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研究発表を行った学会；
・第24回内藤コンファレンス　細胞核ダイナミクスとRNA [ II ]..
　２００９年６月２３日〜２６日（札幌）

タイトル；Spliceosomal U4 snRNA is required for centromeric silencing in fission yeast.
発表者；　知念　まどか　氏
　　　（熊本大学　自然科学研究科　谷研究室）
Abstract；
prp13-1 is one of the mutants isolated in a screen for defective pre-mRNA splicing at a nonpermissive temperature in fission yeast Schizosaccharomyces pombe. prp13-1 shows weak splicing defects in tested pre-mRNAs at the nonpermissive temperature. We isolated the prp13+ gene and found that it encodes U4 snRNA involved in the spliceosome assembly.
Interestingly, in addition to the defects in pre-mRNA splicing, prp13-1 showed aberrant nuclear division and produced elongated cells, a typical phenotype of cell division cycle mutants, at the nonpermissive temperature. Besides these phenotypes, we found that prp13-1 is hypersensitive to the microtuble-destabilizing drug thiabendazole (TBZ), suggesting that prp13-1 is defective in the interaction between kinetochores and microtubules. We then speculated that the formation of centromeric heterochromatin required for the kinetochore functions and directed by the RNAi system might be disturbed in prp13-1. To address that issue, we constructed strains with the prp13-1 mutation and the ura4+ gene inserted into the outermost (otr) or innermost (imr) pericentromeric repeats of chromosome 1. Growth assay on 5-fluoro-orotic acid (5-FOA) plates showed that those strains are sensitive to 5-FOA, which negatively selects against the ura4+ gene expression, indicating that the prp13-1 mutation causes a defect in centromeric silencing. The aberrant expression of the ura4+ gene inserted in the heterochromatic region was also confirmed by the RT-PCR analysis. As far as we tested, no severe splicing defects were observed in pre-mRNAs encoding the factors involved in the RNAi-directed centromeric silencing in prp13-1, suggesting that the silencing abnormality is not a secondary effect of the general splicing defects.
We determined the mutation site in prp13-1 and revealed that G at a position of +35 involved in the formation of a short distal stem in the 5’ stem-loop of U4 snRNA changes to A in prp13-1. To examine the effect of this point mutation on the U4 snRNP assembly, we investigated the interaction between U4 snRNA and Snu13p, which binds directly with the 5’ stem-loop region of U4 snRNA. The results showed that the interaction between U4 snRNA and Snu13p weakened in prp13-1.
In the course of these analyses, Bayne et al. reported that some splicing factors, such as Prp10p, associate with the RNAi machinery and facilitate centromere silencing (Science, 2008). It has been shown that noncoding transcripts derived from centromeric repeats are processed into siRNA that direct the RITS complex to bind the transcripts, recruiting Clr4 to form the heterochromatin. To investigate the relation between the splicing reaction and centromeric silencing through the RNAi pathway, we searched possible splice sites and branch sites in centromeric noncoding transcripts and found an intron-like sequence in the dg transcripts. Using total RNAs from dcr1∆ and RT-PCR, we demonstrated that the intron-like sequence found in the dg transcript is actually spliced. This is a first report that the centromeric noncoding RNAs are spliced. We also found that unprocessed noncoding RNAs are accumulated in prp13-1. Further analysis is now underway to examine if the splicing of the centromeric noncoding RNAs facilitates the formation of siRNAs and centromeric silencing.