Capping of vesicular stomatitis virus pre-mRNA is required for accurate selection of transcription stop-start sites and virus propagation.
Bottom Line: Here, the effects of cap-defective mutations in the HR motif on transcription were analyzed using an in vitro reconstituted transcription system.Cap-defective mutants efficiently produced the leader RNA, but displayed aberrant stop-start transcription using cryptic termination and initiation signals within the first gene, resulting in sequential generation of ∼40-nucleotide transcripts with 5'-ATP from a correct mRNA-start site followed by a 28-nucleotide transcript and long 3'-polyadenylated transcript initiated with non-canonical GTP from atypical start sites.Frequent transcription termination and re-initiation within the first gene significantly attenuated the production of downstream mRNAs.
Affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA firstname.lastname@example.org.Show MeSH
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Mentions: In order to determine the presence or absence of the 3′-poly(A) tail on long transcripts synthesized by the cap-defective mutants, sizes of their transcripts were compared with those after deadenylation with RNase H in the presence of oligo(dT) (Figure 6A and B). Untreated mRNAs synthesized by the wild-type L protein migrated as a broad smear (Figure 6A, lanes 1), while deadenylated mRNAs were separated into three bands with 1.3 (N), 0.8 (P/M) and 1.7 (G) knt (Figure 6A, lanes 2), indicating that these wild-type mRNAs were co-transcriptionally polyadenylated as reported (40). Similarly, as shown in lanes 3–12, long transcripts with heterogeneous lengths (>0.8 knt) synthesized by these HR and R1221 mutants were found to be digested with RNase H in the presence of oligo(dT) into discrete RNA species (e.g. N1, N2, P). In contrast, sizes of short transcripts including the leader RNA, N1–40, N1–38 and N41–68 synthesized by the mutants were not different before and after RNase H treatment in the presence of oligo(dT) (Figure 6B). Thus, it was shown that mRNAs synthesized by the cap-defective mutants are efficiently polyadenylated during transcription.
Affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA email@example.com.