Abstract
The equine infectious anemia virus (EIAV) Rev protein (ERev) negatively regulates its own synthesis by inducing alternative splicing of its mRNA. This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. When Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. The interaction of ERev with its cis-acting RNA response element, the RRE, is also essential for nuclear export of intron-containing viral mRNAs that encode structural and enzymatic gene products. The primary ERev binding site and the manner in which ERev interacts with RNA or cellular proteins to exert its regulatory function have not been defined. We have performed in vitro RNA binding experiments to show that recombinant ERev binds to a 55-nucleotide, purine-rich tract proximal to the 5' splice site of exon 3. Because of its proximity to the 5' splice site and since it contains elements related to consensus exonic splicing enhancer sequences, we asked whether cellular proteins recognize the EIAV RRE. The cellular protein, ASF/SF2, a member of the serine- and arginine-rich family of splicing factors (SR proteins) bound to repeated sequences within the 55-nucleotide RRE region. Electrophoretic mobility shift and UV cross-linking experiments indicated that ERev and SR proteins bind simultaneously to the RRE. Furthermore, in vitro protein-protein interaction studies revealed an association between ERev and SR proteins. These data suggest that EIAV Rev-induced exon skipping observed in vivo may be initiated by simultaneous binding of Rev and SR proteins to the RRE that alter the subsequent assembly or catalytic activity of the spliceosomal complex.
Highlights
The Rev and Rex proteins encoded by lentiviruses and other complex retroviruses mediate the nucleocytoplasmic transport of intron-containing, viral pre-mRNAs that would otherwise be retained in the nucleus [1,2,3,4]
In this report we have identified the binding site for equine infectious anemia virus (EIAV) Rev and show that it is located in a 55-nucleotide region proximal to the 5Ј splice site of exon 3 RNA
Exon 3 RNAs that contained the 55-nucleotide purine-rich tract formed a higher order intermolecular structure and it was this form of RNA, which was bound by EIAV Rev (ERev) in vitro
Summary
Vol 276, No 22, Issue of June 1, pp. 18960 –18967, 2001 Printed in U.S.A. Binding Sites for Rev and ASF/SF2 Map to a 55-Nucleotide Purinerich Exonic Element in Equine Infectious Anemia Virus RNA*. The interaction of ERev with its cis-acting RNA response element, the RRE, is essential for nuclear export of intron-containing viral mRNAs that encode structural and enzymatic gene products. The Rev and Rex proteins encoded by lentiviruses and other complex retroviruses mediate the nucleocytoplasmic transport of intron-containing, viral pre-mRNAs that would otherwise be retained in the nucleus [1,2,3,4] In addition to their roles in RNA transport, some members of this protein family have been shown to affect pre-mRNA splicing [5,6,7,8]. A 255-nucleotide stem-loop structure in the 3Ј long terminal repeat of HTLV-1 functions as the RxRE and contains a 33-nucleotide element, which represents the primary binding site for Rex [24]. ERev and ASF/SF2 interactions with this composite RNA element were not mutually exclusive, but rather cell and virus proteins appeared to bind simultaneously
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