Investigating mRNA-rRNA base pairing in translation initiation

项目总结/摘要 了解核糖体如何选择mRNA上的翻译起始位点是一个重要的问题。 这是生物学中持续的挑战，对于研究细胞功能非常重要。 我们检验了以下假设： mRNA与18 S rRNA中称为530环的保守序列碱基配对， 位于mRNA的核糖体入口通道。我们认为这种碱基配对 有助于将AUG起始密码子定位在核糖体P位点，促进翻译起始。 这一假设是基于观察到530环具有碱基对 与蛋白质开放阅读框中发现的三核苷酸周期性互补 并且在起始序列下游10-20个核苷酸处， 通过肽MS/MS检测的酵母蛋白密码子以及TAP标记的蛋白 在大规模Western分析中表现出高表达。 使用CRISPER-Cas9基因编辑，我们将改变mRNA序列10-20 将起始密码子下游的核苷酸转移到预期不与起始密码子下游的核苷酸互补的序列。 与530循环配对，我们将使用西方分析来测试翻译是否是 正如模型所预测的那样。我们还将测试序列， 阅读框配准碱基配对，以评估是否精确定位 碱基配对的mRNA对于翻译起始是重要的。 在10-20个核苷酸中也发现了相同的三核苷酸周期性的增强 在非标准起始密码子之后，所述非标准起始密码子启动翻译的上游或下游， 注释开始。我们将测试是否减少潜在的碱基配对， 530环的序列下调这些非标准起始位点的起始。 提高对翻译起始的理解将对基因的研究具有重要意义 特别是在表达改变导致基因功能障碍的情况下， 例如在疾病状态下。

PROJECT SUMMARY/ABSTRACT Understanding how ribosomes choose translation initiation sites on mRNAs is an ongoing challenge in biology and is of great importance for investigation of cell function. We test the hypothesis that sequences just downstream of the translation start codon of mRNAs base pair with a conserved sequence in 18S rRNA termed the 530 loop that is located in the ribosome entrance tunnel for mRNAs. We propose that this base pairing helps position the AUG start codon at the ribosome P site facilitating translation initiation. This hypothesis is based on the observation that the 530 loop has base-pair complementarity to a three-nucleotide periodicity found in protein open reading frames and this periodicity is significantly enhanced 10-20 nucleotides downstream of the start codon of yeast proteins detected by peptide MS/MS as well as TAP-tagged proteins exhibiting high expression in large-scale western analysis. Using CRISPER-Cas9 gene editing, we will change the mRNA sequences 10-20 nucleotides downstream of the start codon to sequences that are not expected to base pair with the 530 loop and we will test using western analysis whether translation is depressed as predicted by the model. We will also test sequences that change the reading frame registration of the base pairing to assess whether the precise positioning of the base-paired mRNA is important for translation initiation. Enhancement of the same three-nucleotide periodicity is also found 10-20 nucleotides after non-standard start codons that initiate translation upstream or downstream of the annotated start. We will test whether reducing potential for base pairing by these sequences to the 530 loop down-regulates initiation at these non-standard start sites. Improving our understanding of translation initiation will be important in studying gene expression particularly in contexts where altered expression leads to gene disfunction, such as in disease states.