Overlapping Cis Sites Used for Splicing of HIV-1env/nefand revmRNAs

Axel K Swanson,C Martin Stoltzfus

doi:10.1074/jbc.273.51.34551

Abstract

Alternative splicing is used to generate more than 30 human immunodeficiency virus type 1 (HIV-1) spliced and unspliced mRNAs from a single primary transcript. The abundance of HIV-1 mRNAs is determined by the efficiencies with which its different 5' and 3' splice sites are used. Three splice sites (A4c, A4a, and A4b) are upstream of the rev initiator AUG. RNAs spliced at A4c, A4a, and A4b are used as mRNAs for Rev. Another 3' splice site (A5) is immediately downstream of the rev initiator. RNAs spliced at A5 are used as mRNAs for Env and Nef. In this report, primer extension analysis of splicing intermediates was used to show that there are eight branch points in this region, all of which map to adenosine residues. In addition, cis elements recognized by the cellular splicing machinery overlap; the two most 3' branch points overlap with the AG dinucleotides at rev 3' splice sites A4a and A4b. Competition of the overlapping cis sites for different splicing factors may play a role in maintaining the appropriate balance of mRNAs in HIV-1-infected cells. In support of this possibility, mutations at rev 3' splice site A4b AG dinucleotide dramatically increased splicing of the env/nef 3' splice site A5. This correlated with increased usage of the four most 3' branch points, which include those within the rev 3' splice site AG dinucleotides. Consistent with these results, analysis of a mutant in which three of the four env/nef branch points were inactivated indicated that use of splice site A5 was inhibited and splicing was shifted predominantly to the most 5' rev 3' splice site A4c with preferential use of the two most 5' branch points. Our results suggest that spliceosomes formed at rev A4a-4b, rev A4c, and env/nef A5 3' splice sites each recognize different subsets of the eight branch point sequences.

Highlights

Splicing of mRNA precursors in the nucleus of metazoan cells occurs by the cleavage and joining of 5Ј and 3Ј splice sites
We showed that different sets of branch points are used for splicing at the different human immunodeficiency virus type 1 (HIV-1) rev and env/nef 3Ј splice sites
This implies that separate spliceosome complexes used for splicing at the rev and the env/nef mRNAs form upstream of the different AG dinucleotides

Summary

We have previously shown that the efficiency of splicing at

Splice site A3, which is primarily used for synthesis of tat mRNA, is determined by both a suboptimal polypyrimidine tract and by an exon-splicing silencer element (ESS) [6]. The ESS, which maps approximately 60 nt downstream of the tat splice site, does not significantly inhibit splicing at the rev and env splice sites A4a, A4b, A4c, and A5, which are downstream of the ESS [10] Splicing at these latter closely spaced 3Ј splice sites, which are utilized for approximately 90% of the spliced mRNAs in HIV-1-infected cells [13], may involve the assembly of a single spliceosome complex upstream of the A4c splice site followed by scanning to the four different AGs. The relative usage of the different splice sites would be determined by competition between the AGs based on their position and sequence context [15, 16]. Our results indicate that separate spliceosomes form and that there is overlap of the cis splicing sequences recognized by the cellular splicing machinery

EXPERIMENTAL PROCEDURES

RESULTS

Consensus matchb

DISCUSSION