In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors

Sutandy, F. X. Reymond; Ebersberger, Stefanie; Huang, Lu; Busch, Anke; Bach, Maximilian; Kang, Hyun-Seo; Fallmann, Jörg; Maticzka, Daniel; Backofen, Rolf; Stadler, Peter F.; Zarnack, Kathi; Sattler, Michael; Legewie, Stefan; König, Julian

doi:10.1101/gr.229757.117

Cited by 64 publications

(64 citation statements)

References 67 publications

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“…After the IP, the co‐purified RNA was radioactively labeled and the IPs were run on a SDS–PAGE gel followed by membrane transfer. As a positive control, human U2AF65 (hU2AF65) was used (see Sutandy et al , ). GTSF‐1 protein does not associate with RNA beyond background levels.…”

Section: Resultsmentioning

confidence: 99%

GTSF ‐1 is required for formation of a functional RNA ‐dependent RNA Polymerase complex in Caenorhabditis elegans

Almeida

Dietz²,

Redl

et al. 2018

The EMBO Journal

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Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C-terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Gtsf1 homolog, named it and characterized it in the context of the sRNA pathways of We report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, mutants show a striking depletion of 26G-RNAs, a class of endogenous sRNAs, fully phenocopying mutants. We show, both and , that GTSF-1 interacts with RRF-3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex (ERIC), thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA-mediated silencing activities.

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Section: Resultsmentioning

confidence: 99%

GTSF ‐1 is required for formation of a functional RNA ‐dependent RNA Polymerase complex in Caenorhabditis elegans

Almeida

Dietz²,

Redl

et al. 2018

The EMBO Journal

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“…RBM5 also binds multiple different cis RNA sequences [31,32,40,55]. Recent genome-wide studies suggest that several RNA-binding proteins affect splicing by modulating the binding of U2AF large subunit to alternative 3ʹ SS [59,60]. It is plausible that in C. elegans RBM-5 modulates UAF-1 binding to 3ʹ SS by interacting with the RS domain of UAF-1 or competing for binding the pyrimidine tract preceding 3ʹ SS.…”

Section: Discussionmentioning

confidence: 99%

RBM-5 modulates U2AF large subunit-dependent alternative splicing inC. elegans

Zhou

Gao

et al. 2018

RNA Biology

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A key step in pre-mRNA splicing is the recognition of 3ʹ splicing sites by the U2AF large and small subunits, a process regulated by numerous trans-acting splicing factors. How these trans-acting factors interact with U2AF in vivo is unclear. From a screen for suppressors of the temperature-sensitive (ts) lethality of the C. elegans U2AF large subunit gene uaf-1(n4588) mutants, we identified mutations in the RNA binding motif gene rbm-5, a homolog of the tumor suppressor gene RBM5. rbm-5 mutations can suppress uaf-1(n4588) ts-lethality by loss of function and neuronal expression of rbm-5 was sufficient to rescue the suppression. Transcriptome analyses indicate that uaf-1(n4588) affected the expression of numerous genes and rbm-5 mutations can partially reverse the abnormal gene expression to levels similar to that of wild type. Though rbm-5 mutations did not obviously affect alternative splicing per se, they can suppress or enhance, in a gene-specific manner, the altered splicing of genes in uaf-1(n4588) mutants. Specifically, the recognition of a weak 3ʹ splice site was more susceptible to the effect of rbm-5. Our findings provide novel in vivo evidence that RBM-5 can modulate UAF-1-dependent RNA splicing and suggest that RBM5 might interact with U2AF large subunit to affect tumor formation.

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“…Thereby, our mechanistic model may also explain why combined mutation effects on splicing outcomes can be accurately quantified using additive regression models when expressed as log-fold changes (11,17). In conclusion, exon definition allows for reliable splicing regulation, even though alternative exons are typically influenced by a whole battery of distinct cis-regulatory elements (4,5).…”

Section: Discussionmentioning

confidence: 87%

“…Alternative splicing is commonly regulated by differential recruitment of the U1 and U2 snRNPs. In most cases, such modulation occurs by auxiliary RNA-binding proteins (RBPs) which promote or inhibit U1 or U2 snRNP recruitment by binding to intronic or exonic cis-regulatory elements (1,3,4). Spliceosome assembly may occur by two conceptually different mechanisms: In 'intron definition', the U1 and U2 snRNPs directly assemble across the intron to form a catalytically competent spliceosome.…”

Section: Introductionmentioning

confidence: 99%

Exon definition facilitates reliable control of alternative splicing in the RON proto-oncogene

Enculescu¹,

Braun²,

Setty

et al. 2019

Preprint

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Alternative splicing is a key step in eukaryotic gene expression that allows the production of multiple protein isoforms from the same gene. Even though splicing is perturbed in many diseases, we currently lack insights into regulatory mechanisms promoting its precision and efficiency. Using mechanistic mathematical modeling, we show that alternative splicing control is facilitated if spliceosomes recognize exons as functional units ('exon definition'). We find that exon definition is crucial to prevent the accumulation of partially spliced retention products during alternative splicing regulation. Furthermore, it modularizes splicing control, as multiple regulatory inputs are integrated into a common net input, irrespective of the location and nature of the corresponding cis-regulatory elements in the pre-mRNA. These predictions of our model are qualitatively and quantitatively supported by high-throughput mutagenesis data obtained for an alternatively spliced exon in the proto-oncogene RON (MST1R). Our analysis suggests that exon definition has evolved as the dominant splice-regulatory mechanism in higher organisms to promote robust and reliable splicing outcomes.One Sentence Summary: Exon definition is required for precise alternative splicing control and prevents accumulation of undesired retention isoforms.

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In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors

Cited by 64 publications

References 67 publications

GTSF ‐1 is required for formation of a functional RNA ‐dependent RNA Polymerase complex in Caenorhabditis elegans

GTSF ‐1 is required for formation of a functional RNA ‐dependent RNA Polymerase complex in Caenorhabditis elegans

RBM-5 modulates U2AF large subunit-dependent alternative splicing inC. elegans

Exon definition facilitates reliable control of alternative splicing in the RON proto-oncogene

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