Interactive Fly, Drosophila

transformer 2


EVOLUTIONARY HOMOLOGS (part 2/3)

The role of SR proteins in splicing

SR proteins are essential splicing components that may participate in the maintenance and regulation of cell-specific splicing patterns. SR proteins are constitutive components of the splicing machinery that play important roles during spliceosome assembly by promoting splice site recognition and facilitating interactions between 5' and 3' splice site complexes. Individual SR proteins are able to complement splicing-deficient cytoplasmic S100 extracts, which lack SR proteins but contain other factors necessary for constitutive splicing. In addition, SR proteins can influence alternative splicing in a concentration-dependent manner, either when added to splicing-competent nuclear extracts or when transiently overexpressed in vivo. SR proteins have also been shown to participate in splicing activation of introns containing weak 5' or 3' splice sites by binding to purine-rich exonic splicing enhancers (ESE), frequently situated in downstream exons. Conversely, high-affinity binding sites for the SR proteins ASF/SF2 and SRp40 have been demonstrated to function as ESEs in vitro. These and other studies also show that SR proteins have distinct RNA binding specificities and display substrate specificity in vitro (Tacke, 1998).

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. A genetic system was 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 allow 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, U2AF65, cannot rescue viability and is inactive in in vitro splicing assays, suggesting that this domain performs a distinct function. Depletion of ASF/SF2 affects splicing of specific transcripts in vivo. Although splicing of several simple constitutive introns is not significantly affected, the alternative splicing patterns of two model pre-mRNAs switch in a manner consistent with predictions from previous studies. Unexpectedly, ASF/SF2 depletion results 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 (Wang, 1998).

A genomic clone encoding the Drosophila U1 small nuclear ribonucleoprotein particle 70K protein was isolated by hybridization with a human U1 small nuclear ribonucleoprotein particle 70K protein cDNA. Southern blot and in situ hybridizations show that this U1 70K gene is unique in the Drosophila genome, residing at cytological position 27D1,2. Polyadenylated transcripts of 1.9 and 3.1 kilobases were observed. While the 1.9-kilobase mRNA is always more abundant, the ratio of these two transcripts is developmentally regulated. Analysis of cDNA and genomic sequences indicated that these two RNAs encode an identical protein with a predicted molecular weight of 52,879. Comparison of the U1 70K proteins predicted from Drosophila, human, and Xenopus cDNAs reveals 68% amino acid identity in the most amino-terminal 214 amino acids, which include a sequence motif common to many proteins that bind RNA. The carboxy-terminal half is less well conserved but is highly charged and contains distinctive arginine-rich regions in all three species. These arginine-rich regions contain stretches of arginine-serine dipeptides like those found in Transformer, Transformer-2, and Suppressor-of-white-apricot proteins, all of which have been identified as regulators of mRNA splicing in Drosophila melanogaster (Mancebo, 1990).

A family of six highly conserved proteins that contain domains rich in alternating serine/arginine residues (SR proteins) function in the regulation of splice site selection and are required for splicing. More than 35 proteins were detected in nuclear extracts of HeLa cells that co-fractionate with the defined SR family. Many of these proteins were recognized by three monoclonal antibodies that bind to distinct phosphoepitopes on SR proteins. Two of these SR-related proteins were identified as the nuclear matrix antigens B1C8 and B4A11, which previously have been implicated in splicing. A subset of SR proteins, in their phosphorylated state, are associated with spliceosome complexes through both steps of the splicing reaction, remaining preferentially bound to complexes containing the exon-product. In contrast, other SR-related proteins appear to remain specifically associated with the intron-Lariat complex. The results indicate the existence of a potentially large group of SR-related proteins, and also suggest possible additional functions of SR proteins at a post-splicing level (Blencowe, 1996).

A monoclonal antibody (mAb 16H3) was generated against four SR proteins from a the family of six SR proteins, all known regulators of splice site selection and spliceosome assembly. In addition to the reactive SR proteins (SRp20, SRp40, SRp55, and SRp75), mAb 16H3 also binds approximately 20 distinct nuclear proteins in human, frog, and Drosophila extracts, whereas yeast do not detectably express the epitope. The antigens are shown to be nuclear, nonnucleolar, and concentrated at active sites of RNA polymerase II transcription, suggesting their involvement in pre-mRNA processing. Indeed, most of the reactive proteins observed in nuclear extract are detected in spliceosomes (E and/or B complex) assembled in vitro, including the U1 70K component of the U1 small nuclear ribonucleoprotein particle and both subunits of U2AF. Interestingly, the 16H3 epitope maps to a 40-amino acid polypeptide composed almost exclusively of arginine alternating with glutamate and aspartate. All of the identified antigens, including the human homolog of yeast Prp22 (HRH1), contain a similar structural element characterized by arginine alternating with serine, glutamate, and/or aspartate. These results indicate that many more spliceosomal components contain such arginine-rich domains. Because it is conserved among metazoans, it is proposed that the "alternating arginine" domain recognized by mAb 16H3 may represent a common functional element of pre-mRNA splicing factors (Neugebauer, 1995).

Phosphorylation and the function of SR proteins

ASF/SF2 is a member of a conserved family of splicing factors known as SR proteins. These proteins, which are necessary for splicing in vitro, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repeating Arg-Ser dipeptides (RS domains). Previous studies have suggested that RS domains participate in protein-protein interactions with other RS domain-containing proteins. The RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not sufficient, for binding to the U1 snRNP-specific 70-kD protein (70K) in vitro. An apparent interaction of the isolated RS domain with 70K is observed if contaminating RNA is not removed, suggesting a nonspecific bridging among the basic RS domain, RNA, and 70K. In vitro phosphorylation of recombinant ASF/SF2 significantly enhances binding to 70K and also eliminates the RS domain-RNA interaction. Providing evidence that these interactions are relevant to splicing, ASF/SF2 can bind selectively to U1 snRNP in an RS domain-dependent, phosphorylation-enhanced manner. Conditions are described that reveal for the first time a phosphorylation requirement for ASF/SF2 splicing activity in vitro (Xiao, 1997).

The SR proteins constitute a large family of nuclear phosphoproteins required for constitutive pre-mRNA splicing. These factors also have global, concentration-dependent effects on alternative splicing regulation; this activity is antagonized by members of the hnRNP A/B family of proteins. Whereas some human SR proteins are confined to the nucleus, three of them (SF2/ASF, SRp20, and 9G8) shuttle rapidly and continuously between the nucleus and the cytoplasm. By swapping the corresponding domains between shuttling and nonshuttling SR proteins, it has been shown that the carboxy-terminal arginine/serine-rich (RS) domain is required for shuttling. This domain, however, is not sufficient to promote shuttling of an unrelated protein reporter, suggesting that stable RNA binding mediated by the RNA-recognition motifs may be required for shuttling. Consistent with such a requirement, a double point-mutation in RRM1 of SF2/ASF that impairs RNA binding prevents the protein from shuttling. Phosphorylation of the RS domain affects the shuttling properties of SR proteins. These findings show that different SR proteins have unique intracellular transport properties and suggest that those SR family members that shuttle may have roles not only in nuclear pre-mRNA splicing but also in mRNA transport, cytoplasmic events, and/or processes that involve communication between the nucleus and the cytoplasm (Caceres, 1998).

Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region that is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrates that the sites on ASF/SF2 that phosphorylate in vitro overlap with those that phosphorylate in vivo. A catalytically inactive form of Clk/Sty co-localizes with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase causes a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors (Colwill, 1996).

SR protein subnuclear localization

If pre-mRNA splicing begins during RNA synthesis, then transcriptionally active genes may be expected to contain high concentrations of pre-mRNA splicing factors. However, previous studies have localized splicing factors to a network of "speckles," regions distinct from individual sites of gene transcription where pre-mRNA is spliced. Speckles have been detected with antibodies specific for splicing snRNPs and members of the SR family of splicing factors. Dilution of these probes results in the visualization of hundreds of sites throughout the HeLa cell nucleus, the size and distribution of which are consistent with transcription units viewed with light microscopy. Analysis of the particles detected at varying concentrations of anti-SR reveals that particle dimensions are fairly uniform at a variety of low concentrations of anti-SR, but the diameters increase at higher concentrations. These sites of highest SR protein concentration frequently coincide in three-dimensional space with active sites of RNA polymerase II transcription. A newly developed reagent specific for a single member of the SR family, SRp20, detects a subset (~20%) of these sites (suggesting the gene-specific accumulation of these splicing regulators) that have distinct functions in pre-mRNA splicing. These observations question the view that the nucleus and its functions are highly compartmentalized; instead, they support a model in which the localization of these and possibly other gene regulators is determined primarily by their function (Neugebauer, 1997).

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). The role of the individual domains of these closely related proteins has been studied in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. Striking differences are observed in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, it was found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that has altered specificity in 5' splice site selection, although it is active in alternative splicing. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo (Caceres, 1997).

SR protein interactions

transformer2: Evolutionary homologs part 3/3 | back to part 1/3


transformer 2: Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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