Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins.

Angela Crisci,Jean-Christophe Rain,Monika Raabe,Angela Krämer,Henning Urlaub,Flore Raleff,Ivona Bagdiul

doi:10.1093/nar/gkv952

Abstract

Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions.

Highlights

Pre-mRNA splicing is essential for eukaryotic gene expression and one of the most versatile mechanisms to increase proteome diversity through alternative splice site choices [1]
Five small nuclear ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs) and more than 100 non-snRNP proteins assemble in a step-wise fashion on the pre-mRNA through networks of RNA–RNA, RNA–protein and protein–protein interactions to form the catalytic center of the spliceosome for intron removal
We demonstrate a novel function of Splicing factor 1 (SF1), which is essential for efficient spliceosome assembly, in the initial recruitment of the U2 snRNP through direct interactions with two U2 snRNP-associated proteins

Summary

Introduction

Pre-mRNA splicing is essential for eukaryotic gene expression and one of the most versatile mechanisms to increase proteome diversity through alternative splice site choices [1]. The small U2AF subunit (U2AF35) recognizes the 3 splice site AG dinucleotide [9] Together these interactions assemble the early complex E, which is converted into pre-splicing complex A by incorporation of the U2 snRNP. This is accomplished through interaction of the U2 snRNP-associated SF3b155 with U2AF65 [10], binding of U2 snRNP proteins to and adjacent to the BPS [11,12,13] and base pairing of the U2 snRNA with the BPS. The following steps of spliceosome assembly involve binding of the remaining snRNPs and additional non-snRNP proteins, juxtaposition of the splice sites and dynamic remodeling of the complexes leading to the formation of the catalytic center, followed by intron removal in two catalytic steps [2]

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Conclusion