| Budget Amount *help |
¥63,200,000 (Direct Cost: ¥63,200,000)
Fiscal Year 2006: ¥11,500,000 (Direct Cost: ¥11,500,000)
Fiscal Year 2005: ¥11,500,000 (Direct Cost: ¥11,500,000)
Fiscal Year 2004: ¥13,400,000 (Direct Cost: ¥13,400,000)
Fiscal Year 2003: ¥13,400,000 (Direct Cost: ¥13,400,000)
Fiscal Year 2002: ¥13,400,000 (Direct Cost: ¥13,400,000)
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| Research Abstract |
(A) Enzyme mechanisms of mRNA cap formation in eukaryotes as well as negative-strand RNA viruses have been studied. 1) We have shown that the phosphorylation of RNA polymerase II carboxyl terminal domain (CTD) by TFIIH-associated CTD kinase (CAK) is important for successful capping of nascent RNA chain. Using an in vitro transcription system with immobilized template, we demonstrated that capping occurs when the nascent RNA chain grows to 18-19 nt in length. 2) We demonstrated that SeV RNA-dependent RNA polymerase (RdRP) L protein catalyzes cap methylation of virus specific mRNA. This is to our knowledge the first direct biochemical evidence to show that mononegavirus L protein catalyszes cap G-7-methylation. 3) The influenza virus (FluV) RdRP exhibits a cap-dependent endonuclease activity, which cleaves host mRNAs to produce capped RNA fragments with 11 to13 nucleotides. The resulting capped RNA fragments serve as a primer to initiate viral mRNA synthesis. We found that Flu-A and -B endonucleases exhibited different substrate specificities with regard to the extent as well as the position of cap methylation.
(B) A novel mRNA surveillance for mRNA lacking a termination codon (nonstop mRNA) has been proposed in which Ski7p is thought to recognize stalled ribosomes at the 3' end of mRNA. We found that the level of protein product of nonstop mRNA containing a poly(A) tail was reduced 100-fold, and this reduction was due to rapid mRNA degradation, translation repression and protein destabilization, at least in part, by the proteasome. Insertion of a poly(A) tract upstream of a termination codon resulted in translation repression and protein destabilization but not rapid mRNA decay. We propose that translation of the poly(A) tail plays crucial roles in nonstop mRNA surveillance via translation repression and protein destabilization.
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