Peculis B A
Genetics and Biochemistry Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1766, USA.
RNA. 2001 Feb;7(2):207-19. doi: 10.1017/s1355838201001625.
Several snoRNAs are essential for the sequence of cleavage events required to produce the mature forms of 18S, 5.8S, and 28S rRNA from the large precursor molecule. In the absence of U22, mature 18S rRNA fails to accumulate; U8 snoRNA is essential for accumulation of both 5.8S and 28S rRNA. The mechanisms by which snoRNAs facilitate these cleavage events is not known and might include direct cleavage or assisting the rate or efficiency of ribosome assembly. To learn more about the mechanisms of snoRNA-mediated pre-rRNA processing, an examination of the kinetics of pre-rRNA processing in Xenopus oocytes was undertaken. Correct pre-rRNA processing can be restored in snoRNA-depleted oocytes following cytoplasmic injection of the corresponding in vitro-synthesized snoRNA. Analysis of the kinetics of pre-rRNA processing in these snoRNA-rescue experiments demonstrated that the rate of accumulation of mature rRNAs was slower than that seen in untreated oocytes. The snoRNAs were imported into the nucleus at a rate and overall efficiency less than that of U1 snRNA, used as a control for import. However, sufficient levels of snoRNA were present in the nucleus to yield a functional phenotype (rescue of rRNA processing) several hours before the snoRNAs were directly detectable in the nucleus via autoradiography. This indicated that very low amounts of the snoRNA in the nucleus were sufficient for rescue. Finally, transcriptional inhibitors were used to separate transcription and processing. Failure to rescue snoRNA-mediated processing of pre-accumulated precursors is consistent with a scenario in which U8 and U22 must be present during transcription of pre-rRNA.
几种小核仁RNA（snoRNA）对于从大的前体分子产生成熟形式的18S、5.8S和28S核糖体RNA（rRNA）所需的切割事件序列至关重要。在缺乏U22的情况下，成熟的18S rRNA无法积累；U8 snoRNA对于5.8S和28S rRNA的积累都是必需的。snoRNA促进这些切割事件的机制尚不清楚，可能包括直接切割或协助核糖体组装的速率或效率。为了更多地了解snoRNA介导的前体rRNA加工机制，对非洲爪蟾卵母细胞中前体rRNA加工的动力学进行了研究。在向缺乏snoRNA的卵母细胞细胞质中注射相应的体外合成snoRNA后，可以恢复正确的前体rRNA加工。对这些snoRNA拯救实验中前体rRNA加工动力学的分析表明，成熟rRNA的积累速率比未处理的卵母细胞中慢。snoRNA导入细胞核的速率和总体效率低于用作导入对照的U1小核RNA（snRNA）。然而，在通过放射自显影在细胞核中直接检测到snoRNA之前的几个小时，细胞核中存在足够水平的snoRNA以产生功能性表型（rRNA加工的拯救）。这表明细胞核中非常少量的snoRNA就足以实现拯救。最后，使用转录抑制剂来分离转录和加工。未能拯救snoRNA介导的预积累前体的加工与一种情况一致，即在前体rRNA转录过程中必须存在U8和U22。
