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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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Cap-defective mutations in the HR motif of the L protein impair mRNA synthesis, but not leader RNA synthesis. The wild-type (WT) and HR-RH mutant L proteins were subjected to transcription reactions reconstituted with the N-RNA template and P protein. The HR-RH mutant possesses H1227R and R1228H mutations. 32P-labeled transcripts were deadenylated with RNase H in the presence of oligo(dT) and analyzed by 5% [(A) for the mRNAs] or 20% urea-PAGE [(B) for the leader RNA] followed by autoradiography. Lane 1 indicates no L protein. The positions of the viral mRNAs (G, N and P/M), unknown 1.2-knt RNA, leader RNA (Le), unknown short RNAs (I, II and III) and gel origins (ori.) are indicated. The lower panels show relative RNA synthesis activities of the WT (defined as 100%) and mutant L proteins. Columns and error bars represent the means and standard deviations, respectively, from three independent experiments.
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Figure 1: Cap-defective mutations in the HR motif of the L protein impair mRNA synthesis, but not leader RNA synthesis. The wild-type (WT) and HR-RH mutant L proteins were subjected to transcription reactions reconstituted with the N-RNA template and P protein. The HR-RH mutant possesses H1227R and R1228H mutations. 32P-labeled transcripts were deadenylated with RNase H in the presence of oligo(dT) and analyzed by 5% [(A) for the mRNAs] or 20% urea-PAGE [(B) for the leader RNA] followed by autoradiography. Lane 1 indicates no L protein. The positions of the viral mRNAs (G, N and P/M), unknown 1.2-knt RNA, leader RNA (Le), unknown short RNAs (I, II and III) and gel origins (ori.) are indicated. The lower panels show relative RNA synthesis activities of the WT (defined as 100%) and mutant L proteins. Columns and error bars represent the means and standard deviations, respectively, from three independent experiments.

Mentions: It has been previously shown that the capping activity of the VSV L protein is abolished by mutations in the conserved HR motif (H1227 and R1228) in the step of the L-pRNA intermediate formation (6). Here, to explore the effects of these cap-defective mutations in the VSV L protein on RNA synthesis, transcription reactions were reconstituted with the N–RNA complex, the recombinant P protein and the wild-type or mutant L protein. Internally 32P-labeled transcripts were deadenylated and analyzed by 5% (for mRNAs) or 20% (for the leader RNA) urea-PAGE. Complete results with the previously prepared recombinant L proteins with mutations in the HR motif (called HR mutants) or other residues are available in Supplementary Figure S1. Figure 1 shows a typical result using the HR-RH mutant, which possesses an RH sequence instead of the HR motif (6) and exhibits the highest RNA synthesis activity among the HR mutants, as a representative. As reported (40), the wild-type L protein synthesized 1.7-knt G, 1.3-knt N and 0.8-knt P/M mRNAs (Figure 1A, lane 2) and the leader RNA with ∼50 nt (Figure 1B, lane 2). In contrast, the HR-RH mutant showed an extremely weak mRNA synthesis activity (5% of the wild-type activity) (Figure 1A, lane 3), although its leader RNA synthesis activity was comparable to that of the wild-type L protein (Figure 1B, lane 3). Other HR mutants also showed similar transcription phenotypes to that of the HR-RH mutant (see Supplementary Figure S1B and C). These results indicate that the HR motif is not essential for leader RNA synthesis, but it appears to be required for efficient mRNA synthesis. Interestingly, all these cap-defective HR mutants were found to produce characteristic short RNAs (bands I and II, ∼40 nt; band III, ∼30 nt) and 1.2-knt RNA beside 1.3-knt N mRNA-like RNA (Figure 1 and Supplementary Figure S1).


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)

Cap-defective mutations in the HR motif of the L protein impair mRNA synthesis, but not leader RNA synthesis. The wild-type (WT) and HR-RH mutant L proteins were subjected to transcription reactions reconstituted with the N-RNA template and P protein. The HR-RH mutant possesses H1227R and R1228H mutations. 32P-labeled transcripts were deadenylated with RNase H in the presence of oligo(dT) and analyzed by 5% [(A) for the mRNAs] or 20% urea-PAGE [(B) for the leader RNA] followed by autoradiography. Lane 1 indicates no L protein. The positions of the viral mRNAs (G, N and P/M), unknown 1.2-knt RNA, leader RNA (Le), unknown short RNAs (I, II and III) and gel origins (ori.) are indicated. The lower panels show relative RNA synthesis activities of the WT (defined as 100%) and mutant L proteins. Columns and error bars represent the means and standard deviations, respectively, from three independent experiments.
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Related In: Results  -  Collection

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Figure 1: Cap-defective mutations in the HR motif of the L protein impair mRNA synthesis, but not leader RNA synthesis. The wild-type (WT) and HR-RH mutant L proteins were subjected to transcription reactions reconstituted with the N-RNA template and P protein. The HR-RH mutant possesses H1227R and R1228H mutations. 32P-labeled transcripts were deadenylated with RNase H in the presence of oligo(dT) and analyzed by 5% [(A) for the mRNAs] or 20% urea-PAGE [(B) for the leader RNA] followed by autoradiography. Lane 1 indicates no L protein. The positions of the viral mRNAs (G, N and P/M), unknown 1.2-knt RNA, leader RNA (Le), unknown short RNAs (I, II and III) and gel origins (ori.) are indicated. The lower panels show relative RNA synthesis activities of the WT (defined as 100%) and mutant L proteins. Columns and error bars represent the means and standard deviations, respectively, from three independent experiments.
Mentions: It has been previously shown that the capping activity of the VSV L protein is abolished by mutations in the conserved HR motif (H1227 and R1228) in the step of the L-pRNA intermediate formation (6). Here, to explore the effects of these cap-defective mutations in the VSV L protein on RNA synthesis, transcription reactions were reconstituted with the N–RNA complex, the recombinant P protein and the wild-type or mutant L protein. Internally 32P-labeled transcripts were deadenylated and analyzed by 5% (for mRNAs) or 20% (for the leader RNA) urea-PAGE. Complete results with the previously prepared recombinant L proteins with mutations in the HR motif (called HR mutants) or other residues are available in Supplementary Figure S1. Figure 1 shows a typical result using the HR-RH mutant, which possesses an RH sequence instead of the HR motif (6) and exhibits the highest RNA synthesis activity among the HR mutants, as a representative. As reported (40), the wild-type L protein synthesized 1.7-knt G, 1.3-knt N and 0.8-knt P/M mRNAs (Figure 1A, lane 2) and the leader RNA with ∼50 nt (Figure 1B, lane 2). In contrast, the HR-RH mutant showed an extremely weak mRNA synthesis activity (5% of the wild-type activity) (Figure 1A, lane 3), although its leader RNA synthesis activity was comparable to that of the wild-type L protein (Figure 1B, lane 3). Other HR mutants also showed similar transcription phenotypes to that of the HR-RH mutant (see Supplementary Figure S1B and C). These results indicate that the HR motif is not essential for leader RNA synthesis, but it appears to be required for efficient mRNA synthesis. Interestingly, all these cap-defective HR mutants were found to produce characteristic short RNAs (bands I and II, ∼40 nt; band III, ∼30 nt) and 1.2-knt RNA beside 1.3-knt N mRNA-like RNA (Figure 1 and Supplementary Figure S1).

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