Faculty Collaboration Database

Characterization of Rous sarcoma virus polyadenylation site use in vitro. Virology 2008 May 10;374(2):468-76

Date

02/15/2008

Pubmed ID

18272196

Pubmed Central ID

PMC2413101

DOI

10.1016/j.virol.2008.01.012

Scopus ID

2-s2.0-42649140232 (requires institutional sign-in at Scopus site)   11 Citations

Abstract

Polyadenylation of Rous sarcoma virus (RSV) RNA is inefficient, as approximately 15% of RSV RNAs represent read-through transcripts that use a downstream cellular polyadenylation site (poly(A) site). Read-through transcription has implications for the virus and the host since it is associated with oncogene capture and tumor induction. To explore the basis of inefficient RSV RNA 3'-end formation, we characterized RSV polyadenylation in vitro using HeLa cell nuclear extracts and HEK293 whole cell extracts. RSV polyadenylation substrates composed of the natural 3' end of viral RNA and various lengths of upstream sequence showed little or no polyadenylation, indicating that the RSV poly(A) site is suboptimal. Efficiently used poly(A) sites often have identifiable upstream and downstream elements (USEs and DSEs) in close proximity to the conserved AAUAAA signal. The sequences upstream and downstream of the RSV poly(A) site deviate from those found in efficiently used poly(A) sites, which may explain inefficient RSV polyadenylation. To assess the quality of the RSV USEs and DSEs, the well-characterized SV40 late USEs and/or DSEs were substituted for the RSV elements and vice versa, which showed that the USEs and DSEs from RSV are suboptimal but functional. CstF interacted poorly with the RSV polyadenylation substrate, and the inactivity of the RSV poly(A) site was at least in part due to poor CstF binding since tethering CstF to the RSV substrate activated polyadenylation. Our data are consistent with poor polyadenylation factor binding sites in both the USE and DSE as the basis for inefficient use of the RSV poly(A) site and point to the importance of additional elements within RSV RNA in promoting 3' end formation.

Author List

Maciolek NL, McNally MT

Author

Mark T. McNally PhD Associate Professor in the Microbiology and Immunology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

3' Untranslated Regions
Binding Sites
Cell Line
Cleavage Stimulation Factor
Enhancer Elements, Genetic
HeLa Cells
Humans
Polyadenylation
RNA Splicing
RNA, Messenger
RNA, Viral
Rous sarcoma virus
Substrate Specificity