Results SRSF7 overexpression induces auto-regulation.To investigate the mechanisms of SRSF7 homeostasis, we generated cell lines SRSF7 is an essential RNA-binding protein whose misexpression promotes cancer. Here, we describe how SRSF7 maintains its protein homeostasis in murine P19 cells using an intricate negative feedback mechanism. SRSF7 binding to its premessenger RNA promotes inclusion of a poison cassette exon and transcript degradation via nonsense-mediated decay (NMD). However, elevated SRSF7 levels inhibit NMD and promote translation of two protein halves, termed Split-ORFs, from the bicistronic SRSF7-PCE transcript. The first half acts as dominant-negative isoform suppressing poison cassette exon inclusion and instead promoting the retention of flanking introns containing repeated SRSF7 binding sites. Massive SRSF7 binding to these sites and its oligomerization promote the assembly of large nuclear bodies, which sequester SRSF7 transcripts at their transcription site, preventing their export and restoring normal SRSF7 protein levels. We further show that hundreds of human and mouse NMD targets, especially RNA-binding proteins, encode potential Split-ORFs, some of which are expressed under specific cellular conditions. SRSF7 binding promotes splicing of NMD-sensitive and -resistant SRSF7 isoforms. To understand the mechanism of SRSF7 auto-regulation, we examined the binding of SRSF7 protein to SRSF7 transcripts using individual-nucleotide resolution ultraviolet (UV) crosslinking and immunoprecipitation (iCLIP). We used normalized significant crosslink events (X-links, false discovery rate (FDR) < 0.05) from SRSF3 and SRSF7 iCLIP datasets of P19 cell lines without OE 8 . Similar to SRSF3, which promotes the inclusion of the PCE in SRSF7 transcripts 17 , SRSF7 showed enriched crosslinks in an extended region encompassing the PCE, its flanking introns 3a and 3b, and exons 3, 4 and 5 ( Fig. 1d). Quantification revealed that SRSF7 binds ~50-fold more to SRSF7 transcripts than SRSF3 ( Supplementary Table 1), indicating that SRSF7 has an unusual preference for its own transcripts.RNA-seq followed by quantification of junction reads revealed that SRSF7 OE promotes inclusion of either the complete PCE (460 nucleotides (nt)) or a partial PCE (107 nt) in SRSF7 transcripts ( Fig. 1e). Additionally, both PCE-flanking introns (3a and 3b) and intron 5 displayed increased read coverage, indicating that they are partly retained upon SRSF7 OE. Semiquantitative reverse transcription PCR and sequencing confirmed that SRSF7 OE caused the appearance of transcripts containing the entire PCE (SRSF7-PCE, orange asterisk), the partial PCE (SRSF7-PCE 1/4 , yellow asterisk) or the PCE in combination with both flanking introns (SRSF7-I3a+b, red asterisk) or with only intron 3b (SRSF7-I3b, green asterisk; Fig. 1f and Extended Data Fig. 1b,c). Identical isoforms were detected for endogenous and SRSF7-GFP reporter transcripts, indicating that auto-regulation operates similarly on both.All these transcripts contain PTCs and should be s...
Hypoxia induces massive changes in alternative splicing (AS) to adapt cells to the lack of oxygen. Here, we identify the splicing factor SRSF6 as a key factor in the AS response to hypoxia. The SRSF6 level is strongly reduced in acute hypoxia, which serves a dual purpose: it allows for exon skipping and triggers the dispersal of nuclear speckles. Our data suggest that cells use dispersal of nuclear speckles to reprogram their gene expression during hypoxic adaptation and that SRSF6 plays an important role in cohesion of nuclear speckles. Down-regulation of SRSF6 is achieved through inclusion of a poison cassette exon (PCE) promoted by SRSF4. Removing the PCE 3′ splice site using CRISPR/Cas9 abolishes SRSF6 reduction in hypoxia. Aberrantly high SRSF6 levels in hypoxia attenuate hypoxia-mediated AS and impair dispersal of nuclear speckles. As a consequence, proliferation and genomic instability are increased, while the stress response is suppressed. The SRSF4–PCE–SRSF6 hypoxia axis is active in different cancer types, and high SRSF6 expression in hypoxic tumors correlates with a poor prognosis. We propose that the ultra-conserved PCE of SRSF6 acts as a tumor suppressor and that its inclusion in hypoxia is crucial to reduce SRSF6 levels. This may prevent tumor cells from entering the metastatic route of hypoxia adaptation.
There is no control over the information provided with sequences when they are deposited in the sequence databases. Consequently mistakes can seed the incorrect annotation of other sequences. Grouping genes into families and applying controlled annotation overcomes the problems of incorrect annotation associated with individual sequences. Two databases (http://www.mendel.ac.uk) were created to apply controlled annotation to plant genes and plant ESTs: Mendel-GFDb is a database of plant protein (gene) families based on gapped-BLAST analysis of all sequences in the SWISS-PROT family of databases. Sequences are aligned (ClustalW) and identical and similar residues shaded. The families are visually curated to ensure that one or more criteria, for example overall relatedness and/or domain similarity relate all sequences within a family. Sequence families are assigned a 'Gene Family Number' and a unified description is developed which best describes the family and its members. If authority exists the gene family is assigned a 'Gene Family Name'. This information is placed in Mendel-GFDb. Mendel-ESTS is primarily a database of plant ESTs, which have been compared to Mendel-GFDb, completely sequenced genomes and domain databases. This approach associated ESTs with individual sequences and the controlled annotation of gene families and protein domains; the information being placed in Mendel-ESTS. The controlled annotation applied to genes and ESTs provides a basis from which a plant transcription database can be developed.
Nuclear RNA binding proteins (RBPs) are difficult to study because they often belong to large protein families and form extensive networks of auto- and cross-regulation. They are highly abundant and often localize to condensates with a slow turnover, requiring long depletion times or knockouts that cannot distinguish between direct and indirect or compensatory effects. Here, we developed a system that is optimized for the rapid degradation of nuclear RBPs, called hGRAD. It comes as a 'one-fits-all' plasmid, and integration into any cell line that expresses endogenously GFP-tagged proteins allows an inducible, rapid and complete knockdown. We show that the nuclear RBPs SRSF3, SRSF5, SRRM2 and NONO are completely cleared from nuclear speckles and paraspeckles within two hours. hGRAD works in various cell types, is more efficient than other methods and does not require the expression of exogenous ubiquitin ligases. Combining SRSF5 hGRAD degradation with Nascent-seq uncovered highly dynamic transient transcript changes, compensatory mechanisms and that SRSF5 promotes transcript stability.
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