The essential pre-mRNA splicing factor SF2 influences 5′ splice site selection by activating proximal sites
454
Citation
46
Reference
10
Related Paper
Citation Trend
Keywords:
splice
Splicing factor
Splice site mutation
SR protein
Precursor mRNA
Exonic splicing enhancer
Protein splicing
Exon skipping
Splicing factor
Exonic splicing enhancer
SR protein
Protein splicing
Spliceosome
Polypyrimidine tract
Precursor mRNA
Cite
Citations (49)
The SR proteins constitute a family of splicing factors, highly conserved in metazoans, that contain one or two amino-terminal RNA-binding domains (RBDs) and a region enriched in arginine/serine repeats (RS domain) at the carboxyl terminus. Previous studies have shown that SR proteins possess distinct RNA-binding specificities that likely contribute to their unique functions, but it is unclear whether RS domains have specific roles in vivo. Here, we used a genetic system developed in the chicken B cell line DT40 to address this question. Expression of chimeric proteins generated by fusion of the RS domains of heterologous SR proteins, or a human TRA-2 protein, with the RBDs of ASF/SF2 allowed cell growth following genetic inactivation of endogenous ASF/SF2, indicating that RS domains are interchangeable for all functions required to maintain cell viability. However, a chimera containing the RS domain from a related splicing factor, U2AF 65 , could not rescue viability and was inactive in in vitro splicing assays, suggesting that this domain performs a distinct function. We also used the DT40 system to show that depletion of ASF/SF2 affects splicing of specific transcripts in vivo. Although splicing of several simple constitutive introns was not significantly affected, the alternative splicing patterns of two model pre-mRNAs switched in a manner consistent with predictions from previous studies. Unexpectedly, ASF/SF2 depletion resulted in a substantial increase in splicing of an HIV-1 tat pre-mRNA substrate, indicating that ASF/SF2 can repress tat splicing in vivo. These results provide the first demonstration that an SR protein can influence splicing of specific pre-mRNAs in vivo.
SR protein
Protein splicing
Splicing factor
Exonic splicing enhancer
Minigene
Heterogeneous ribonucleoprotein particle
Cite
Citations (100)
Two distinct functions have been proposed for the serine–arginine (SR)-rich family of splicing factors. First, SR proteins are essential splicing factors and are thought to function by mediating protein–protein interactions within the intron during spliceosome assembly. Second, SR proteins bind to exonic enhancer sequences and recruit spliceosome components to adjacent introns. The latter activity is required for splice-site recognition and alternative splicing. Until now it has not been possible to determine whether the requirement for SR proteins in the basic splicing reaction is a secondary consequence of their exon-dependent recruitment function. Here we show that RNA substrates containing only 1 nt of exon sequence can undergo the first step of the splicing reaction in vitro and that this activity requires SR proteins. Thus, we provide direct evidence that SR proteins have both exon-independent and exon-dependent functions in pre-mRNA splicing.
Exonic splicing enhancer
SR protein
Spliceosome
Splicing factor
Protein splicing
Polypyrimidine tract
Minigene
Cite
Citations (47)
Spliceosome
SR protein
Protein splicing
Exonic splicing enhancer
Dephosphorylation
Splicing factor
Minigene
Cite
Citations (221)
Q Sun, A Mayeda, R K Hampson, A R Krainer, and F M Rottman Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4960.
Splicing factor
Exonic splicing enhancer
Minigene
SR protein
Protein splicing
Precursor mRNA
Cite
Citations (286)
Serine/arginine-rich (SR) proteins are essential splicing factors with one or two RNA-recognition motifs (RRMs) and a C-terminal arginine- and serine-rich (RS) domain. SR proteins bind to exonic splicing enhancers via their RRM(s), and from this position are thought to promote splicing by antagonizing splicing silencers, recruiting other components of the splicing machinery through RS-RS domain interactions, and/or promoting RNA base-pairing through their RS domains. An RS domain tethered at an exonic splicing enhancer can function as a splicing activator, and RS domains play prominent roles in current models of SR protein functions. However, we previously reported that the RS domain of the SR protein SF2/ASF is dispensable for in vitro splicing of some pre-mRNAs. We have now extended these findings via the identification of a short inhibitory domain at the SF2/ASF N-terminus; deletion of this segment permits splicing in the absence of this SR protein's RS domain of an IgM pre-mRNA substrate previously classified as RS-domain-dependent. Deletion of the N-terminal inhibitory domain increases the splicing activity of SF2/ASF lacking its RS domain, and enhances its ability to bind pre-mRNA. Splicing of the IgM pre-mRNA in S100 complementation with SF2/ASF lacking its RS domain still requires an exonic splicing enhancer, suggesting that an SR protein RS domain is not always required for ESE-dependent splicing activation. Our data provide additional evidence that the SF2/ASF RS domain is not strictly required for constitutive splicing in vitro, contrary to prevailing models for how the domains of SR proteins function to promote splicing.
SR protein
Exonic splicing enhancer
Protein splicing
Splicing factor
Cite
Citations (28)
SR proteins are well known to promote exon inclusion in regulated splicing through exonic splicing enhancers.SR proteins have also been reported to cause exon skipping, but little is known about the mechanism.We previously characterized SRSF1 (SF2/ASF)-dependent exon skipping of the CaMKII␦ gene during heart remodeling.By using mouse embryo fibroblasts derived from conditional SR protein knockout mice, we now show that SR protein-induced exon skipping depends on their prevalent actions on a flanking constitutive exon and requires collaboration of more than one SR protein.These findings, coupled with other established rules for SR proteins, provide a theoretical framework to understand the complex effect of SR protein-regulated splicing in mammalian cells.We further demonstrate that heart-specific CaMKII␦ splicing can be reconstituted in fibroblasts by downregulating SR proteins and upregulating a RBFOX protein and that SR protein overexpression impairs regulated CaMKII␦ splicing and neuronal differentiation in P19 cells, illustrating that SR protein-dependent exon skipping may constitute a key strategy for synergism with other splicing regulators in establishing tissue-specific alternative splicing critical for cell differentiation programs.
Exon skipping
Exonic splicing enhancer
SR protein
Splicing factor
Minigene
Cite
Citations (80)
SR proteins are essential pre-mRNA splicing factors that have been shown to bind a number of exonic splicing enhancers where they function to stimulate the splicing of adjacent introns. Members of the SR protein family contain one or two N-terminal RNA binding domains, as well as a C-terminal arginine-serine (RS) rich domain. The RS domains mediate protein-protein interactions with other RS domain containing proteins and are essential for many, but not all, SR protein functions. Hybrid proteins containing an RS domain fused to the bacteriophage MS2 coat protein are sufficient to activate enhancer-dependent splicing in HeLa cell nuclear extract when bound to the pre-mRNA. Here we report progress towards determining the protein sequence requirements for RS domain function. We show that the RS domains from non-SR proteins can also function as splicing activation domains when tethered to the pre-mRNA. Truncation experiments with the RS domain of the human SR protein 9G8 identified a 29 amino acid segment, containing 26 arginine or serine residues, that is sufficient to activate splicing when fused to MS2. We also show that synthetic domains composed solely of RS dipeptides are capable of activating splicing, although their potency is proportional to their size.
SR protein
Protein splicing
Exonic splicing enhancer
Minigene
Splicing factor
Cite
Citations (41)
SR protein
Protein splicing
Exonic splicing enhancer
Splicing factor
Cite
Citations (13)