Ser/Arg-rich Protein-mediated Communication between U1 and U2 Small Nuclear Ribonucleoprotein Particles

Lisa A Boukis,Nan Liu,Suzanne Furuyama,James P Bruzik

doi:10.1074/jbc.m313209200

Lisa A Boukis, Nan Liu + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m313209200

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Abstract

Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a downstream 5' splice site, can positively influence utilization of an upstream 3' splice site via exon definition in both trans- and cis-splicing systems. Although exon definition results in the enhancement of splicing of an upstream intron, the nature of the factors involved has remained elusive. We assayed the interaction of U1 snRNP as well as the positive effect of a downstream 5' splice site on trans-splicing in nematode extracts containing either inactive (early in development) or active (later in development) serine/arginine-rich splicing factors (SR proteins). We have determined that U1 snRNP interacts with the 5' splice site in the downstream exon even in the absence of active SR proteins. In addition, we determined that U1 snRNP-directed loading of U2 snRNP onto the branch site as well as efficient trans-splicing in these inactive extracts could be rescued upon the addition of active SR proteins. Identical results were obtained when we examined the interaction of U1 snRNP as well as the requirement for SR proteins in communication across a cis-spliced intron. Weakening of the 3' splice site uncovered distinct differences, however, in the ability of U1 snRNP to promote U2 addition, dependent upon its position relative to the branch site. These results demonstrate that SR proteins are required for communication between U1 and U2 snRNPs whether this interaction is across introns or exons.

Highlights

Pre-mRNA splicing is a process whereby introns are removed, and the exons are linked together to produce mature mRNA molecules
We examined the interaction between U1 small nuclear ribonucleoprotein particle (snRNP) and a 5Ј splice site involved in exon definition in inactive two-cell-stage extracts
U1 snRNP Interacts with a 5Ј Splice Site-like Enhancer Element in the Absence of Functional SR Proteins—Many groups have documented the ability of SR proteins to participate in U1 addition to the 5Ј splice site [38, 43,44,45,46]

Summary

SR Proteins Mediate Interactions across Exons and Introns

We examined the interaction between U1 snRNP and a 5Ј splice site involved in exon definition in inactive two-cell-stage extracts. Even though this extract is not competent for splicing due to the phosphorylation state of the SR proteins, U1 snRNP interacted with the 5Ј splice site-like enhancer without supplementation with active SR proteins. Having established that U1 binds to the 5Ј splice site-like enhancer in inactive two-cell-stage extracts, we examined the loading of U2 snRNP onto the branch site. U1 was bound to the 5Ј splice site, robust U2 interaction with the branch point sequence was only observed in two-cell-stage extracts upon the addition of active SR proteins. Our results demonstrate that SR proteins mediate the interaction between U1 and U2 snRNPs whether U1 is bound in an upstream or downstream position

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DISCUSSION