选择性RNA剪切和保护调控纤维小体化学计量的分子机制研究

Anaerobic and cellulolytic microorganisms digest cellulose very efficiently via a multienzyme complex called cellulosome, which represents a major paradigm for efficient biological degradation of cellulosic biomass. Functional cellulosome usually requires coordinated expression of multiplegenes that are usually encoded within a single operon so as to produce the many protein subunits at the appropriate stoichiometry. However, little is known about the regulatory mechanism of the stoichiometry of cellulosome.This has been on one of the key challenges in engineering highly efficient designer cellulosomes for industrial applications such as those for cellulosic biofuel production. Our recent discovery based on differential RNA-seq (dRNA-seq) suggested that selective RNA processing and stabilization (SRPS) regulate the stoichiometry of cellulosome at the post-transcriptional level in the cellulolytic model bacteria Clostridium cellulolyticum. Here we proposed to reveal the molecular mechanism and the genetic machineries that underpin this novel process. Firstly, we will address how the cip-cel mRNA is cleaved by specific intracellular enzyme activities. Secondly, we will identify those factors controlling the stability of processed mRNA. These aims will be accomplished by identifying the endoribonucleases cleaving cip-cel mRNA and the processed sites that was recognized, and by unveiling the conserved sequences and the evolutionary pattern of the stem-loop structures that stabilize processed RNA. The molecular mechanism of SRPS-based regulation of cellulsome stoichiometry elucidated in this project should provide a novel approach for rational design and assembly of designer cellulosome in vivo and moreover will lead to novel synthetic biology device and methods for precise control of gene expression at the post-transcriptional level.

纤维小体是细菌分泌的高效降解木质纤维素的多酶复合体，其各亚基维持一定的化学计量比对其功能的发挥至关重要。由于缺乏对控制纤维小体各亚基比例机制的了解，限制了其工业化改造。最近，项目申请人基于差异RNA测序技术（dRNA-seq）在纤维素降解菌Clostridium cellulolyticum中发现纤维小体基因簇cip-cel中各个基因的差异表达是通过选择性RNA剪切保护机制（SRPS）精确控制的。为探讨该调控过程的分子机制，本项目将以纤维素降解菌为模式，围绕RNA如何被剪切，以及哪些因素控制剪切后RNA的稳定性这两个核心问题，通过发现剪切cip-cel mRNA的核糖核酸内切酶及其作用位点、挖掘控制RNA稳定的颈环结构特征等，揭示纤维小体化学计量转录后水平调控的分子机制。这些努力不仅为设计和体内装配超级纤维小体提供新思路，也将开拓在转录后水控制基因相对表达丰度的合成生物学新元件与新方法。

纤维小体是细菌分泌的高效降解木质纤维素的多酶复合体，其各亚基维持一定的化学计量比对其功能的发挥至关重要。课题组前期基于差异RNA测序技术（dRNA-seq）在纤维素降解菌Clostridium cellulolyticum中发现纤维小体基因簇cip-cel中各个基因的差异表达是通过选择性RNA剪切保护机制（SRPS）在转录后水平进行精确控制的。因此，本项目以纤维素降解梭菌为研究体系对该调控过程的分子机制进行了深入研究。首先，通过对cip-cel的基因间隔区进行系统功能分析，发现底部富含GC的颈环结构是RNA剪切发生的关键元件，而且剪切后RNA的3'和5'端的二级结构决定其稳定性。其次，通过基因敲除和过表达等方法对核糖核酸内切酶的功能进行分析鉴定，初步发现RNase III潜在作用于cip-cel。最后，通过对同源Clostridium papyrosolvens的纤维小体表达调控机制研究，发现SRPS在纤维小体表达调控方面具有保守性。总之，通过本项目的实施，为开发控制基因化学计量性质的SRPS合成生物学方法提供理论基础并提供一系列合成生物学标准元器件。同时，本项目以纤维小体为切入点，为纤维小体基因表达调控机制提供新的理论基础和实验依据，从而为体内改造或体外人工设计、装配高效降解纤维素的超级纤维小体奠定基础。