Mutational analysis of a viral RNA element that counteracts rapid RNA decay by interaction with the polyadenylate tail

Nicholas K. Conrad, Mei Di Shu, Katherine E. Uyhazi, Joan A. Steitz

Research output: Contribution to journalArticle

40 Scopus citations

Abstract

We previously demonstrated that the Kaposi's sarcoma-associated herpesvirus polyadenylated nuclear RNA contains a 79-nt cis-acting element, the ENE, which allows intronless polyadenylated transcripts to accumulate to high nuclear levels by protecting them from rapid degradation. We proposed a model based on the predicted structure of the ENE in which a U-rich internal loop hybridizes with the 3′-polyadenylate (polyA) tail to sequester it from exonucleolytic attack. We have tested this model by mutational analysis of the ENE. Point mutations in the predicted U-rich internal loop and in the flanking stems abolish the ENE's ability to (i) interact with the polyA tail, (ii) inhibit deadenylation in vitro, and (iii) stabilize transcripts in vivo. In all but one case, compensatory mutations in the flanking stems restore ENE activities, demonstrating the importance of these stems and uncovering a unique role for the loop-proximal G-C base pair in the lower stem. Increasing the U content of the U-rich internal loop surprisingly decreases stability in vivo but does not affect deadenylation in vitro, comparable to the effects of deleting certain "unstructured" regions of the ENE. Taken together, our data support the formation of the proposed ENE secondary structure in vivo and argue that the specific ENE structure inhibits rapid RNA decay in cis by engaging in a limited set of base-pairing interactions with the polyA tail.

Original languageEnglish (US)
Pages (from-to)10412-10417
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number25
DOIs
StatePublished - Jun 19 2007

Keywords

  • Deadenylation
  • Polyadenylated nuclear RNA
  • Polyadenylation
  • RNA degradation
  • RNA structure

ASJC Scopus subject areas

  • General

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