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

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.

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

10.1101/2020.02.16.951657 ◽

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

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

10.1101/2020.09.21.305912 ◽

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.

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

PLoS Genetics ◽

10.1371/journal.pgen.1009439 ◽

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.

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Alternative polyadenylation: methods, mechanism, function, and role in cancer

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01852-7 ◽

2021 ◽

Vol 40 (1) ◽

Cited By ~ 1

Author(s):

Yi Zhang ◽

Lian Liu ◽

Qiongzi Qiu ◽

Qing Zhou ◽

Jinwang Ding ◽

...

Keyword(s):

Gene Regulation ◽

Rna Binding ◽

Rna Binding Proteins ◽

Single Gene ◽

Alternative Polyadenylation ◽

Length Change ◽

Suppressor Gene ◽

Gene Expressions ◽

Related Factors ◽

Mrna Maturation

AbstractOccurring in over 60% of human genes, alternative polyadenylation (APA) results in numerous transcripts with differing 3’ends, thus greatly expanding the diversity of mRNAs and of proteins derived from a single gene. As a key molecular mechanism, APA is involved in various gene regulation steps including mRNA maturation, mRNA stability, cellular RNA decay, and protein diversification. APA is frequently dysregulated in cancers leading to changes in oncogenes and tumor suppressor gene expressions. Recent studies have revealed various APA regulatory mechanisms that promote the development and progression of a number of human diseases, including cancer. Here, we provide an overview of four types of APA and their impacts on gene regulation. We focus particularly on the interaction of APA with microRNAs, RNA binding proteins and other related factors, the core pre-mRNA 3’end processing complex, and 3’UTR length change. We also describe next-generation sequencing methods and computational tools for use in poly(A) signal detection and APA repositories and databases. Finally, we summarize the current understanding of APA in cancer and provide our vision for future APA related research.

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Regulation, diversity and function of MECP2 exon and 3′UTR isoforms

Human Molecular Genetics ◽

10.1093/hmg/ddaa154 ◽

2020 ◽

Vol 29 (R1) ◽

pp. R89-R99

Author(s):

Deivid Carvalho Rodrigues ◽

Marat Mufteev ◽

James Ellis

Keyword(s):

Dna Binding ◽

Rna Binding ◽

Rna Binding Proteins ◽

Alternative Polyadenylation ◽

Cell Types ◽

Protein Isoforms ◽

Translation Efficiency ◽

Mrna Isoforms ◽

Exon 2 ◽

Messenger Ribonucleic Acid

Abstract The methyl-CpG-binding protein 2 (MECP2) is a critical global regulator of gene expression. Mutations in MECP2 cause neurodevelopmental disorders including Rett syndrome (RTT). MECP2 exon 2 is spliced into two alternative messenger ribonucleic acid (mRNA) isoforms encoding MECP2-E1 or MECP2-E2 protein isoforms that differ in their N-termini. MECP2-E2, isolated first, was used to define the general roles of MECP2 in methyl-deoxyribonucleic acid (DNA) binding, targeting of transcriptional regulatory complexes, and its disease-causing impact in RTT. It was later found that MECP2-E1 is the most abundant isoform in the brain and its exon 1 is also mutated in RTT. MECP2 transcripts undergo alternative polyadenylation generating mRNAs with four possible 3′untranslated region (UTR) lengths ranging from 130 to 8600 nt. Together, the exon and 3′UTR isoforms display remarkable abundance disparity across cell types and tissues during development. These findings indicate discrete means of regulation and suggest that protein isoforms perform non-overlapping roles. Multiple regulatory programs have been explored to explain these disparities. DNA methylation patterns of the MECP2 promoter and first intron impact MECP2-E1 and E2 isoform levels. Networks of microRNAs and RNA-binding proteins also post-transcriptionally regulate the stability and translation efficiency of MECP2 3′UTR isoforms. Finally, distinctions in biophysical properties in the N-termini between MECP2-E1 and E2 lead to variable protein stabilities and DNA binding dynamics. This review describes the steps taken from the discovery of MECP2, the description of its key functions, and its association with RTT, to the emergence of evidence revealing how MECP2 isoforms are differentially regulated at the transcriptional, post-transcriptional and post-translational levels.

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An autoregulation loop in fust-1 for circular RNA regulation in Caenorhabditis elegans

10.1101/2021.03.22.436400 ◽

2021 ◽

Author(s):

Dong Cao

Keyword(s):

Caenorhabditis Elegans ◽

Rna Binding ◽

Rna Binding Proteins ◽

Exon Skipping ◽

Circular Rna ◽

Circular Rnas ◽

Rna Regulation ◽

Fused In Sarcoma ◽

Specific Factors ◽

Isoform B

Circular RNAs (circRNAs) are always expressed tissue-specifically, suggestive of specific factors that regulate their biogenesis. Here, taking advantage of available mutation strains of RNA binding proteins (RBPs) in Caenorhabditis elegans, I performed a screening of circRNA regulation in thirteen conserved RBPs. Among them, loss of FUST-1, the homolog of FUS (Fused in Sarcoma), caused downregulation of multiple circRNAs. By rescue experiments, I confirmed FUST-1 as a circRNA regulator. Further, I showed that FUST-1 regulates circRNA formation without affecting the levels of the cognate linear mRNAs. When recognizing circRNA pre-mRNAs, FUST-1 can affect both exon-skipping and circRNA in the same genes. Moreover, I identified an autoregulation loop in fust-1, where FUST-1, isoform a promotes the skipping of exon 5 of its own pre-mRNA, which produces FUST-1, isoform b with different N-terminal sequences. FUST-1, isoform a is the functional isoform in circRNA regulation. Although FUST-1, isoform b has the same functional domains as isoform a, it cannot regulate either exon-skipping or circRNA formation.

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Crosstalk Between mRNA 3'-End Processing and Epigenetics

Frontiers in Genetics ◽

10.3389/fgene.2021.637705 ◽

2021 ◽

Vol 12 ◽

Author(s):

Lindsey V. Soles ◽

Yongsheng Shi

Keyword(s):

Histone Modifications ◽

Dynamic Process ◽

Rna Binding ◽

Rna Binding Proteins ◽

Alternative Polyadenylation ◽

Regulatory Mechanisms ◽

Epigenetic Mechanisms ◽

Cis Elements ◽

Mrna Isoforms ◽

Eukaryotic Genes

The majority of eukaryotic genes produce multiple mRNA isoforms by using alternative poly(A) sites in a process called alternative polyadenylation (APA). APA is a dynamic process that is highly regulated in development and in response to extrinsic or intrinsic stimuli. Mis-regulation of APA has been linked to a wide variety of diseases, including cancer, neurological and immunological disorders. Since the first example of APA was described 40 years ago, the regulatory mechanisms of APA have been actively investigated. Conventionally, research in this area has focused primarily on the roles of regulatory cis-elements and trans-acting RNA-binding proteins. Recent studies, however, have revealed important functions for epigenetic mechanisms, including DNA and histone modifications and higher-order chromatin structures, in APA regulation. Here we will discuss these recent findings and their implications for our understanding of the crosstalk between epigenetics and mRNA 3'-end processing.

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The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation

eLife ◽

10.7554/elife.72377 ◽

2021 ◽

Vol 10 ◽

Author(s):

Laura Arribas-Hernández ◽

Sarah Rennie ◽

Michael Schon ◽

Carlotta Porcelli ◽

Balaji Enugutti ◽

...

Keyword(s):

Indirect Effects ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Expression Profiles ◽

Alternative Polyadenylation ◽

Target Mrna ◽

Target Mrnas ◽

Yth Domain ◽

Target Sets

Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3 and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

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Predominance of spliceosomal complex formation over polyadenylation site selection in TDP-43 autoregulation

Nucleic Acids Research ◽

10.1093/nar/gkt1343 ◽

2013 ◽

Vol 42 (5) ◽

pp. 3362-3371 ◽

Cited By ~ 20

Author(s):

Sara Bembich ◽

Jeremias S. Herzog ◽

Laura De Conti ◽

Cristiana Stuani ◽

S. Eréndira Avendaño-Vázquez ◽

...

Keyword(s):

Complex Formation ◽

Messenger Rna ◽

Protein Production ◽

Rna Binding ◽

Rna Binding Proteins ◽

Alternative Polyadenylation ◽

Cellular Viability ◽

Protein Levels ◽

Polyadenylation Sites ◽

Nucleocytoplasmic Distribution

AbstractTDP-43 is a nuclear protein involved in many aspects of RNA metabolism. To ensure cellular viability, its expression levels within cells must be tightly regulated. We have previously demonstrated that TDP-43 autoregulation occurs through the activation of a normally silent intron in its 3′-UTR sequence that results in the use of alternative polyadenylation sites. In this work, we analyse which is the dominant event in autoregulation: the recognition of the splice sites of 3′-UTR intron 7 or the intrinsic quality of the alternative polyadenylation sites. A panel of minigene constructs was tested for autoregulation functionality, protein production and subcellular messenger RNA localization. Our data clearly indicate that constitutive spliceosome complex formation across intron 7 does not lead to high protein production but, on the contrary, to lower TDP-43 messenger RNA and protein levels. This is due to altered nucleocytoplasmic distribution of the RNA that is mostly retained in the nucleus and degraded. This study provides a novel in-depth characterization of how RNA binding proteins can autoregulate their own levels within cells, an essential regulatory process in maintaining cellular viability.

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