cis-Acting and trans-acting modulation of equine infectious anemia virus alternative RNA splicing.
Equine infectious anemia virus (EIAV), a lentivirus distantly related to HIV-1, encodes regulatory proteins, EIAV Tat (ETat) and Rev (ERev), from a four-exon mRNA. Exon 3 of the tat/rev mRNA contains a 30-nucleotide purine-rich element (PRE) which binds both ERev and SF2/ASF, a member of the SR family of RNA splicing factors. To better understand the role of this element in the regulation of EIAV pre-mRNA splicing, we quantified the effects of mutation or deletion of the PRE on exon 3 splicing in vitro and on alternative splicing in vivo. We also determined the branch point elements upstream of exons 3 and 4. In vitro splicing of exon 3 to exon 4 was not affected by mutation of the PRE, and addition of purified SR proteins enhanced splicing independently of the PRE. In vitro splicing of exon 2 to exon 3 was dependent on the PRE; under conditions of excess SR proteins, either the PRE or the 5' splice site of exon 3 was sufficient to activate splicing. We applied isoform-specific primers in real-time RT-PCR reactions to quantitatively analyze alternative splicing in cells transfected with rev-minus EIAV provirus constructs. In the context of provirus with wild-type exon 3, greater than 80% of the viral mRNAs were multiply spliced, and of these, less than 1% excluded exon 3. Deletion of the PRE resulted in a decrease in the relative amount of multiply spliced mRNA to about 40% of the total and approximately 39% of the viral mRNA excluded exon 3. Ectopic expression of ERev caused a decrease in the relative amount of multiply spliced mRNA to approximately 50% of the total and increased mRNAs that excluded exon 3 to about 4%. Over-expression of SF2/ASF in cells transfected with wild-type provirus constructs inhibited splicing but did not significantly alter exon 3 skipping.
Liao
Huey-Jane
HJ
Basic Research Program, SAIC-Frederick, Frederick, MD 21702, USA.
Baker
Carl C
CC
Princler
Gerald L
GL
Derse
David
D
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D004742:Enhancer Elements, Genetic; D005091:Exons; D015967:Gene Expression Regulation, Viral; D015694:Gene Products, rev / Q000235:genetics / Q000378:metabolism; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D011687:Purines / Q000737:chemistry; D012322:RNA Precursors; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors
D015694:Gene Products, rev; D015696:Gene Products, tat; D009687:Nuclear Proteins; D011687:Purines; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors