-untranslated region (3’-UTR) of mRNA produces transcripts with shorter 3’-UTR. Often, 3’-UTR serves as a binding platform for microRNAs and RNA-binding proteins, which affect the fate of the mRNA transcript. Thus, 3’-UTR APA provides a means to regulate gene expression at the post-transcriptional level and is known to promote translation. Current bioinformatics pipelines have limited capability in profiling 3’-UTR APA events due to incomplete annotations and a low-resolution analyzing power: widely available bioinformatics pipelines do not reference actionable polyadenylation (cleavage) sites but simulate 3’-UTR APA only using RNA-seq read coverage, causing false positive identifications. To overcome these limitations, we developed APA-Scan, a robust program that identifies 3’-UTR APA events and visualizes the RNA-seq short-read coverage with gene annotations. APA-Scan utilizes either predicted or experimentally validated actionable polyadenylation signals as a reference for polyadenylation sites and calculates the quantity of long and short 3’-UTR transcripts in the RNA-seq data. The performance of APA-Scan was validated by qPCR.ImplementationAPA-Scan is implemented in Python. Source code and a comprehensive user’s manual are freely available at https://github.com/compbiolabucf/APA-Scan

Regulation of the ELAV target ewg: insights from an evolutionary perspective

2008 ◽  
Vol 36 (3) ◽  
pp. 502-504 ◽  
Author(s):  
Matthias Soller ◽  
Min Li ◽  
Irmgard U. Haussmann
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Processing ◽  
Untranslated Regions ◽  
Cellular Environment ◽  
Specific Regulation ◽  
Abnormal Visual ◽  
Recent Sequencing

ELAV (embryonic lethal abnormal visual system)/Hu family proteins are prototype RNA-binding proteins with binding preferences for AU-rich regions. Due to frequent occurrence of AU-rich motifs in introns and untranslated regions, it is poorly understood how gene-specific RNA-binding proteins, such as ELAV/Hu family members, recognize their complement of target RNAs in a complex cellular environment. The powerful genetic tools of Drosophila make the fruitfly an excellent model to study alternative mRNA processing in vivo in a developing organism. Recent sequencing of 12 Drosophila genomes will provide a novel resource to enhance our understanding of how gene-specific regulation of mRNA processing is achieved by ELAV/Hu family proteins.

scholarly journals Characterization of RBP9 and RBP10, two developmentally regulated RNA-binding proteins in Trypanosoma brucei

Open Biology ◽  
2017 ◽  
Vol 7 (4) ◽  
pp. 160159 ◽  
Author(s):  
Luis Miguel De Pablos ◽  
Steve Kelly ◽  
Janaina de Freitas Nascimento ◽  
Jack Sunter ◽  
Mark Carrington
Keyword(s):  
Trypanosoma Brucei ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ectopic Expression ◽  
Developmentally Regulated ◽  
Mrna Metabolism ◽  
Ribonucleoprotein Complexes ◽  
Processing Body ◽  
External Signals

The fate of an mRNA is determined by its interaction with proteins and small RNAs within dynamic complexes called ribonucleoprotein complexes (mRNPs). In Trypanosoma brucei and related kinetoplastids, responses to internal and external signals are mainly mediated by post-transcriptional processes. Here, we used proximity-dependent biotin identification (BioID) combined with RNA-seq to investigate the changes resulting from ectopic expression of RBP10 and RBP9, two developmentally regulated RNA-binding proteins (RBPs). Both RBPs have reduced expression in insect procyclic forms (PCFs) compared with bloodstream forms (BSFs). Upon overexpression in PCFs, both proteins were recruited to cytoplasmic foci, co-localizing with the processing body marker SCD6. Further, both RBPs altered the transcriptome from a PCF- to a BSF-like pattern. Notably, upon expression of BirA*-RBP9 and BirA*-RBP10, BioID yielded more than 200 high confidence protein interactors (more than 10-fold enriched); 45 (RBP9) and 31 (RBP10) were directly related to mRNA metabolism. This study validates the use of BioID for investigating mRNP components but also illustrates the complexity of mRNP function.

scholarly journals Concentration and localization of co-expressed ELAV/Hu proteins control specificity of mRNA processing

2015 ◽  
pp. MCB.00473-15 ◽  
Author(s):  
Emanuela Zaharieva ◽  
Irmgard U. Haussmann ◽  
Ulrike Bräuer ◽  
Matthias Soller
Keyword(s):  
Cellular Localization ◽  
Binding Proteins ◽  
Target Genes ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mrna Processing ◽  
Hu Proteins ◽  
Sex Lethal ◽  
Specific Regulation

Neuronally co-expressed ELAV/Hu proteins comprise a family of highly related RNA binding proteins, which bind to very similar cognate sequences. How this redundancy is linked to in vivo function and how gene specific regulation is achieved, has not been clear. Analysis of mutants inDrosophilaELAV/Hu family proteins ELAV, FNE and RBP9, and genetic interactions among them, indicates mostly independent roles in neuronal development and function, but convergence in the regulation of synaptic plasticity. Conversely, ELAV, FNE, RBP9 and human HuR bind ELAV target RNA in vitro with similar affinity. Likewise, all can regulate alternative splicing of ELAV target genes in non-neuronal wing-disc cells and substitute ELAV in eye development with artificially increased expression, but can also substantially restore ELAV's biological functions, when expressed under the control of theelavgene. Furthermore, ELAV related Sex-lethal can regulate ELAV targets and ELAV/Hu proteins can interfere with sexual differentiation. An ancient relationship to Sex-lethal is revealed by gonadal expression of RBP9 providing a maternal failsafe for dosage compensation. Our results indicate that highly related ELAV/Hu RNA binding proteins select targets for mRNA processing based on expression levels and sub-cellular localization, but only minimally by altered RNA binding specificity.

scholarly journals Dynamic Variations of 3′UTR Length Reprogram the mRNA Regulatory Landscape

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1560
Author(s):  
Estanislao Navarro ◽  
Adrián Mallén ◽  
Miguel Hueso
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Exon Skipping ◽  
Alternative Polyadenylation ◽  
Point Of View ◽  
Cleavage Sites ◽  
Dynamic Variations ◽  
Human Disorders ◽  
Functional Features ◽  
Control Stability

This paper concerns 3′-untranslated regions (3′UTRs) of mRNAs, which are non-coding regulatory platforms that control stability, fate and the correct spatiotemporal translation of mRNAs. Many mRNAs have polymorphic 3′UTR regions. Controlling 3′UTR length and sequence facilitates the regulation of the accessibility of functional effectors (RNA binding proteins, miRNAs or other ncRNAs) to 3′UTR functional boxes and motifs and the establishment of different regulatory landscapes for mRNA function. In this context, shortening of 3′UTRs would loosen miRNA or protein-based mechanisms of mRNA degradation, while 3′UTR lengthening would strengthen accessibility to these effectors. Alterations in the mechanisms regulating 3′UTR length would result in widespread deregulation of gene expression that could eventually lead to diseases likely linked to the loss (or acquisition) of specific miRNA binding sites. Here, we will review the mechanisms that control 3′UTR length dynamics and their alterations in human disorders. We will discuss, from a mechanistic point of view centered on the molecular machineries involved, the generation of 3′UTR variability by the use of alternative polyadenylation and cleavage sites, of mutually exclusive terminal alternative exons (exon skipping) as well as by the process of exonization of Alu cassettes to generate new 3′UTRs with differential functional features.

scholarly journals Post-transcriptional regulation of human endogenous retroviruses by RNA-Binding Motif Protein 4, RBM4

2020 ◽  
Author(s):  
Amir K. Foroushani ◽  
Bryan Chim ◽  
Madeline Wong ◽  
Andre Rastegar ◽  
Kent Barbian ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Human Genome ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ectopic Expression ◽  
Endogenous Retroviruses ◽  
Binding Motif ◽  
Computational Pipeline ◽  
Post Transcriptional Regulation

AbstractThe human genome encodes for over 1,500 RNA-binding proteins (RBPs), which coordinate regulatory events on RNA transcripts (Gerstberger et al., 2014). Most studies of RBPs concentrate on their action on mRNAs that encode protein, which constitute a minority of the transcriptome. A widely neglected subset of our transcriptome derives from integrated retroviral elements termed endogenous retroviruses (ERVs) that comprise ~8% of the human genome. Some ERVs have been shown to be transcribed under physiological and pathological conditions suggesting that sophisticated regulatory mechanisms to coordinate and prevent their ectopic expression exist. However, it is unknown whether RBPs and ERV transcripts directly interact to provide a post-transcriptional layer of regulation. Here, we implemented a computational pipeline to determine the correlation of expression between individual RBPs and ERVs from single-cell or bulk RNA sequencing data. One of our top candidates for an RBP negatively regulating ERV expression was RNA-Binding Motif Protein 4 (RBM4). We used PAR-CLIP to demonstrate that RBM4 indeed bound ERV transcripts at CGG consensus elements. Loss of RBM4 resulted in elevated transcript level of bound ERVs of the HERV-K and -H families, as well as increased expression of HERV-K envelope protein. We pinpointed RBM4 regulation of HERV-K to a CGG-containing element that is conserved in the long terminal repeats (LTRs) of HERV-K-10 and -K-11, and validated the functionality of this site using reporter assays. In summary, we identified RBPs as potential regulators of ERV function and demonstrate a new role for RBM4 in controlling ERV expression.Significance StatementThe expression of endogenous retroviruses (ERVs) appears to have broad impact on human biology. Nevertheless, only a handful of transcriptional regulators of ERV expression are known and to our knowledge no RNA-binding proteins (RBPs) were yet implicated in positive or negative post-transcriptional regulation of ERVs. We implemented a computational pipeline that allowed us to identify RBPs that modulate ERV expression levels. Experimental validation of one of the prime candidates we identified, RBM4, showed that it indeed bound RNAs made from ERVs and negatively regulated the levels of those RNAs. We hereby identify a new layer of ERV regulation by RBPs.

scholarly journals Evolutionarily Conserved Polyadenosine RNA Binding Protein Nab2 Cooperates with Splicing Machinery To Regulate the Fate of Pre-mRNA

2016 ◽  
Vol 36 (21) ◽  
pp. 2697-2714 ◽  
Author(s):  
Sharon Soucek ◽  
Yi Zeng ◽  
Deepti L. Bellur ◽  
Megan Bergkessel ◽  
Kevin J. Morris ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Tail Length ◽  
Mrna Processing ◽  
Evolutionarily Conserved ◽  
Mrna Maturation

Numerous RNA binding proteins are deposited onto an mRNA transcript to modulate posttranscriptional processing events ensuring proper mRNA maturation. Defining the interplay between RNA binding proteins that couple mRNA biogenesis events is crucial for understanding how gene expression is regulated. To explore how RNA binding proteins control mRNA processing, we investigated a role for the evolutionarily conserved polyadenosine RNA binding protein, Nab2, in mRNA maturation within the nucleus. This study reveals thatnab2mutant cells accumulate intron-containing pre-mRNAin vivo. We extend this analysis to identify genetic interactions between mutant alleles ofnab2and genes encoding a splicing factor,MUD2, and RNA exosome,RRP6, within vivoconsequences of altered pre-mRNA splicing and poly(A) tail length control. As further evidence linking Nab2 proteins to splicing, an unbiased proteomic analysis of vertebrate Nab2, ZC3H14, identifies physical interactions with numerous components of the spliceosome. We validated the interaction between ZC3H14 and U2AF2/U2AF65. Taking all the findings into consideration, we present a model where Nab2/ZC3H14 interacts with spliceosome components to allow proper coupling of splicing with subsequent mRNA processing steps contributing to a kinetic proofreading step that allows properly processed mRNA to exit the nucleus and escape Rrp6-dependent degradation.

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