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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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Related in: MedlinePlus

Cap-defective mutant L proteins produce large amounts of short N mRNA fragments including N1–40 and N41–68 RNAs. As illustrated in panel (A), short transcripts, synthesized by the wild-type (WT) or mutant L protein, were analyzed by northern blotting to detect the leader RNA (B), N1–40 RNA (C) and N41–68 RNA (D) with 32P-labeled oligo-DNA probes complementary to their 5′-terminal 28-nt sequences. Lane 1 indicates no L protein. Putative N1–28 and N1–30 RNAs are marked by arrowheads (C). Asterisks represent unidentified transcripts (∼70 nt) detected with the (−)N41–68 probe (D).
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Figure 3: Cap-defective mutant L proteins produce large amounts of short N mRNA fragments including N1–40 and N41–68 RNAs. As illustrated in panel (A), short transcripts, synthesized by the wild-type (WT) or mutant L protein, were analyzed by northern blotting to detect the leader RNA (B), N1–40 RNA (C) and N41–68 RNA (D) with 32P-labeled oligo-DNA probes complementary to their 5′-terminal 28-nt sequences. Lane 1 indicates no L protein. Putative N1–28 and N1–30 RNAs are marked by arrowheads (C). Asterisks represent unidentified transcripts (∼70 nt) detected with the (−)N41–68 probe (D).

Mentions: In order to investigate whether other cap-defective mutants produce N1–40, N1–38 and N41–68 RNAs in addition to the leader RNA, short transcripts synthesized by selected mutants (H1227R, R1228H, HR-RH, R1221A and R1221K) were analyzed by northern blotting with 32P-labeled oligo-DNA probes complementary to the 5′-terminal 28 nt of the leader RNA, N1–40 and N41–68 [designated as (−)Le1–28, (−)N1–28 and (−)N41–68, respectively] (Figure 3A). Although these cap-defective mutants exhibited leader RNA synthesis activities comparable to or lower than that of the wild-type L protein (Figure 3B; see Supplementary Figure S1C), they produced significantly higher amounts of N1–40, N1–38 (Figure 3C, lanes 3–7) and N41–68 (Figure 3D, lanes 3–7) than the wild-type L protein (Figure 3C and D, lane 2). Note that these mutants also yielded unidentified minor products including 28–30-nt RNAs and ∼70-nt RNAs, which were detected with the (−)N1–28 (Figure 3C, marked by arrowheads) and (−)N41–68 (Figure 3D, marked by asterisks) probes, respectively. These results indicate that the cap-defective mutations cause aberrant stop–start transcription to produce short RNAs using cryptic transcription signals in the N gene.


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 mutant L proteins produce large amounts of short N mRNA fragments including N1–40 and N41–68 RNAs. As illustrated in panel (A), short transcripts, synthesized by the wild-type (WT) or mutant L protein, were analyzed by northern blotting to detect the leader RNA (B), N1–40 RNA (C) and N41–68 RNA (D) with 32P-labeled oligo-DNA probes complementary to their 5′-terminal 28-nt sequences. Lane 1 indicates no L protein. Putative N1–28 and N1–30 RNAs are marked by arrowheads (C). Asterisks represent unidentified transcripts (∼70 nt) detected with the (−)N41–68 probe (D).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231761&req=5

Figure 3: Cap-defective mutant L proteins produce large amounts of short N mRNA fragments including N1–40 and N41–68 RNAs. As illustrated in panel (A), short transcripts, synthesized by the wild-type (WT) or mutant L protein, were analyzed by northern blotting to detect the leader RNA (B), N1–40 RNA (C) and N41–68 RNA (D) with 32P-labeled oligo-DNA probes complementary to their 5′-terminal 28-nt sequences. Lane 1 indicates no L protein. Putative N1–28 and N1–30 RNAs are marked by arrowheads (C). Asterisks represent unidentified transcripts (∼70 nt) detected with the (−)N41–68 probe (D).
Mentions: In order to investigate whether other cap-defective mutants produce N1–40, N1–38 and N41–68 RNAs in addition to the leader RNA, short transcripts synthesized by selected mutants (H1227R, R1228H, HR-RH, R1221A and R1221K) were analyzed by northern blotting with 32P-labeled oligo-DNA probes complementary to the 5′-terminal 28 nt of the leader RNA, N1–40 and N41–68 [designated as (−)Le1–28, (−)N1–28 and (−)N41–68, respectively] (Figure 3A). Although these cap-defective mutants exhibited leader RNA synthesis activities comparable to or lower than that of the wild-type L protein (Figure 3B; see Supplementary Figure S1C), they produced significantly higher amounts of N1–40, N1–38 (Figure 3C, lanes 3–7) and N41–68 (Figure 3D, lanes 3–7) than the wild-type L protein (Figure 3C and D, lane 2). Note that these mutants also yielded unidentified minor products including 28–30-nt RNAs and ∼70-nt RNAs, which were detected with the (−)N1–28 (Figure 3C, marked by arrowheads) and (−)N41–68 (Figure 3D, marked by asterisks) probes, respectively. These results indicate that the cap-defective mutations cause aberrant stop–start transcription to produce short RNAs using cryptic transcription signals in the N gene.

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