In higher eukaryotes, the coding regions (exons) of nearly all genes are split, and the intervening sequences (introns) must be precisely and efficiently removed during splicing to allow correct protein expression (7
). To add more complexity, a great number of genes contain multiple exons and introns and the choice of exon selection can vary in a tissue- or development-specific fashion (20
). It has been estimated that about 60% of human genes undergo alternative splicing (6
), but even this number is likely to be an underestimate since cDNA databases are not complete and alternative splicing events in noncoding regions are relatively underreported.
Alternative splicing can be regulated by both cis
-acting RNA elements, such as splicing enhancers and splicing silencers, and trans
-acting factors, of which the SR protein family is perhaps the best studied. SR proteins are characterized by one or two RNA recognition motifs at the N terminus and a C-terminal region rich in arginine-serine dipeptides (RS domain). They are essential splicing factors that participate in multiple steps of splicing (19
). In early spliceosome formation (E complex), SR proteins enhance the binding of U1 snRNP to 5′ splice sites through interaction with U1 snRNP U1-70K (30
). At later steps, SR proteins escort the U4/U6•U5 tri-snRNP into the spliceosome (50
) and they play important roles in forming bridge complexes across exons and introns by mediating essential protein-protein interaction across splice sites (1
). For alternative splicing, SR proteins most often bind enhancer elements to facilitate the recognition and activation of weak splice sites (5
The functions of SR proteins partially overlap in that most individual SR proteins can complement splicing-deficient S100 extracts. Consistent with partial redundancy, RNA interference-mediated knockdown of several SR proteins in Caenorhabditis elegans
led to no observable phenotype (36
). However, targeted disruption of ASF/SF2 in chicken DT40 cells (64
), RNA interference with ASF/SF2 in C
), and null alleles of Drosophila
SR protein B52 all resulted in lethality (48
). In addition, SR proteins display substrate specificity and have distinct functions in alternative splicing (19
). Therefore, SR proteins do not simply have redundant functions but can act in a variety of ways to regulate splicing.
As key regulators of splicing, SR proteins themselves need to be regulated. The expression levels of some SR proteins are transcriptionally regulated (2
), but posttranscriptional control is also important. Many SR protein genes are themselves subject to alternative splicing, often in an autoregulatory manner (25
). Phosphorylation-dephosphorylation of the RS domain is also known to affect the activity and subnuclear localization of SR proteins (9
). In addition, the effects of SR proteins can be counteracted by the action of other proteins, including the hnRNP A/B proteins (16
), p32 (44
), RSF1 (33
), and two recently identified SR superfamily members, SRrp35 and SRrp40 (13
). These proteins inhibit SR proteins either by interfering with RNA binding or by disrupting crucial protein-protein interactions. The important point is that the activity of SR proteins is tightly controlled at multiple levels.
We have been studying SRrp86, a novel SR-related protein that can regulate the activity of other SR proteins both negatively and positively (3
). In both in vitro and in vivo splicing assays, SRrp86 inhibits ASF/SF2, SC35, and SRp55 while activating SRp20. Domain analysis revealed that the C-terminal RS-EK-RS domain is required for full activity while the unique EK domain acts as a splicing inhibitor (4
). It appears that SRrp86 regulates SR proteins through direct protein-protein interaction, and we have identified some of the specific interaction targets, but the full range of proteins that interact with SRrp86 remain unknown.
To identify proteins that interact with SRrp86 in vivo, we performed a yeast two-hybrid library screen and coimmunoprecipitation experiments coupled to mass spectrometry. Consistent with the regulation of other SR proteins, we found that all of the core SR family members associated with SRrp86. In contrast, only SRp20 and SRp75 associated with a construct lacking the EK domain, indicating that this domain is an important modulator of protein-protein interaction. Besides SR proteins, other splicing factors and RNA-processing factors were also found to associate with SRrp86. In this study, five such proteins, SAF-B (scaffold attachment factor B), hnRNP G, YB-1, p72, and 9G8, were further investigated because they have been previously implicated in splicing regulation (11
). By using a CD44 v5 minigene as a splicing reporter, we show that SAF-B, hnRNP G, and 9G8 antagonize the ability of SRrp86 to activate v5 inclusion. In the same assay, YB1 and p72 were found to have no effect. Our findings support the hypothesis that protein-protein interaction underlies the function of SRrp86 and reinforce the notion that combinatorial control of splicing by multiple factors allows precise regulation of alternative splicing.