The start sites of sequences of 18S and 25S rRNA found to be exonuclease resistant as indicated by oligo attachment
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Abstract Background We have previously reported 18S and 25S ribosomal RNA molecules in Candida albicans resistant to processive 5´→3´ exonuclease, appearing as cells approached stationary growth phase. Initial analysis pointed to extra phosphate(s) at their 5’- end raising the possibility that they were newly transcribed. Here we report on additional experiments exploring this possibility and try to establish which of the RNA polymerases may be transcribing them. Results Oligo-ligation and primer extension again showed the presence of extra phosphate at 5’-end at or near the reported processing sites for both 18S and 25S ribosomal RNA components. Inhibition of Pol I with BMH-21 increased the presence of the molecules. Quantitation with an Agilent Bioanalyzer showed that resistant 18S and 25S molecules are primarily produced in the nucleus. Utilizing an RNA cap specific antibody, a signal could be detected on these molecules via immunoblotting; such signal could be eliminated by decapping reaction. Both the cap specific antibody and eIF4E cap-binding protein, increased fold enrichment upon quantitative amplification. Antibodies specific for the RNA Polymerase II c-terminal domain and TFIIB initiator factor showed the presence of Pol II on DNA sequences for both 18S and 25S molecules in chromatin precipitation and qPCR assays. Rapamycin inhibition of TOR complex also resulted in an increase of resistant 18S and 25S molecules. Conclusions These data raise the possibility of a role for RNA Polymerase II in the production of 18S and 25S molecules and indicate that efforts for more direct proof may be worthwhile. If definitively proven it will establish an additional role for RNA Polymerase II in ribosomal production.
摘要 背景 我们此前曾报道，白色念珠菌（Candida albicans）中存在耐持续性5´→3´核酸外切酶（processive 5´→3´ exonuclease）的18S和25S核糖体RNA（ribosomal RNA）分子，此类分子会在细胞接近静止生长期（stationary growth phase）时出现。初步分析显示其5´端带有额外磷酸基团，提示它们可能为新转录产物。本研究开展了一系列额外实验以验证这一假说，并尝试明确参与此类分子转录的RNA聚合酶种类。 结果 寡核苷酸连接（oligo-ligation）与引物延伸（primer extension）实验再次证实，在18S和25S核糖体RNA组分的已报道加工位点或其附近，其5´端存在额外磷酸基团。使用BMH-21抑制RNA聚合酶I（RNA Polymerase I, Pol I）后，此类耐药分子的丰度显著升高。通过安捷伦生物分析仪（Agilent Bioanalyzer）定量分析发现，耐药性18S和25S RNA分子主要在细胞核内生成。利用帽结构特异性抗体，通过免疫印迹（immunoblotting）可在这类分子上检测到特异性信号，且该信号可通过脱帽反应（decapping reaction）被消除。无论是帽结构特异性抗体还是eIF4E帽结合蛋白（eIF4E cap-binding protein），在经过定量扩增后均表现出富集倍数提升。针对RNA聚合酶II（RNA Polymerase II）羧基末端结构域（c-terminal domain, CTD）以及TFIIB起始因子（TFIIB initiator factor）的抗体，在染色质沉淀（chromatin precipitation）与定量聚合酶链式反应（qPCR）实验中均可检测到Pol II结合于18S和25S分子对应的DNA序列。雷帕霉素（Rapamycin）抑制TOR复合物（TOR complex）后，耐药性18S和25S RNA分子的丰度同样升高。 结论 本研究数据提示RNA聚合酶II可能参与了18S和25S RNA分子的生成，同时表明开展更直接的验证实验具有研究价值。若该假说得到确证，将拓展RNA聚合酶II在核糖体生物生成过程中的功能范围。
