Exon junction complex-associated multi-adapter RNPS1 nucleates splicing regulatory complexes to maintain transcriptome surveillance

The exon junction complex (EJC) is an RNA-binding multi-protein complex with critical functions in post-transcriptional gene regulation. It is deposited on the mRNA during splicing and regulates diverse processes including pre-mRNA splicing, mRNA export, mRNA translation, and nonsense-mediated mRNA decay (NMD) via various interacting peripheral proteins. The EJC-binding protein RNPS1 might serve two functions: it suppresses mis-splicing of cryptic splice sites and activates NMD in the cytoplasm. When analyzing the transcriptome-wide effects of EJC and RNPS1 knockdowns in different human cell lines, we find no evidence for RNPS1 being a globally essential NMD factor. However, various aberrant splicing events strongly suggest that the main function of RNPS1 is splicing regulation. Rescue analyses revealed that about half of these RNPS1-dependent splicing events was fully or partially rescued by the expression of the isolated RRM domain of RNPS1, whereas other splicing events are regulated by its C-terminal domain. We identified many splicing-regulatory factors, including SR proteins and U1 snRNP components, that specifically interact with the C-terminus or with the RRM of RNPS1. Thus, RNPS1 emerges as a multifunctional splicing regulator that promotes correct and efficient splicing of different vulnerable splicing events via the formation of diverse splicing-promoting complexes.

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PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions

10.1101/074823 ◽

2016 ◽

Author(s):

Alex M. Tamburino ◽

Ebru Kaymak ◽

Shaleen Shrestha ◽

Amy D. Holdorf ◽

Sean P. Ryder ◽

...

Keyword(s):

Protein Interactions ◽

Small Rna ◽

Rna Binding ◽

Mrna Translation ◽

Single Experiment ◽

Novel Interactions ◽

Transcriptional Gene Regulation ◽

Rna Interaction ◽

Eukaryotic Genomes

SUMMARYInteractions between RNA binding protein (RBP) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using ‘protein-centered’ (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest – in a ‘gene-centered’ manner, yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA ‘bait’ can be tested versus multiple RBP ‘preys’ in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3′UTRs. PRIMA faithfully recapitulates numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.

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Activation of Pre-mRNA Splicing by Human RNPS1 Is Regulated by CK2 Phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.25.4.1446-1457.2005 ◽

2005 ◽

Vol 25 (4) ◽

pp. 1446-1457 ◽

Cited By ~ 22

Author(s):

Janeen H. Trembley ◽

Sawako Tatsumi ◽

Eiji Sakashita ◽

Pascal Loyer ◽

Clive A. Slaughter ◽

...

Keyword(s):

Nuclear Localization ◽

Phosphorylation Site ◽

Mrna Splicing ◽

Exon Junction Complex ◽

Exon Junction ◽

In Vivo Experiments ◽

Splicing Regulator ◽

Major Phosphorylation Site

ABSTRACT Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation.

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The RNA-binding profile of Acinus, a peripheral component of the exon junction complex, reveals its role in splicing regulation

RNA ◽

10.1261/rna.057158.116 ◽

2016 ◽

Vol 22 (9) ◽

pp. 1411-1426 ◽

Cited By ~ 17

Author(s):

Julie Rodor ◽

Qun Pan ◽

Benjamin J. Blencowe ◽

Eduardo Eyras ◽

Javier F. Cáceres

Keyword(s):

Rna Binding ◽

Exon Junction Complex ◽

Splicing Regulation ◽

Binding Profile ◽

Exon Junction

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Association of the Breast Cancer Protein MLN51 with the Exon Junction Complex via Its Speckle Localizer and RNA Binding Module

Journal of Biological Chemistry ◽

10.1074/jbc.m402754200 ◽

2004 ◽

Vol 279 (32) ◽

pp. 33702-33715 ◽

Cited By ~ 67

Author(s):

Sébastien Degot ◽

Hervé Le Hir ◽

Fabien Alpy ◽

Valérie Kedinger ◽

Isabelle Stoll ◽

...

Keyword(s):

Breast Cancer ◽

Rna Binding ◽

Exon Junction Complex ◽

Exon Junction

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The exon junction complex and intron removal prevent re-splicing of mRNA

PLoS Genetics ◽

10.1371/journal.pgen.1009563 ◽

2021 ◽

Vol 17 (5) ◽

pp. e1009563

Author(s):

Brian Joseph ◽

Eric C. Lai

Keyword(s):

Gene Expression ◽

Splice Site ◽

Site Selection ◽

Exon Junction Complex ◽

Splice Sites ◽

Splice Site Selection ◽

Exon Junction ◽

Intron Removal ◽

Cryptic Splice Sites

Accurate splice site selection is critical for fruitful gene expression. Recently, the mammalian EJC was shown to repress competing, cryptic, splice sites (SS). However, the evolutionary generality of this remains unclear. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic SS, even though most bear weak splicing motifs and are seemingly incompetent. Mechanistically, the EJC directly conceals cryptic splicing elements by virtue of its position-specific recruitment, preventing aberrant SS definition. Unexpectedly, we discover the EJC inhibits scores of regenerated 5’ and 3’ recursive SS on segments that have already undergone splicing, and that loss of EJC regulation triggers faulty resplicing of mRNA. An important corollary is that certain intronless cDNA constructs yield unanticipated, truncated transcripts generated by resplicing. We conclude the EJC has conserved roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments.

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The Exon Junction Complex and intron removal prevents resplicing of mRNA

10.1101/2020.07.31.231498 ◽

2020 ◽

Author(s):

Brian Joseph ◽

Eric C. Lai

Keyword(s):

Gene Expression ◽

Splice Site ◽

Site Selection ◽

Exon Junction Complex ◽

Splice Sites ◽

Splice Site Selection ◽

Exon Junction ◽

Exon Junctions ◽

Intron Removal ◽

Cryptic Splice Sites

AbstractAccurate splice site selection is critical for fruitful gene expression. Here, we demonstrate the Drosophila EJC suppresses hundreds of functional cryptic splice sites (SS), even though majority of these bear weak splicing motifs and appear incompetent. Mechanistically, the EJC directly conceals splicing elements through position-specific recruitment, preventing SS definition. We note that intron removal using strong, canonical SS yields AG|GU signatures at exon-exon junctions. Unexpectedly, we discover that scores of these minimal exon junction sequences are in fact EJC-suppressed 5’ and 3’ recursive SS, and that loss of EJC regulation from such transcripts triggers faulty mRNA resplicing. An important corollary is that intronless cDNA expression constructs from aforementioned targets yield high levels of unanticipated, truncated transcripts generated by resplicing. Consequently, we conclude the EJC has ancestral roles to defend transcriptome fidelity by (1) repressing illegitimate splice sites on pre-mRNAs, and (2) preventing inadvertent activation of such sites on spliced segments.

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The transcriptome-wide landscape and modalities of EJC binding in adult Drosophila

10.1101/459354 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ales Obrdlik ◽

Gen Lin ◽

Nejc Haberman ◽

Jernej Ule ◽

Anne Ephrussi

Keyword(s):

Binding Sites ◽

Rna Binding ◽

Exon Junction Complex ◽

Splice Sites ◽

Rna Structures ◽

Rna Surveillance ◽

Exon Junction ◽

Higher Eukaryotes ◽

Exon Junctions ◽

Adult Drosophila

AbstractSplicing-dependent assembly of the exon junction complex (EJC) at canonical sites −20 to −24 nucleotides upstream of exon-exon junctions in mRNAs occurs in all higher eukaryotes and affects most major regulatory events in the life of a transcript. In mammalian cell cytoplasm, EJC is essential for efficient RNA surveillance, while in Drosophila the most essential cytoplasmic EJC function is in localization of oskar mRNA. Here we developed a method for isolation of protein complexes and associated RNA-targets (ipaRt), which provides a transcriptome-wide view of RNA binding sites of the fully assembled EJC in adult Drosophila. We find that EJC binds at canonical positions, with highest occupancy on mRNAs from genes comprising multiple splice sites and long introns. Moreover, the occupancy is highest at junctions adjacent to strong splice sites, CG-rich hexamers and RNA structures. These modalities have not been identified by previous studies in mammals, where more binding was seen at non-canonical positions. The most highly occupied transcripts in Drosophila have increased tendency to be maternally localized, and are more likely to derive from genes involved in differentiation or development. Taken together, we identify the RNA modalities that specify EJC assembly in Drosophila on a biologically coherent set of transcripts.

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Zinc Binds to RRM2 Peptide of TDP-43

International Journal of Molecular Sciences ◽

10.3390/ijms21239080 ◽

2020 ◽

Vol 21 (23) ◽

pp. 9080

Author(s):

Andrey V. Golovin ◽

Francois Devred ◽

Dahbia Yatoui ◽

Andrei Yu. Roman ◽

Arthur O. Zalevsky ◽

...

Keyword(s):

Motor Neurons ◽

Rna Binding ◽

Titration Calorimetry ◽

Ionization Mass ◽

Dna And Rna ◽

Rrm Domain ◽

C Terminus ◽

Zinc Binding Site ◽

Nuclear Ribonucleoprotein ◽

Dna And Rna Binding

Transactive response DNA and RNA binding protein 43 kDa (TDP-43) is a highly conserved heterogeneous nuclear ribonucleoprotein (hnRNP), which is involved in several steps of protein production including transcription and splicing. Its aggregates are frequently observed in motor neurons from amyotrophic lateral sclerosis patients and in the most common variant of frontotemporal lobar degeneration. Recently it was shown that TDP-43 is able to bind Zn2+ by its RRM domain. In this work, we have investigated Zn2+ binding to a short peptide 256–264 from C-terminus of RRM2 domain using isothermal titration calorimetry, electrospray ionization mass spectrometry, QM/MM simulations, and NMR spectroscopy. We have found that this peptide is able to bind zinc ions with a Ka equal to 1.6 × 105 M−1. Our findings suggest the existence of a zinc binding site in the C-terminal region of RRM2 domain. Together with the existing structure of the RRM2 domain of TDP-43 we propose a model of its complex with Zn2+ which illustrates how zinc might regulate DNA/RNA binding.

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Surveying the global landscape of post-transcriptional regulators

10.1101/2021.08.09.455688 ◽

2021 ◽

Author(s):

Kendra Reynaud ◽

Anna M McGeachy ◽

David Noble ◽

Zuriah A Meacham ◽

Nicholas Ingolia

Keyword(s):

Rna Binding ◽

Mrna Translation ◽

Transcriptional Regulators ◽

Regulatory Activity ◽

Modular Architecture ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Binding Domains ◽

Fluorescence Measurements ◽

Transcriptional Gene Regulation

Numerous proteins regulate gene expression by modulating mRNA translation and decay. In order to uncover the full scope of these post-transcriptional regulators, we conducted an unbiased survey that quantifies regulatory activity across the budding yeast proteome and delineates the protein domains responsible for these effects. Our approach couples a tethered function assay with quantitative single-cell fluorescence measurements to analyze ~50,000 protein fragments and determine their effects on a tethered mRNA. We characterize hundreds of strong regulators, which are enriched for canonical and unconventional mRNA-binding proteins. Regulatory activity typically maps outside the RNA-binding domains themselves, highlighting a modular architecture that separates mRNA targeting from post-transcriptional regulation. Activity often aligns with intrinsically disordered regions that can interact with other proteins, even in core mRNA translation and degradation factors. Our results thus reveal networks of interacting proteins that control mRNA fate and illuminate the molecular basis for post-transcriptional gene regulation.

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Stop codon-proximal 3′UTR introns in vertebrates can elicit EJC-dependent Nonsense-Mediated mRNA Decay

10.1101/677666 ◽

2019 ◽

Author(s):

Pooja Gangras ◽

Thomas L. Gallagher ◽

Robert D. Patton ◽

Zhongxia Yi ◽

Michael A. Parthun ◽

...

Keyword(s):

Motor Neuron ◽

Mrna Decay ◽

Rna Binding ◽

Stop Codon ◽

Embryonic Stem ◽

Nervous System Development ◽

Exon Junction Complex ◽

Loss Of Function ◽

Exon Junction ◽

Nonsense Mediated Mrna Decay

AbstractThe Exon Junction Complex (EJC) regulates many steps in post-transcriptional gene expression and is essential for cellular function and organismal development; however, EJC-regulated genes and genetic pathways during development remain largely unknown. To study EJC function during zebrafish development, we first established that zebrafish EJCs mainly bind ∼24 nucleotides upstream of exon-exon junctions, and are also detected at more distant non-canonical positions. We then generated mutations in two zebrafish EJC core genes, rbm8a and magoh, and observed that homozygous mutant embryos show paralysis, muscle disorganization, neural cell death, and motor neuron outgrowth defects. Coinciding with developmental defects, mRNAs subjected to Nonsense-Mediated mRNA Decay (NMD) due to translation termination ≥ 50 nts upstream of the last exon-exon junction are upregulated in EJC mutant embryos. Surprisingly, several transcripts containing 3′UTR introns (3′UI) < 50 nts downstream of a stop codon are also upregulated in EJC mutant embryos. These proximal 3′UI-containing transcripts are also upregulated in NMD-compromised zebrafish embryos, cultured human cells, and mouse embryonic stem cells. Loss of function of foxo3b, one of the upregulated proximal 3′UI-containing genes, partially rescues EJC mutant motor neuron outgrowth. In addition to foxo3b, 166 other genes contain a proximal 3′UI in zebrafish, mouse and humans, and these genes are enriched in nervous system development and RNA binding functions. A proximal 3′UI-containing 3′UTR from one of these genes, HNRNPD, is sufficient to reduce steady state transcript levels when fused to a β-globin reporter in HeLa cells. Overall, our work shows that genes with stop codon-proximal 3′UIs encode a new class of EJC-regulated NMD targets with critical roles during vertebrate development.

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