Analysis and perturbation of C/D box sRNA-guided 2'-O-methylation patterns of Sulfolobus acidocaldarius ribosomal RNAs

在古细菌和真核生物中，存在含有小C/D盒sno样RNA（sRNA）的核糖核蛋白复合物。这些sRNA含有两个指导序列，其可以表现出与核糖体RNA（rRNA）内的靶位点的碱基对互补性。靶核苷酸经历2 '-O-核糖甲基化，这有助于新生rRNA分子的折叠和稳定。超嗜热古菌的基因组编码大量的C/D盒sRNA基因，这表明在极端生长温度下rRNA稳定化的必要性增加。我们使用Illumina RNA-Seq方法鉴定了来自七种古细菌的完整C/D盒sRNA。从指导序列推断它们的潜在靶位点。这些分析表明，C/D盒sRNA可以作为RNA伴侣，在rRNA折叠过程中使用它们的两个引导区连接靶位点。在我们提出的研究中，我们计划验证细胞2 '-O-核糖甲基化模式，以便能够将C/D盒sRNA指导内的靶向潜力与实验观察到的修饰进行比较。分析将在遗传上易于处理的极端嗜热古菌酸热硫化叶菌中进行，这使我们能够干扰C/D盒sRNA引导机制。我们的目的是建立一种方法，结合在2 '-O-核糖甲基化位点（RTL-P）的逆转录酶终止分析与RNA-Seq方法。这些全球性研究将使我们能够检测单个C/D盒sRNA基因缺失对核糖体RNA甲基化模式的影响。我们的目标是删除RNA的C/D盒sRNA基因，（i）已被证明靶向最保守的古细菌rRNA甲基化位点，（ii）建议包含可用于协调rRNA折叠的连接指导序列。最后，我们将研究酸热硫化叶菌生长温度的变化是否影响C/D盒sRNA/rRNA退火的严格性，这应该会影响核糖体的异质性。

In archaea and eukaryotes, ribonucleoprotein complexes exist containing small C/D box sno-like RNAs (sRNAs). These sRNAs contain two guide sequences that can exhibit base pair complementarity to target sites within ribosomal RNA (rRNA). The target nucleotides are subjected to 2'-O-ribose methylation, which aids in the folding and stabilization of nascent rRNA molecules. The genomes of hyperthermophilic archaea encode large numbers of C/D box sRNA genes, suggesting an increased necessity for rRNA stabilization at extreme growth temperatures. We have identified the complete sets C/D box sRNAs from seven archaea using Illumina RNA-Seq methodology. Their potential target sites were deduced from the guide sequences. These analyses revealed that C/D box sRNAs could acts as RNA chaperones that use their two guide regions to link target sites during rRNA folding. In our proposed research we plan to verify cellular 2'-O-ribose methylation patterns to be able to compare the targeting potential within the C/D box sRNA guides with experimentally observed modifications. Analyses will be performed in the genetically tractable hyperthermophilic archaeon Sulfolobus acidocaldarius, which allows us to perturb the C/D box sRNA-guide machinery. We aim to establish a method that combines the analysis of reverse transcriptase termination at 2'-O-ribose methylation sites (RTL-P) with RNA-Seq methodology. These global studies will allow us to detect the effects of individual C/D box sRNA gene deletions on the methylation patterns of ribosomal RNAs. We aim to delete C/D box sRNA genes for RNAs that (i) were shown to target the most conserved archaeal rRNA methylation sites and (ii) are proposed to contain linked guide sequences that could be used to coordinate rRNA folding. Finally, we will investigate if changes in the Sulfolobus acidocaldarius growth temperature affect stringency of C/D box sRNA/rRNA annealing, which should influence ribosome heterogeneity.