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Journal of Virology, August 2007, p. 7974-7984, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.00714-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Distance-Dependent Processing of Adeno-Associated Virus Type 5 RNA Is Controlled by 5' Exon Definition

Jianming Qiu, Fang Cheng, and David Pintel^*

Department of Molecular Microbiology and Immunology, University of Missouri—Columbia, School of Medicine, Life Sciences Center, Columbia, Missouri 65212

Received 3 April 2007/ Accepted 8 May 2007

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.

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* Corresponding author. Mailing address: 471f Life Sciences Center, University of Missouri—Columbia, 1201 Rollins Rd., Columbia, MO 65212. Phone: (573) 882-3920. Fax: (573) 884-9676. E-mail: pinteld{at}missouri.edu

Published ahead of print on 16 May 2007.

Present address: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160.

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Journal of Virology, August 2007, p. 7974-7984, Vol. 81, No. 15
0022-538X/07/$08.00+0     doi:10.1128/JVI.00714-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Li, L., Qiu, J., Pintel, D. J. (2009). The Choice of Translation Initiation Site of the Rep Proteins from Goose Parvovirus P9-Generated mRNA Is Governed by Splicing and the Nature of the Excised Intron. J. Virol. 83: 10264-10268 [Abstract] [Full Text]  
• Guan, W., Cheng, F., Yoto, Y., Kleiboeker, S., Wong, S., Zhi, N., Pintel, D. J., Qiu, J. (2008). Block to the Production of Full-Length B19 Virus Transcripts by Internal Polyadenylation Is Overcome by Replication of the Viral Genome. J. Virol. 82: 9951-9963 [Abstract] [Full Text]