Molecular biology and coevolution

分子生物学和共同进化

• 批准号: RGPIN-2018-03878
• 负责人: Xia, Xuhua
• 金额: $ 7.29万
• 依托单位: University of Ottawa
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=745595
• 关键词: Molecular; biology; coevolution

Efficient biosynthesis depends much on efficient RNA processing and translation. My research program focuses on 1) coevolution between protein-coding genes and translation machinery in bacteria, and 2) intron-splicing in eukaryotes. In bacterial translation, coevolution occurs between 1) Shine-Dalgarno (SD) sequences on mRNA and anti-SD (aSD) sequences on small subunit (SSU) rRNA, 2) sense codon usage and differential tRNA abundance, and 3) stop codon usage and differential abundance of release factors (RF1 and RF2). All these involve a motif signal and its decoders, i.e., SD decoded by aSD, sense codon by tRNA, and stop codon by release factors. Coevolution between the signal motifs and their decoders are confounded by many other factors. SDs, as well as start and stop codons, can all be embedded in a secondary structure and become invisible to their respective decoders. The effect of selection is more visible in highly expressed genes than in lowly expressed genes. Requirement of SD/aSD pairing differs between the first gene in an operon and the following genes. Different nucleotides downstream of stop codons affect translation terminate efficiency. Stop codon can be misread by tRNAs and sense codons misread by release factors. We need accurate quantification of motif signals and decoders to achieve an integrated understanding of their relationships. We have been using RNA-Seq data to characterize the abundance of mRNA, tRNA and SSU rRNA with different 3' tails generated by continuous degradation of 3' exoribonucleases, and statistical modeling to quantify their relationships Intron splicing signals consist of 5' and 3' splice sites and branchpoint sites, as well as exonic and intronic splice enhancers. Their signal strength also depends on secondary structure, flanking nucleotides, and gene expression. Many yeast spliceosomes are recruited by intronless mRNAs because these mRNAs harbor false splice site signals, although such false signals are selected against in highly transcribed genes. We will 1) characterize splicing signal strength by using position weight matrix and self-organizing map algorithms, 2) measure splicing efficiency (SE) by using RNA-Seq data to obtain abundance of exon-exon junctions (AEE), exon-intron junctions (AEI), and total mRNA (AT) so that SE = AEE/AT and AT = AEE + AEI, and 3) model how SE is affected by factors such as splicing strength, secondary structure, presence of splicing enhancers, etc. We will apply this method first to yeast (Saccharomyces cerevisiae) where most intron-containing genes have just one intron, and then extend the method to study genes with multiple introns in multicellular eukaryotes. Our research will not only advance knowledge of translation and splicing, but also lead to better design of biosynthesis in industry. Two PhD, 4 MSc and 4 undergrad student currently participate in the program.

有效的生物合成在很大程度上取决于有效的RNA加工和翻译。我的研究项目主要集中在1）细菌中蛋白质编码基因和翻译机制之间的协同进化，以及2）真核生物中的内含子剪接。在细菌翻译中，1）mRNA上的Shine-Dalgarno（SD）序列和小亚基（SSU）rRNA上的抗SD（aSD）序列之间发生协同进化，2）有义密码子使用和差异tRNA丰度，以及3）终止密码子使用和释放因子（RF 1和RF 2）的差异丰度。所有这些都涉及基序信号及其解码器，即，SD由aSD解码，正义密码子由tRNA解码，终止密码子由释放因子解码。 信号模体和它们的解码器之间的协同进化受到许多其他因素的干扰。SD以及起始和终止密码子都可以嵌入二级结构中，并且对于它们各自的解码器来说是不可见的。选择的影响在高表达基因中比在低表达基因中更明显。SD/aSD配对的要求在操纵子中的第一个基因和随后的基因之间不同。终止密码子下游的不同核苷酸影响翻译终止效率。终止密码子可被tRNA误读，有义密码子可被释放因子误读。 我们需要准确的量化基序信号和解码器，以实现对它们之间关系的综合理解。我们一直在使用RNA-Seq数据来表征mRNA、tRNA和SSU rRNA的丰度，这些mRNA、tRNA和SSU rRNA具有由3'外切核糖核酸酶连续降解产生的不同3'尾，并且使用统计建模来量化它们之间的关系。内含子剪接信号包括5'和3'剪接位点和分支点位点，以及外显子和内含子剪接增强子。它们的信号强度还取决于二级结构、侧翼核苷酸和基因表达。许多酵母剪接体被无内含子mRNA募集，因为这些mRNA含有假剪接位点信号，尽管这种假信号在高度转录的基因中被选择。我们将1）通过使用位置权重矩阵和自组织映射算法来表征剪接信号强度，2）通过使用RNA-Seq数据来测量剪接效率（SE），以获得外显子-外显子连接（AEE）、外显子-内含子连接（AEI）和总mRNA（AT）的丰度，使得SE = AEE/AT和AT = AEE + AEI，以及3）对SE如何受剪接强度等因素的影响进行建模，二级结构，剪接增强子的存在等，我们将首先应用这种方法，酵母（酿酒酵母），其中大多数含内含子的基因只有一个内含子，然后扩展的方法来研究多细胞真核生物中的多个内含子的基因。 我们的研究不仅将促进翻译和剪接的知识，而且还将导致更好的设计工业生物合成。两名博士，4名硕士和4名本科生目前参加该计划。