Mutational analysis of a viral RNA element that counteracts rapid RNA decay by interaction with the polyadenylate tail.
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.
Conrad
Nicholas K
NK
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, New Haven, CT 06536, USA.
Shu
Mei-Di
MD
Uyhazi
Katherine E
KE
Steitz
Joan A
JA
D020029:Base Pairing; D001483:Base Sequence; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011061:Poly A / Q000378:metabolism*; D026723:Polyadenylation; D020871:RNA Stability; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism*; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*
D012367:RNA, Viral; D038621:Regulatory Sequences, Ribonucleic Acid; D011061:Poly A