SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.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 developmentspecific fashion (20,43,54). It has been estimated that about 60% of human genes undergo alternative splicing (6, 12), 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 (19,21,38). In early spliceosome formation (E complex), SR proteins enhance the binding of U1 snRNP to 5Ј splice sites through interaction with U1 snRNP U1-70K (30, 73). 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 (1,29,49,66). 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 wi...
Previously published online as a RNA Biology E-publication: http://www.landesbioscience.com/journals/rnabiology/abstract.php?id=3017 KEY WORDS Research PaperThe Splicing Factor PSF is Part of a Large Complex that Assembles in the Absence of pre-mRNA and Contains All 5 snRNPs ABSTRACT PSF (PTB-associated splicing factor) is a large nuclear protein that has been implicated in numerous processes including transcription and RNA splicing. It has been shown to directly associate with U5 snRNA and has also been found within numerous purified splicing complexes. Here, we show that when HeLa nuclear extracts are adjusted to splicing conditions, PSF is found as part of a large complex that contains all five snRNPs and most known splicing factors. Formation of the complex does not require addition of exogenous pre-mRNA substrate and occurs at 4˚C but is salt sensitive. Sedimentation experiments and identification of individual components by mass spectrometry revealed association with multiple nuclear factors, most of which overlap with spliceosome components.
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