Interactive Fly, Drosophila

transformer 2


SR protein interactions

Although transcription and pre-mRNA processing are colocalized in eukaryotic nuclei, molecules linking these processes have not previously been described. Four novel rat proteins have been identified by their ability to interact with the repetitive C-terminal domain (CTD) of RNA polymerase II in a yeast two-hybrid assay. A yeast homolog of one of the rat proteins has also been shown to interact with the CTD. These CTD-binding proteins are all similar to the SR (serine/arginine-rich) family of proteins that have been shown to be involved in constitutive and regulated splicing. In addition to alternating Ser-Arg domains, these proteins each contain discrete N-terminal or C-terminal CTD-binding domains. SR-related proteins have been identified in a complex that can be immunoprecipitated from nuclear extracts with antibodies directed against RNA polymerase II. In addition, in vitro splicing is inhibited either by an antibody directed against the CTD or by wild-type but not mutant CTD peptides. Thus, these results suggest that the CTD and a set of CTD-binding proteins may act to physically and functionally link transcription and pre-mRNA processing (Yuryev, 1996).

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are abundant nuclear polypeptides, most likely involved in different steps of pre-mRNA processing. Protein A1 (34 kDa), a prominent member of the hnRNP family, seems to act by modulating the RNA secondary structure and by antagonizing some splicing factors (SR proteins) in splice-site selection and exon skipping/inclusion. A role of A1 in the nucleo-cytoplasmic transport of RNA has also been proposed. These activities might depend not only on the RNA-binding properties of the protein but also on specific protein-protein interactions. A1 can indeed selectively interact, in vitro, both with itself and with other hnRNP basic "core" proteins. Such selective binding is mediated exclusively by the Gly-rich C-terminal domain, where a novel protein-binding motif constituted by hydrophobic repeats can be envisaged. The same domain is necessary and sufficient to promote specific interaction in vivo, as assayed by the yeast two-hybrid assay. Moreover, an in vitro interaction with some SR proteins is also observed. These observations suggest that diverse and specific protein-protein interactions might contribute to the different functions of the hnRNP A1 protein in mRNA maturation (Cartegni, 1996).

An in vitro splicing assay has been devised in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. This assay was used to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB can be partially reversed if pre-mRNAs are preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affects the joining of exons 1-3 with no dramatic increases in 1-2 splicing detected. Preincubation of pre-mRNAs with SR proteins leads to small increases in 1-2 splicing. However, if pre-mRNAs are preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there is a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus multiple pairwise and sometimes antagonizing interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection (Lin, 1995).

SR phosphorylation and activity

Serine/arginine-rich splicing factors (SR proteins) are substrates for serine phosphorylation that can regulate SR protein function. Changes in the phosphorylation of splicing components occurs at the level of the individual reaction as well as in a broader, cell-wide manner. Phosphatase inhibitors have been used to demonstrate that dephosphorylation is important for single splicing reactions. Specific phosphatases including protein phosphatase 1 (PP1) have been shown to affect in vitro splicing. In addition, thiophosphorylation of splicing factors interferes with pre-mRNA splicing. More directed experiments have demonstrated that the state of phosphorylation of SR proteins in particular can be linked to splicing activity in vitro. Several SR protein kinases have been described, including SR protein kinase 1 (SRPK1), Clk/Sty, SRPK2, and a kinase activity of DNA topoisomerase I. Hyperphosphorylation with SRPK1 can inhibit splicing, as can dephosphorylation of SR proteins; thus, an intermediate level of phosphorylation is required for splicing in vitro. In addition, dephosphorylation of SR proteins appears to be required during constitutive splicing reactions but not for their ability to act as splicing activators in regulated splicing. Broad changes in SR protein phosphorylation are also seen in response to events that affect the entire cell. Hyperphosphorylation of SR proteins occurs during M phase of the cell cycle, in which splicing is quiescent. Large-scale dephosphorylation of SR proteins is observed in late adenovirus-infected HeLa cells. In Drosophila, defects in SR protein phosphorylation have been correlated with discrete phenotypes in the sex determination pathway (Du, 1998). Changes in SR protein phosphorylation have also altered SR protein subnuclear localization, with increased expression of either SRPK1 or Clk/Sty leading to diffusion of speckles. PP1 also alters the speckled morphology. In addition, hyperphosphorylation of SF2/ASF leads to its accumulation in the cytoplasm (Sanford, 1999 and references).

Gross changes have been observed in SR protein phosphorylation during early development coincident with major zygotic gene activation in the nematode Ascaris lumbricoides. These differences correlate with large-scale changes in SR protein activity in the promotion of both trans- and cis-splicing. Importantly, inactive early stage extracts can be made splicing competent with the addition of later stage SR proteins. To assess their phosphorylation state, SR proteins at these different stages were phosphorylated with the SR protein-specific kinase SRPK1 and [32Pgamma]ATP. Over this developmental span, the SR proteins become progressively better substrates for SRPK1. SR proteins that are hyperphosphorylated do not incorporate label, whereas the same proteins that have undergone partial dephosphorylation are labeled more efficiently. To demonstrate that this difference is the result of limited dephosphorylation and not a mass effect, phosphorylation reactions were performed after first dephosphorylating the SR preparations with PP1. Under these conditions, there is an equivalent amount of phosphate incorporated into SR proteins prepared from each of three developmental stages. In summary, each of these assays indicates that SR proteins undergo dephosphorylation concomitant with the major activation of gene expression. These data suggest that changes in SR protein phosphorylation have a role in the activation of pre-mRNA splicing during early development (Sanford, 1999).

The splicing reaction that removes introns from pre-messenger RNAs requires the assembly of the spliceosome on the nascent transcript, proper folding of the substrate-enzyme complex, and finally, two transesterification reactions. These stages in the splicing reaction must require careful orchestration. The sequential phosphorylation and dephosphorylation of SR proteins marks the transition between stages in the splicing reaction. Much evidence has already led to the idea that phosphorylation of SR proteins could modulate their activity. Dephosphorylation of these proteins abrogates their activity in a reaction measuring conversion of pre-spliceosomes to spliceosomes Phosphorylated ASF/SF2, but not mock-phosphorylated ASF/SF2, activates the splicing of HIV tat pre-mRNA in reactions challenged with excess random RNA. These two findings are confirmed and extended by this study. Phosphorylated ASF/SF2 efficiently complements an SR protein-deficient HeLa S100 extract in promoting the splicing of an adenovirus-2-derived pre-messenger RNA, whereas unphosphorylated ASF/ SF2 does not. Whereas unphosphorylated ASF/SF2 inhibits splicing in HeLa nuclear extracts, phosphorylation of the ASF/SF2 reverses the inhibition and enhances splicing. Data is presented that shows that dephosphorylation of ASF/SF2 is required for the first transesterification reaction once the spliceosome has assembled. Thiophosphorylated ASF/SF2, which cannot be readily dephosphorylated, can promote spliceosome assembly, but cannot promote the first transesterification reaction. These data, together with other observations, indicate for the first time a requirement for SR protein dephosphorylation in pre-messenger RNA splicing in vitro (Cao, 1997).

SR proteins are a family of essential splicing factors required for early recognition of splice sites during spliceosome assembly. They also function as alternative RNA splicing factors when overexpressed in vivo or added in excess to extracts in vitro. SR proteins are highly phosphorylated in vivo, a modification that is required for their function in spliceosome assembly and splicing catalysis. SR proteins purified from late adenovirus-infected cells are inactivated as splicing enhancer or splicing repressor proteins by virus-induced dephosphorylation. The virus-encoded protein E4-ORF4 activates dephosphorylation by protein phosphatase 2A of HeLa SR proteins and converts their splicing properties into those of SR proteins purified from late adenovirus-infected cells. Taken together, these results suggest that E4-ORF4 is an important factor controlling the temporal shift in adenovirus alternative RNA splicing. It is concluded that alternative pre-mRNA splicing, like many other biological processes, is regulated by reversible protein phosphorylation (Kanopka, 1998).

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

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