Inter-domain Flexibility of Human Ser/Arg-Rich Splicing Factor 1 Allows Variable Spacer Length in Cognate RNA’s Bipartite Motifs

Silva, Naiduwadura Ivon Upekala De; Fargason, Talia; Zhang, Zihan; Wang, Ting; Zhang, Jun

doi:10.1021/acs.biochem.2c00565

Cited by 4 publications

(8 citation statements)

References 57 publications

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“…Others and our studies have identified the SRSF1 residues for RNA binding, such as Y19, F56, F58, W134, and Q135 (Fig. 3A) (26,28). In our previous study, we have confirmed that mutation of these residues does not perturb the protein structure (28).…”

Section: Identification Of Key Srsf1 Residues Responsible For Arpc2 G...supporting

confidence: 73%

“…3A) (26,28). In our previous study, we have confirmed that mutation of these residues does not perturb the protein structure (28). Therefore, we compared binding affinities of the wild-type RRM tandem with its mutants.…”

Section: Resultsmentioning

confidence: 99%

“…1A). Consequently, SRSF1's RRM tandem recognizes purine-rich RNA motifs with upstream or downstream C motifs (25,28). Unlike SRSF1, SRSF3's RRM recognizes pyrimidine-rich RNA sequences (29).…”

Section: Introductionmentioning

confidence: 99%

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Unearthing SRSF1’s Novel Function in Binding and Unfolding of RNA G-Quadruplexes

De Silva,

Lehman,

Fargason

et al. 2023

Preprint

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SRSF1 governs splicing of over 1,500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity. With our success in solving the insolubility problem of SRSF1, we can explore the unknown RNA-binding landscape of SRSF1. We find that SRSF1 RS prefers purine over pyrimidine. Moreover, SRSF1 binds to the G-quadruplex (GQ) from the ARPC2 mRNA, with both RRMs and RS being crucial. Our binding assays show that the traditional RNA-binding sites on the RRM tandem and the Arg in RS are responsible for GQ binding. Interestingly, our FRET and circular dichroism data reveal that SRSF1 unfolds the ARPC2 GQ, with RS leading unfolding and RRMs aiding. Our saturation transfer difference NMR results discover that Arg residues in SRSF1 RS interact with the guanine base but other nucleobases, underscoring the uniqueness of the Arg/guanine interaction. Our luciferase assays confirm that SRSF1 can alleviate the inhibitory effect of GQ on gene expression in the cell. Given the prevalence of RNA GQ and SR proteins, our findings unveil unexplored SR protein functions with broad implications in RNA splicing and translation.

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Section: Identification Of Key Srsf1 Residues Responsible For Arpc2 G...supporting

confidence: 73%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Unearthing SRSF1’s Novel Function in Binding and Unfolding of RNA G-Quadruplexes

De Silva,

Lehman,

Fargason

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…These motifs are also present in ARPC2 GQ. Others and our studies have identified the SRSF1 residues for RNA binding, such as Y19, F56, F58, W134 and Q135 (Figure 3A and B ) ( 26 , 28 ). In our previous study, we have confirmed that mutation of these residues does not perturb the protein structure ( 28 ).…”

Section: Resultsmentioning

confidence: 62%

“…RRM1 prefers C-containing RNA motifs ( 25 ), while RRM2 prefers GGA motifs ( 26 , 27 ) (Figure 1A ). Consequently, SRSF1’s RRM tandem recognizes purine-rich RNA motifs with upstream or downstream C motifs ( 25 , 28 ). Unlike SRSF1, SRSF3’s RRM recognizes pyrimidine-rich RNA sequences ( 29 ).…”

Section: Introductionmentioning

confidence: 99%

Unearthing a novel function of SRSF1 in binding and unfolding of RNA G-quadruplexes

De Silva,

Lehman,

Fargason

et al. 2024

Nucleic Acids Research

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SRSF1 governs splicing of over 1500 mRNA transcripts. SRSF1 contains two RNA-recognition motifs (RRMs) and a C-terminal Arg/Ser-rich region (RS). It has been thought that SRSF1 RRMs exclusively recognize single-stranded exonic splicing enhancers, while RS lacks RNA-binding specificity. With our success in solving the insolubility problem of SRSF1, we can explore the unknown RNA-binding landscape of SRSF1. We find that SRSF1 RS prefers purine over pyrimidine. Moreover, SRSF1 binds to the G-quadruplex (GQ) from the ARPC2 mRNA, with both RRMs and RS being crucial. Our binding assays show that the traditional RNA-binding sites on the RRM tandem and the Arg in RS are responsible for GQ binding. Interestingly, our FRET and circular dichroism data reveal that SRSF1 unfolds the ARPC2 GQ, with RS leading unfolding and RRMs aiding. Our saturation transfer difference NMR results discover that Arg residues in SRSF1 RS interact with the guanine base but not other nucleobases, underscoring the uniqueness of the Arg/guanine interaction. Our luciferase assays confirm that SRSF1 can alleviate the inhibitory effect of GQ on gene expression in the cell. Given the prevalence of RNA GQ and SR proteins, our findings unveil unexplored SR protein functions with broad implications in RNA splicing and translation.

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The U1‐70K and SRSF1 interaction is modulated by phosphorylation during the early stages of spliceosome assembly

Paul,

Zhang,

Zhang

et al. 2024

Protein Science

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In eukaryotes, pre‐mRNA splicing is vital for RNA processing and orchestrated by the spliceosome, whose assembly starts with the interaction between U1‐70K and SR proteins. Despite the significance of the U1‐70K/SR interaction, the dynamic nature of the complex and the challenges in obtaining soluble U1‐70K have impeded a comprehensive understanding of the interaction at the structural level for decades. We overcome the U1‐70K solubility issues, enabling us to characterize the interaction between U1‐70K and SRSF1, a representative SR protein. We unveil specific interactions: phosphorylated SRSF1 RS with U1‐70K BAD1, and SRSF1 RRM1 with U1‐70K RRM. The RS/BAD1 interaction plays a dominant role, whereas the interaction between the RRM domains further enhances the stability of the U1‐70K/SRSF1 complex. The RRM interaction involves the C‐terminal extension of U1‐70K RRM and the conserved acid patches on SRSF1 RRM1 that is involved in SRSF1 phase separation. Our circular dichroism spectra reveal that BAD1 adapts an α‐helical conformation and RS is intrinsically disordered. Intriguingly, BAD1 undergoes a conformation switch from α‐helix to β‐strand and random coil upon RS binding. In addition to the regulatory mechanism via SRSF1 phosphorylation, the U1‐70K/SRSF1 interaction is also regulated by U1‐70K BAD1 phosphorylation. We find that U1‐70K phosphorylation inhibits the U1‐70K and SRSF1 interaction. Our structural findings are validated through in vitro splicing assays and in‐cell saturated domain scanning using the CRISPR method, providing new insights into the intricate regulatory mechanisms of pre‐mRNA splicing.

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Inter-domain Flexibility of Human Ser/Arg-Rich Splicing Factor 1 Allows Variable Spacer Length in Cognate RNA’s Bipartite Motifs

Cited by 4 publications

References 57 publications

Unearthing SRSF1’s Novel Function in Binding and Unfolding of RNA G-Quadruplexes

Unearthing SRSF1’s Novel Function in Binding and Unfolding of RNA G-Quadruplexes

Unearthing a novel function of SRSF1 in binding and unfolding of RNA G-quadruplexes

The U1‐70K and SRSF1 interaction is modulated by phosphorylation during the early stages of spliceosome assembly

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