Opposing effects of inhibiting cap addition and cap methylation on polyadenylation during vesicular stomatitis virus mRNA synthesis.
J Virol
; 83(4): 1930-40, 2009 Feb.
Article
em En
| MEDLINE
| ID: mdl-19073725
ABSTRACT
The multifunctional large (L) polymerase protein of vesicular stomatitis virus (VSV) contains enzymatic activities essential for RNA synthesis, including mRNA cap addition and polyadenylation. We previously mapped amino acid residues G1154, T1157, H1227, and R1228, present within conserved region V (CRV) of L, as essential for mRNA cap addition. Here we show that alanine substitutions to these residues also affect 3'-end formation. Specifically, the cap-defective polymerases produced truncated transcripts that contained A-rich sequences at their 3' termini and predominantly terminated within the first 500 nucleotides (nt) of the N gene. To examine how the cap-defective polymerases respond to an authentic VSV termination and reinitiation signal present at each gene junction, we reconstituted RNA synthesis using templates that contained genes inserted (I) at the leader-N gene junction. The I genes ranged in size from 382 to 1,098 nt and were typically transcribed into full-length uncapped transcripts. In addition to lacking a cap structure, the full-length I transcripts synthesized by the cap-defective polymerases lacked an authentic polyadenylate tail and instead contained 0 to 24 A residues. Moreover, the cap-defective polymerases were also unable to copy efficiently the downstream gene. Thus, single amino acid substitutions in CRV of L protein that inhibit cap addition also inhibit polyadenylation and sequential transcription of the genome. In contrast, an amino acid substitution, K1651A, in CRVI of L protein that completely inhibits cap methylation results in the hyperpolyadenylation of mRNA. This work reveals that inhibiting cap addition and cap methylation have opposing effects on polyadenylation during VSV mRNA synthesis and provides evidence in support of a link between correct 5' cap formation and 3' polyadenylation.
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Base de dados:
MEDLINE
Assunto principal:
Capuzes de RNA
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RNA Mensageiro
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RNA Viral
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Proteínas não Estruturais Virais
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Vesiculovirus
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Poliadenilação
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article