Determinants that govern alternative splicing of the large intron of minute virus of mice p4-generated PRE-mRNA

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Following transfection of murine fibroblasts, MVMi does not efficiently produce progeny single-strand DNA (ssDNA). However, changing a single nucleotide in the MVMi 3' splice site to that found in the fibrotropic strain MVMp enabled full DNA replication and production of ssDNA. This change enhanced excision of the large intron and the production of NS2, likely by improving interaction with the branch point-binding U2 snRNA. The defect in production of MVMi ssDNA in fibroblasts can also be overcome by introducing a mutation in MVMi NS2 that enhances its interaction with CRM1. In addition to the sequences within the 3' splice site, excision of the large intron is also controlled by the 5' nonconsensus donor and the sequences surrounding the 5' splice site. We identified U-rich and A-rich intronic sequences (ISE) immediately downstream of the 5' splice site of the large intron. This ISE is required for efficient usage of the 5' nonconsensus donor and subsequent large intron excision. We demonstrated cellular factors TIA-1 and TIAR directly bind with this ISE and helps efficient splicing of the large intron. In addition, we also have identified exonic enhancer elements within the shared NS1/2 exon that affects excision of large intron.