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Capping of vesicular stomatitis virus pre-mRNA is required for accurate selection of transcription stop-start sites and virus propagation.

Ogino T - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA tomoaki.ogino@case.edu.

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The cap-defective mutant L proteins polyadenylate N1 and N2 RNAs at their 3′-ends. (A, B) 32P-labeled transcripts, synthesized by the wild-type (WT) or mutant L protein, were treated with or without RNase H and oligo(dT) and analyzed by 5% (A) or 15% (B) urea-PAGE followed by autoradiography. In panel (A), lanes 1 and 2, the sample volume of transcripts synthesized by the WT L protein for the urea-PAGE analysis was 20-fold smaller than those of transcripts synthesized by the other mutants. (C, D) N1 (N mRNA) and/or N2 RNAs, synthesized by either the WT or HR-RH mutant L protein, were cleaved 200 nt upstream of the polyadenylation site with RNase H in the presence of an oligo-DNA complementary to positions +1107 to +1126 of N mRNA [(−)N1107–1126]. The resulting 3′-end fragments with poly(A) tails were detected by northern blotting with a 32P-labeled antisense oligo-DNA [(−)N1241–1268] [(D) lanes 3 and 7]. To estimate lengths of poly(A) tails on the 3′-end fragments (0.2 knt) of N1 and N2 RNAs, the positions of the marker RNAs (see the M lane) are shown on the right with lengths calculated by subtracting 0.2 knt from their actual lengths (D). Poly(A) tails were digested with RNase H in the presence of oligo(dT) with (lanes 4 and 8) or without (lanes 2 and 6) the (−)N1107–1126 oligo-DNA.
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Figure 6: The cap-defective mutant L proteins polyadenylate N1 and N2 RNAs at their 3′-ends. (A, B) 32P-labeled transcripts, synthesized by the wild-type (WT) or mutant L protein, were treated with or without RNase H and oligo(dT) and analyzed by 5% (A) or 15% (B) urea-PAGE followed by autoradiography. In panel (A), lanes 1 and 2, the sample volume of transcripts synthesized by the WT L protein for the urea-PAGE analysis was 20-fold smaller than those of transcripts synthesized by the other mutants. (C, D) N1 (N mRNA) and/or N2 RNAs, synthesized by either the WT or HR-RH mutant L protein, were cleaved 200 nt upstream of the polyadenylation site with RNase H in the presence of an oligo-DNA complementary to positions +1107 to +1126 of N mRNA [(−)N1107–1126]. The resulting 3′-end fragments with poly(A) tails were detected by northern blotting with a 32P-labeled antisense oligo-DNA [(−)N1241–1268] [(D) lanes 3 and 7]. To estimate lengths of poly(A) tails on the 3′-end fragments (0.2 knt) of N1 and N2 RNAs, the positions of the marker RNAs (see the M lane) are shown on the right with lengths calculated by subtracting 0.2 knt from their actual lengths (D). Poly(A) tails were digested with RNase H in the presence of oligo(dT) with (lanes 4 and 8) or without (lanes 2 and 6) the (−)N1107–1126 oligo-DNA.

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.


Capping of vesicular stomatitis virus pre-mRNA is required for accurate selection of transcription stop-start sites and virus propagation.

Ogino T - Nucleic Acids Res. (2014)

The cap-defective mutant L proteins polyadenylate N1 and N2 RNAs at their 3′-ends. (A, B) 32P-labeled transcripts, synthesized by the wild-type (WT) or mutant L protein, were treated with or without RNase H and oligo(dT) and analyzed by 5% (A) or 15% (B) urea-PAGE followed by autoradiography. In panel (A), lanes 1 and 2, the sample volume of transcripts synthesized by the WT L protein for the urea-PAGE analysis was 20-fold smaller than those of transcripts synthesized by the other mutants. (C, D) N1 (N mRNA) and/or N2 RNAs, synthesized by either the WT or HR-RH mutant L protein, were cleaved 200 nt upstream of the polyadenylation site with RNase H in the presence of an oligo-DNA complementary to positions +1107 to +1126 of N mRNA [(−)N1107–1126]. The resulting 3′-end fragments with poly(A) tails were detected by northern blotting with a 32P-labeled antisense oligo-DNA [(−)N1241–1268] [(D) lanes 3 and 7]. To estimate lengths of poly(A) tails on the 3′-end fragments (0.2 knt) of N1 and N2 RNAs, the positions of the marker RNAs (see the M lane) are shown on the right with lengths calculated by subtracting 0.2 knt from their actual lengths (D). Poly(A) tails were digested with RNase H in the presence of oligo(dT) with (lanes 4 and 8) or without (lanes 2 and 6) the (−)N1107–1126 oligo-DNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 6: The cap-defective mutant L proteins polyadenylate N1 and N2 RNAs at their 3′-ends. (A, B) 32P-labeled transcripts, synthesized by the wild-type (WT) or mutant L protein, were treated with or without RNase H and oligo(dT) and analyzed by 5% (A) or 15% (B) urea-PAGE followed by autoradiography. In panel (A), lanes 1 and 2, the sample volume of transcripts synthesized by the WT L protein for the urea-PAGE analysis was 20-fold smaller than those of transcripts synthesized by the other mutants. (C, D) N1 (N mRNA) and/or N2 RNAs, synthesized by either the WT or HR-RH mutant L protein, were cleaved 200 nt upstream of the polyadenylation site with RNase H in the presence of an oligo-DNA complementary to positions +1107 to +1126 of N mRNA [(−)N1107–1126]. The resulting 3′-end fragments with poly(A) tails were detected by northern blotting with a 32P-labeled antisense oligo-DNA [(−)N1241–1268] [(D) lanes 3 and 7]. To estimate lengths of poly(A) tails on the 3′-end fragments (0.2 knt) of N1 and N2 RNAs, the positions of the marker RNAs (see the M lane) are shown on the right with lengths calculated by subtracting 0.2 knt from their actual lengths (D). Poly(A) tails were digested with RNase H in the presence of oligo(dT) with (lanes 4 and 8) or without (lanes 2 and 6) the (−)N1107–1126 oligo-DNA.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA tomoaki.ogino@case.edu.

Show MeSH
Related in: MedlinePlus