PDF(Pubmed)
Abstract：
Ribosomal RNA (rRNA) processing and maturation are fundamentally important for ribosome biogenesis, but the mechanisms in archaea, the third form of life, remains largely elusive. This study aimed to investigate the rRNA maturation process in Methanococcus maripaludis, a representative archaeon lacking known 3\'-5\' exonucleases. Through cleavage site identification and enzymatic assays, the splicing endonuclease EndA was determined to process the bulge-helix-bulge (BHB) motifs in 16S and 23S rRNA precursors. After splicing, the circular processing intermediates were formed and this was confirmed by quantitative RT-PCR and Northern blot. Ribonuclease assay revealed a specific cleavage at a 10-nt A/U-rich motif at the mature 5\' end of pre-16S rRNA, which linearized circular pre-16S rRNA intermediate. Further 3\'-RACE and ribonuclease assays determined that the endonuclease Nob1 cleaved the 3\' extension of pre-16S rRNA, and so generated the mature 3\' end. Circularized RT-PCR (cRT-PCR) and 5\'-RACE identified two cleavage sites near helix 1 at the 5\' end of 23S rRNA, indicating that an RNA structure-based endonucleolytic processing linearized the circular pre-23S rRNA intermediate. In the maturation of pre-5S rRNA, multiple endonucleolytic processing sites were determined at the 10-nt A/U-rich motif in the leader and trailer sequence. This study demonstrates that endonucleolytic processing, particularly at the 10-nt A/U-rich motifs play an essential role in the pre-rRNA maturation of M. maripaludis, indicating diverse pathways of rRNA maturation in archaeal species.
摘要：
核糖体RNA（rRNA）加工和成熟对于核糖体生物发生至关重要，但是古细菌的机制，生命的第三种形式，在很大程度上仍然难以捉摸。本研究旨在探讨海洋甲烷球菌rRNA的成熟过程，缺乏已知的3'-5'外切核酸酶的代表性古细菌。通过切割位点鉴定和酶分析，确定剪接核酸内切酶EndA以处理16S和23SrRNA前体中的凸起-螺旋-凸起（BHB）基序。拼接后,形成环状加工中间体，并通过定量RT-PCR和Northern印迹证实。核糖核酸酶分析显示，在16SrRNA的成熟5'末端，在富含10-ntA/U的基序处进行了特异性切割，线性化环状前16SrRNA中间体。进一步的3'-RACE和核糖核酸酶测定确定核酸内切酶Nob1切割了前16SrRNA的3'延伸，并因此产生了成熟的3'结束。环化RT-PCR（cRT-PCR）和5'-RACE在23SrRNA的5'末端确定了螺旋1附近的两个切割位点，表明基于RNA结构的核酸内切处理使环状pre-23SrRNA中间体线性化。在5S前rRNA的成熟过程中，在前导序列和尾部序列中的富含10-ntA/U的基序处确定了多个核酸内切处理位点。这项研究表明，核酸内切处理，特别是在富含10-ntA/U的基序中，在M.maripaludis的pre-rRNA成熟中起着至关重要的作用，表明古细菌物种rRNA成熟的不同途径。
