SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly

Abstract

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...

Perspective in Alternative Splicing Coupled to Nonsense-Mediated mRNA Decay

“…Particularly, this SR protein targets its own pre-mRNA to induce both, inclusion of a poison cassette exon and intron retention in the 3′UTR [ 74 ], the latter forcing the normal termination codon to be recognized as premature, thus being subjected to NMD. This negative autoregulation has also been documented in other human classical SR proteins, such as SRSF1, SRSF3, SRSF4, SRSF5, or SRSF7 ( Table 1 ), due to different splicing events [ 71 , 75 , 77 , 78 , 80 , 81 , 89 , 90 , 91 ]. For example, in the case of SRSF5, the usage of the proximal 3′ splice site in the exon 6 includes an in-frame stop codon that induces NMD [ 81 ].…”

Poison Exon Splicing Regulates a Coordinated Network of SR Protein Expression during Differentiation and Tumorigenesis

“…For example, binding of the TRA2b protein to TRA2b-PE enhances its inclusion (Stoilov et al, 2004). Similar auto-regulatory patterns that link PE splicing to changes in protein levels have been demonstrated experimentally for only five other SR proteins (Jumaa and Nielsen, 1997;Kö nigs et al, 2020;Sun et al, 2010;Sureau et al, 2001;Yang et al, 2018). A few examples of cross-regulation have been uncovered, where an SR protein influences the inclusion of PEs in other SFs, including for TRA2b and its paralog TRA2a (Best et al, 2014) and SRSF3 on SRSF5 or SRSF7 (Ä nkö et al, 2012;Yang et al, 2018).…”

SRSF3 and SRSF7 modulate 3′UTR length through suppression or activation of proximal polyadenylation sites and regulation of CFIm levels

“…Both proteins are very closely related and share a similar domain architecture with one RNA recognition motif (RRM, 80% identical), a short linker region for NXF1 interaction, and a region enriched in arginine-serine di-peptides called RS domain [ 36 – 38 ]. Unlike SRSF3, SRSF7 contains an additional CCHC-type zinc (Zn) knuckle domain that together with the RRM determines its distinct RNA-binding specificity [ 39 – 41 ]. In addition, the RS domain of SRSF7 is 66 amino acids longer and harbors a distinct hydrophobic stretch [ 41 ].…”

“…Unlike SRSF3, SRSF7 contains an additional CCHC-type zinc (Zn) knuckle domain that together with the RRM determines its distinct RNA-binding specificity [ 39 – 41 ]. In addition, the RS domain of SRSF7 is 66 amino acids longer and harbors a distinct hydrophobic stretch [ 41 ]. RS domains mediate protein-protein interactions and, through phosphorylation and dephosphorylation of their serine residues, the splicing activity, nuclear import, subnuclear localization, and mRNA export activity of SR proteins are regulated [ 42 ].…”