Distance-Dependent Processing of Adeno-Associated Virus Type 5 RNA Is Controlled by 5′ Exon Definition
Open Access
- 1 August 2007
- journal article
- research article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 81 (15), 7974-7984
- https://doi.org/10.1128/jvi.00714-07
Abstract
Adeno-associated virus type 5 (AAV5) is unique among human AAV serotypes in that it uses a polyadenylation site [(pA)p] within the single small intron in the center of the genome. We previously reported that inhibition of polyadenylation at (pA)p, necessary for read-through of P41-generated capsid gene pre-mRNAs which are subsequently spliced, requires binding of U1 snRNP to the upstream donor. Inhibition was reduced as the distance between the cap site and the donor was increased (increasing the size of the 5′ exon). Here, we have demonstrated that U1-70K is a key component of U1 snRNP that mediates inhibition of polyadenylation at (pA)p. Furthermore, introduction of a U-rich stretch, predicted to target TIA-1 and thus increase the affinity of U1 snRNP binding to the intervening donor site, significantly augmented inhibition of (pA)p, while depletion of TIA-1 by siRNA increased (pA)p read-through. Finally, artificially tethering the cap binding complex (CBC) components CBP80 and CBP20 upstream of the intron donor increased inhibition of polyadenylation at (pA)p. Our results suggest that interaction with the CBC strengthens U1 snRNP binding to the downstream intron donor in a manner inversely proportional to the size of the 5′ exon, thus governing the competition between intron splicing and polyadenylation at (pA)p. This competition must be optimized to program both the levels of polyadenylation of P7- and P19-generated RNA at (pA)p required to produce proper levels of the essential Rep proteins and the splicing of P41-generated RNAs to produce the proper ratio of capsid proteins during AAV5 infection.Keywords
This publication has 63 references indexed in Scilit:
- The Structure and Biochemical Properties of the Human Spliceosomal Protein U1CJournal of Molecular Biology, 2004
- Alternative Polyadenylation of Adeno-associated Virus Type 5 RNA within an Internal Intron Is Governed by the Distance between the Promoter and the Intron and Is Inhibited by U1 Small Nuclear RNP Binding to the Intervening DonorJournal of Biological Chemistry, 2004
- Splicing of a Cap-proximal Human Papillomavirus 16 E6E7 Intron Promotes E7 Expression, but can be Restrained by Distance of the Intron from its RNA 5′ CapJournal of Molecular Biology, 2004
- Alternative Polyadenylation of Adeno-Associated Virus Type 5 RNA within an Internal Intron Is Governed by both a Downstream Element within the Intron 3′ Splice Acceptor and an Element Upstream of the P41 Initiation SiteJournal of Virology, 2004
- U1 snRNP-Dependent Function of TIAR in the Regulation of Alternative RNA Processing of the Human Calcitonin/CGRP Pre-mRNAMolecular and Cellular Biology, 2003
- Characterization of the Transcription Profile of Adeno-Associated Virus Type 5 Reveals a Number of Unique Features Compared to Previously Characterized Adeno-Associated VirusesJournal of Virology, 2002
- TIA-1 and TIAR Activate Splicing of Alternative Exons with Weak 5′ Splice Sites followed by a U-rich Stretch on Their Own Pre-mRNAsJournal of Biological Chemistry, 2001
- The human U1A snRNP protein regulates polyadenylation via a direct interaction with poly(A) polymeraseCell, 1994
- Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicingCell, 1989
- A compensatory base change in U1 snRNA suppresses a 5′ splice site mutationCell, 1986