A noncanonical poly(A) signal, UAUAAA, and flanking elements in Epstein-Barr virus DNA polymerase mRNA function in cleavage and polyadenylation assays.
Two forms of the Epstein-Barr virus DNA polymerase (pol) mRNA (3.7 and 5.1 kb) have been detected, neither of which contains a canonical poly(A) signal. The 5.1-kb pol mRNA, which contains a rare poly(A) signal, UAUAAA, studied only in transcripts of Hepadnaviridae and a plant pararetrovirus, was analyzed in cleavage and polyadenylation assays. Incubation of the pol transcript in cell extracts produced relatively low efficiency of cleavage (12 to 14%), which was improved by conversion of the poly(A) signal to AAUAAA. Deletion of the UAUAAA signal abolished cleavage and polyadenylation. An auxiliary element, UUUGUA, 3-8 nt upstream of the poly(A) signal and two downstream core elements, a GU-rich sequence 36-46 nt, and an AUUUGUGU sequence 47-53 nt downstream of the signal (8-19 nt and 20-28 nt downstream of cleavage site) facilitated processing of pol mRNA. Replacement of sequences near the cleavage/poly(A) site affected cleavage accuracy. Binding of the 64-kDa cleavage stimulatory factor to the U-rich as well as the GU-rich elements correlated with cleavage efficiency. Thus the UAUAAA hexanucleotide plus the other cis-acting elements are clearly functional in the native pol mRNA, but are relatively inefficient. Implications of the use of an anomalous poly(A) signal and its elements are discussed.
Silver Key
S C
SC
Department of Microbiology, UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 27599, USA.
Pagano
J S
JS
D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells; D004854:Herpesvirus 4, Human / Q000201:enzymology*; D006801:Humans; D011061:Poly A / Q000378:metabolism*; D011233:Precipitin Tests; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D011061:Poly A; C083202:BALF5 protein, Epstein-barr virus; D004259:DNA-Directed DNA Polymerase