Interactive Fly, Drosophila

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Interaction of U1 snRNP with SR proteins: the role of SR proteins in splicing

SR proteins are required for the first step of spliceosome assembly: the interaction of the U1 small nuclear ribonucleoprotein complex (U1 snRNP) with the 5' splice site of the pre-mRNA. Individual SR proteins have been shown to be distinctly capable of promoting the interaction of U1 snRNP with alternative 5' splice junctions. These results suggest that SR proteins direct 5' splice site selection by regulation of U1 snRNP assembly onto the pre-mRNA (Zahler, 1995).

Addition of SR proteins to in vitro splicing extracts results in a significant increase in assembly of the earliest prespliceosomal complex E and a corresponding decrease in assembly of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex H. In addition, SR proteins promote formation of the E5' and E3' complexes that assemble (respectively) on RNAs containing only 5' and 3' splice sites. It is concluded that SR proteins promote the earliest specific recognition of both the 5' and 3' splice sites and are limiting factors for this function in HeLa nuclear extracts. Specific, splice site-dependent RNA-protein interactions of SR proteins can be demonstrated in the E, E5', and E3' complexes. SR proteins do not UV cross-link in the H complex, and conversely, hnRNP cross-linking is largely excluded from the E-type complexes. A discrete complex resembling the E5' complex assembles on both purine-rich and non-purine-rich exonic splicing enhancers. This complex, which has been designated the Enhancer complex, contains U1 small nuclear RNP and is associated with different SR protein family members, depending on the sequence of the enhancer. It is propose that both downstream 5' splice site enhancers and exonic enhancers function by establishing a network of pre-mRNA-protein and protein-protein interactions involving U1 snRNP, SR proteins, and U2AF that is similar to the interactions that bring the 5' and 3' splice sites together in the E complex (Staknis, 1994).

SR proteins are essential splicing factors that also influence 5' splice site choice. Addition of excess mixed SR proteins to a HeLa in vitro splicing system stimulates utilization of a novel 5' splice site (site 125) within the intron of the standard adenovirus pre-mRNA substrate. When U1 snRNPs are debilitated by sequestering the 5' end of U1 snRNA with a 2'-O-methyl oligoribonucleotide, excess SR proteins not only rescue splicing at the normal site and site 125 but also activate yet another 5' splice site (site 47) in the adenovirus intron. One SR protein, SC35, is sufficient to exhibit the above activities. The possibility that excess SR proteins recruit residual unblocked U1 snRNPs to participate in 5' splice site recognition has been ruled out by psoralen cross-linking studies, which demonstrate that the 2'-O-methyl oligoribonucleotide effectively blocks 5' splice site/U1 interaction. Native gel analysis reveals a nearly normal splicing complex profile in the 2'-O-methyl oligoribonucleotide pretreated, SR protein-supplemented extract. These results indicate that SR proteins can replace some functions of the U1 snRNP but underscore the contribution of U1 to the fidelity of 5' splice site selection (Tarn, 1994).

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). The U1 snRNP in HeLa cell nuclear extract is perturbed by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of the 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for the 5' splice site selection. Accordingly, it was found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. These results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection (Tarn, 1995).

A class of pre-mRNAs has been identified that are spliced in HeLa extracts depleted for U1 snRNP (delta U1 extracts). Previously, pre-mRNAs were described that can be spliced in delta U1 extracts only when high concentrations of SR splicing factors are added. In contrast, the substrates characterized here are efficiently processed in delta U1 extracts without the addition of excess SR proteins. The members of this class comprise both a naturally occurring pre-mRNA, from the Drosophila fushi tarazu gene, and a chimera containing sequences from two different pre-mRNAs that individually are dependent upon either U1 snRNP or excess SR proteins. Several sequence elements account for the variations in dependence on U1 snRNP and SR proteins for splicing. In one pre-mRNA, a single element was identified adjacent to the branch site. In the other, two elements flanking the 5' splice site were found to be critical. This U1-independent splicing reaction may provide a mechanism for cells to control the extent of processing of different classes of pre-mRNAs in response to altered activities of SR proteins, and furthermore suggests that U1 snRNP-independent splicing may not be uncommon (Crispino, 1996).

Members of the SR family of proteins, can collaborate with U1 snRNP in the recognition of 5' splice sites in pre-messenger RNAs. Purified U1 snRNP and ASF/SF2 form a ternary complex with pre-mRNA, which is dependent on a functional 5' splice site. Sequences in the pre-mRNA, domains in ASF/SF2 and components of the U1 snRNP particle are shown to be required for complex formation. Sequences at the 5' splice site are necessary and sufficient for complex formation. One functional RNA binding domain and the RS domain are both required for ASF/SF2 to participate in complex formation. The RNA binding domains were redundant in this assay, suggesting that either domain can interact with the pre-messenger RNA. There is no function for the U1-specific A protein in complex formation, whereas a function for U1-specific C protein is strongly suggested (Jamison, 1995).

Exactly how specific splice sites are recognized during the processing of complex precursor messenger RNAs is not clear. Small nuclear ribonucleoprotein particles (snRNPs) are involved, but are not sufficient by themselves to define splice sites. Now a human protein essential for splicing in vitro, called alternative splicing factor/splicing factor 2, is shown to cooperate with the U1 snRNP particle in binding pre-mRNA. This cooperation is probably achieved by specific interactions between the arginine/serine-rich domain of the splicing factor and a similar region in a U1 snRNP-specific protein (Kohtz, 1994).

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 70K subunit of U1 snRNP

Continued: see sans fille Evolutionary homologs part 3/3 | back to Evolutionary homologs part 1/3 |

sans fille : Biological Overview | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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