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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 mutations in the L protein cause aberrant stop–start transcription. The cap-defective mutant L protein (lower) as well as the wild-type L protein (upper) together with the P protein synthesizes the leader RNA from the 3′-end of the genomic RNA. After synthesis of the leader RNA, the wild-type L protein productively synthesizes 5′-capped and 3′-polyadenylated N mRNA using the gene-start and gene-end sequences as transcription initiation and termination/polyadenylation signals, respectively. In contrast, the cap-defective mutant L proteins (e.g. HR-RH, H1227R, R1228H) frequently use cryptic transcription signals within the N gene, resulting in the generation of large amounts of 5′-uncapped short transcripts (e.g. N1–40, N41–68) and a small amount of 5′-uncapped and 3′-polyadenylated N2 RNA. GDN and HR indicate active site motifs for the RNA-dependent RNA polymerase (RdRp) and polyribonucleotidyltransferase (PRNTase) domains, respectively.
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Figure 7: The cap-defective mutations in the L protein cause aberrant stop–start transcription. The cap-defective mutant L protein (lower) as well as the wild-type L protein (upper) together with the P protein synthesizes the leader RNA from the 3′-end of the genomic RNA. After synthesis of the leader RNA, the wild-type L protein productively synthesizes 5′-capped and 3′-polyadenylated N mRNA using the gene-start and gene-end sequences as transcription initiation and termination/polyadenylation signals, respectively. In contrast, the cap-defective mutant L proteins (e.g. HR-RH, H1227R, R1228H) frequently use cryptic transcription signals within the N gene, resulting in the generation of large amounts of 5′-uncapped short transcripts (e.g. N1–40, N41–68) and a small amount of 5′-uncapped and 3′-polyadenylated N2 RNA. GDN and HR indicate active site motifs for the RNA-dependent RNA polymerase (RdRp) and polyribonucleotidyltransferase (PRNTase) domains, respectively.

Mentions: The cap-defective mutations in the HR motif or its adjacent R1221 residue of the VSV L protein significantly decreased the accuracy of selection of transcription termination and initiation signals for mRNA synthesis during in vitro transcription together with the P protein, leading to sequential production of non-overlapping 5′-triphosphorylated N mRNA fragments, such as N1–40, N41–68 and N2 (3′-polyadenylated N157–1326) RNAs, as depicted in Figure 7. In the presence of the P protein, the wild-type L protein produced the leader RNA and N mRNA at a molar ratio of 1:0.4 under the standard transcription conditions, whereas the HR-RH mutant synthesized the leader, N1–38/N1–40, N41–68, N1 (N mRNA) and N2 RNAs at a molar ratio of 1:0.7:0.3:0.02:0.01. Erroneous transcription termination and re-initiation using the cryptic signals within the N gene at higher frequencies resulted in a marked decrease in full-length N mRNA synthesis and attenuation of downstream mRNA production.


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 mutations in the L protein cause aberrant stop–start transcription. The cap-defective mutant L protein (lower) as well as the wild-type L protein (upper) together with the P protein synthesizes the leader RNA from the 3′-end of the genomic RNA. After synthesis of the leader RNA, the wild-type L protein productively synthesizes 5′-capped and 3′-polyadenylated N mRNA using the gene-start and gene-end sequences as transcription initiation and termination/polyadenylation signals, respectively. In contrast, the cap-defective mutant L proteins (e.g. HR-RH, H1227R, R1228H) frequently use cryptic transcription signals within the N gene, resulting in the generation of large amounts of 5′-uncapped short transcripts (e.g. N1–40, N41–68) and a small amount of 5′-uncapped and 3′-polyadenylated N2 RNA. GDN and HR indicate active site motifs for the RNA-dependent RNA polymerase (RdRp) and polyribonucleotidyltransferase (PRNTase) domains, respectively.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 7: The cap-defective mutations in the L protein cause aberrant stop–start transcription. The cap-defective mutant L protein (lower) as well as the wild-type L protein (upper) together with the P protein synthesizes the leader RNA from the 3′-end of the genomic RNA. After synthesis of the leader RNA, the wild-type L protein productively synthesizes 5′-capped and 3′-polyadenylated N mRNA using the gene-start and gene-end sequences as transcription initiation and termination/polyadenylation signals, respectively. In contrast, the cap-defective mutant L proteins (e.g. HR-RH, H1227R, R1228H) frequently use cryptic transcription signals within the N gene, resulting in the generation of large amounts of 5′-uncapped short transcripts (e.g. N1–40, N41–68) and a small amount of 5′-uncapped and 3′-polyadenylated N2 RNA. GDN and HR indicate active site motifs for the RNA-dependent RNA polymerase (RdRp) and polyribonucleotidyltransferase (PRNTase) domains, respectively.
Mentions: The cap-defective mutations in the HR motif or its adjacent R1221 residue of the VSV L protein significantly decreased the accuracy of selection of transcription termination and initiation signals for mRNA synthesis during in vitro transcription together with the P protein, leading to sequential production of non-overlapping 5′-triphosphorylated N mRNA fragments, such as N1–40, N41–68 and N2 (3′-polyadenylated N157–1326) RNAs, as depicted in Figure 7. In the presence of the P protein, the wild-type L protein produced the leader RNA and N mRNA at a molar ratio of 1:0.4 under the standard transcription conditions, whereas the HR-RH mutant synthesized the leader, N1–38/N1–40, N41–68, N1 (N mRNA) and N2 RNAs at a molar ratio of 1:0.7:0.3:0.02:0.01. Erroneous transcription termination and re-initiation using the cryptic signals within the N gene at higher frequencies resulted in a marked decrease in full-length N mRNA synthesis and attenuation of downstream mRNA production.

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