Activation of Cryptic 3' Splice-Sites by SRSF2 Contributes to Cassette Exon Skipping.

Jiyeon Ha,Heegyum Moon,Haihong Shen,Namjeong Choi,Yongchao Liu,Ha Na Jang,Xuexiu Zheng,Jagyeong Oh

doi:10.3390/cells8070696

Abstract

Here we show that the serine/arginine rich splicing factor 2 (SRSF2) promotes cryptic 3′ splice-site (3′AG′) usage during cassette exon exclusion in survival of motor neuron (SMN2) minigenes. Deletion of the 3′AG′ (3′AG′1), its associated branch point (BP′) and polypyrimidine tract (PPT′) sequences directs SRSF2 to promote a second 3′AG′ (3′AG′2) with less conserved associated region for intron splicing. Furthermore, deletion of both 3′AG′1 and 3′AG′2 and their associated sequences triggered usage of a third 3′AG′3 that has very weak associated sequences. Interestingly, when intron splicing was directed to the 3′AG′ cryptic splice-sites, intron splicing from the canonical 3′AG splice-site was reduced along with a decrease in cassette exon inclusion. Moreover, multiple SRSF2 binding sites within the intron are responsible for 3′AG′ activation. We conclude that SRSF2 facilitates exon exclusion by activating a cryptic 3′AG′ and inhibiting downstream intron splicing.

Highlights

Splicing occurs at the consensus sequences near the 50 and 30 ends of introns, known as 50 and 30 splice-sites (50 ss and 30 ss) by a large, dynamic RNA-protein complex called the spliceosome [1]
We demonstrate that serine/arginine rich splicing factor 2 (SRSF2) significantly stimulates intron splicing at a cryptic splice-site (30 AG0 1) with the most conserved associated region located at upstream of exon 7 in a SMN2 minigene
We have previously demonstrated that SRSF2 promotes a cryptic 30 splice-site (30 AG0 ) that is located at 682 nt upstream of 30 AG at exon 7 while simultaneously suppressing splicing at the 30 AG

Summary

Introduction

Splicing occurs at the consensus sequences near the 50 and 30 ends of introns, known as 50 and 30 splice-sites (50 ss and 30 ss) by a large, dynamic RNA-protein complex called the spliceosome [1]. Small nuclear ribonucleoproteins (snRNPs) and several other proteins are recruited to the pre-mRNA [2]. The assembly of spliceosome, U1 small nuclear ribonucleoprotein (snRNP) is recruited to the 50 ss, U2 snRNP is directed to branch point (BP) sequence of intron. Alternative splicing generates diversity of mRNA isoforms and protein variants by selecting different combinations of 50 and 30 splice-site pairs to mediate gene regulation. High-throughput and genome-wide technologies indicate that alternative splicing occurs within transcripts from ~95% human multi-exon genes [10]

Methods

Results

Conclusion