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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 HR-HR mutant L protein produces aberrant short transcripts using cryptic signals within the N gene. (A) Transcripts, synthesized by the wild-type (WT), cap-defective (HR-RH) or transcription-defective (D714A) L protein, were post-labeled by vaccinia virus capping enzyme in the presence of [α-32P]GTP and analyzed by 20% urea-PAGE. Lane 1 indicates no L protein. (B) Cap-labeled short RNAs [Le, leader RNA; bands I–III, see panel (A)] were purified and digested with nuclease P1. The resulting digests were analyzed by PEI-cellulose TLC. The positions of standard cap analogs are shown on the right. (C–G) Purified cap-labeled short RNAs (lane 1) were partially digested with RNase T1 (lane 2) or RNase A (lane 3). The resulting digests were analyzed by 20% urea-PAGE followed by autoradiography. The OH− lanes indicate alkaline RNA ladders. The positions of digestion products with 3′-terminal Gp or Yp (Up or Cp) are indicated on the right. (H) Transcripts synthesized by the HR-RH mutant L protein were capped with vaccinia virus capping enzyme in the presence of [α-32P]GTP (lane 1, Vaccinia) or with the transcription-defective D714A mutant L protein in the presence of [α-32P]GDP (lane 2, VSV), and analyzed by 20% urea-PAGE. Short RNAs capped with the D714A mutant were grouped into i (∼40 nt) and ii (∼30 nt). (I) Capped short RNAs [groups i and ii; see panel (H)] were purified and digested with nuclease P1. The digests were analyzed as in panel (B).
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Figure 2: The cap-defective HR-HR mutant L protein produces aberrant short transcripts using cryptic signals within the N gene. (A) Transcripts, synthesized by the wild-type (WT), cap-defective (HR-RH) or transcription-defective (D714A) L protein, were post-labeled by vaccinia virus capping enzyme in the presence of [α-32P]GTP and analyzed by 20% urea-PAGE. Lane 1 indicates no L protein. (B) Cap-labeled short RNAs [Le, leader RNA; bands I–III, see panel (A)] were purified and digested with nuclease P1. The resulting digests were analyzed by PEI-cellulose TLC. The positions of standard cap analogs are shown on the right. (C–G) Purified cap-labeled short RNAs (lane 1) were partially digested with RNase T1 (lane 2) or RNase A (lane 3). The resulting digests were analyzed by 20% urea-PAGE followed by autoradiography. The OH− lanes indicate alkaline RNA ladders. The positions of digestion products with 3′-terminal Gp or Yp (Up or Cp) are indicated on the right. (H) Transcripts synthesized by the HR-RH mutant L protein were capped with vaccinia virus capping enzyme in the presence of [α-32P]GTP (lane 1, Vaccinia) or with the transcription-defective D714A mutant L protein in the presence of [α-32P]GDP (lane 2, VSV), and analyzed by 20% urea-PAGE. Short RNAs capped with the D714A mutant were grouped into i (∼40 nt) and ii (∼30 nt). (I) Capped short RNAs [groups i and ii; see panel (H)] were purified and digested with nuclease P1. The digests were analyzed as in panel (B).

Mentions: To define a defective step(s) in mRNA synthesis by the HR mutants, short transcripts produced by these mutants (see Figure 1B, bands I, II and III) were further analyzed. First, in order to investigate whether these short transcripts have a 5′-di- or tri-phosphate end, transcripts were synthesized with the HR-RH mutant in the presence of unlabeled NTPs and then capped in the presence of [α-32P]GTP with vaccinia virus capping enzyme. This conventional capping enzyme with the GTase and RTPase domains is known to cap 5′-di- or tri-phosphorylated RNAs without strict RNA sequence specificity (47). As shown in Figure 2A (lane 2), the leader RNA in transcripts synthesized by the wild-type L protein was mainly capped with vaccinia virus capping enzyme, indicating that the leader RNA is a major RNA product with a 5′-di- or tri-phosphate end. In the case of transcripts synthesized by the HR-RH mutant (lane 3), three prominent RNA products with ∼40 nt (bands I and II) and ∼30 nt (band III) were found to be capped with vaccinia virus capping enzyme in addition to the leader RNA. As expected, the transcription-defective D714A mutant (3) did not produce any guanylyl acceptor substrates for vaccinia virus capping enzyme (lane 4).


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 HR-HR mutant L protein produces aberrant short transcripts using cryptic signals within the N gene. (A) Transcripts, synthesized by the wild-type (WT), cap-defective (HR-RH) or transcription-defective (D714A) L protein, were post-labeled by vaccinia virus capping enzyme in the presence of [α-32P]GTP and analyzed by 20% urea-PAGE. Lane 1 indicates no L protein. (B) Cap-labeled short RNAs [Le, leader RNA; bands I–III, see panel (A)] were purified and digested with nuclease P1. The resulting digests were analyzed by PEI-cellulose TLC. The positions of standard cap analogs are shown on the right. (C–G) Purified cap-labeled short RNAs (lane 1) were partially digested with RNase T1 (lane 2) or RNase A (lane 3). The resulting digests were analyzed by 20% urea-PAGE followed by autoradiography. The OH− lanes indicate alkaline RNA ladders. The positions of digestion products with 3′-terminal Gp or Yp (Up or Cp) are indicated on the right. (H) Transcripts synthesized by the HR-RH mutant L protein were capped with vaccinia virus capping enzyme in the presence of [α-32P]GTP (lane 1, Vaccinia) or with the transcription-defective D714A mutant L protein in the presence of [α-32P]GDP (lane 2, VSV), and analyzed by 20% urea-PAGE. Short RNAs capped with the D714A mutant were grouped into i (∼40 nt) and ii (∼30 nt). (I) Capped short RNAs [groups i and ii; see panel (H)] were purified and digested with nuclease P1. The digests were analyzed as in panel (B).
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Figure 2: The cap-defective HR-HR mutant L protein produces aberrant short transcripts using cryptic signals within the N gene. (A) Transcripts, synthesized by the wild-type (WT), cap-defective (HR-RH) or transcription-defective (D714A) L protein, were post-labeled by vaccinia virus capping enzyme in the presence of [α-32P]GTP and analyzed by 20% urea-PAGE. Lane 1 indicates no L protein. (B) Cap-labeled short RNAs [Le, leader RNA; bands I–III, see panel (A)] were purified and digested with nuclease P1. The resulting digests were analyzed by PEI-cellulose TLC. The positions of standard cap analogs are shown on the right. (C–G) Purified cap-labeled short RNAs (lane 1) were partially digested with RNase T1 (lane 2) or RNase A (lane 3). The resulting digests were analyzed by 20% urea-PAGE followed by autoradiography. The OH− lanes indicate alkaline RNA ladders. The positions of digestion products with 3′-terminal Gp or Yp (Up or Cp) are indicated on the right. (H) Transcripts synthesized by the HR-RH mutant L protein were capped with vaccinia virus capping enzyme in the presence of [α-32P]GTP (lane 1, Vaccinia) or with the transcription-defective D714A mutant L protein in the presence of [α-32P]GDP (lane 2, VSV), and analyzed by 20% urea-PAGE. Short RNAs capped with the D714A mutant were grouped into i (∼40 nt) and ii (∼30 nt). (I) Capped short RNAs [groups i and ii; see panel (H)] were purified and digested with nuclease P1. The digests were analyzed as in panel (B).
Mentions: To define a defective step(s) in mRNA synthesis by the HR mutants, short transcripts produced by these mutants (see Figure 1B, bands I, II and III) were further analyzed. First, in order to investigate whether these short transcripts have a 5′-di- or tri-phosphate end, transcripts were synthesized with the HR-RH mutant in the presence of unlabeled NTPs and then capped in the presence of [α-32P]GTP with vaccinia virus capping enzyme. This conventional capping enzyme with the GTase and RTPase domains is known to cap 5′-di- or tri-phosphorylated RNAs without strict RNA sequence specificity (47). As shown in Figure 2A (lane 2), the leader RNA in transcripts synthesized by the wild-type L protein was mainly capped with vaccinia virus capping enzyme, indicating that the leader RNA is a major RNA product with a 5′-di- or tri-phosphate end. In the case of transcripts synthesized by the HR-RH mutant (lane 3), three prominent RNA products with ∼40 nt (bands I and II) and ∼30 nt (band III) were found to be capped with vaccinia virus capping enzyme in addition to the leader RNA. As expected, the transcription-defective D714A mutant (3) did not produce any guanylyl acceptor substrates for vaccinia virus capping enzyme (lane 4).

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