The CMR complex for prokaryotic RNA silencing

用于原核RNA沉默的CMR复合物

• 批准号: BB/K000314/1
• 负责人: Malcolm White
• 金额: $ 51.94万
• 依托单位: University of St Andrews
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK000314%2F1
• 关键词: CMR; complex; prokaryotic; RNA; silencing

The battle between viruses and cells is brutal and often compared to an "Arms Race". It is the most ancient of wars - as old as life itself. Viral infections cause millions of human casualties globally every year. The extreme evolutionary pressure exerted on cells by viruses, and vice versa, has been and remains a key driving force in evolution. For humankind, there are clear imperatives to explore the possibilities of virus-mediated cell killing for pathogenic bacteria and, conversely, to harness bacterial virus immunity systems in nature to the benefit of food production. Arguably, most exciting discovery in prokaryotic molecular biology in the past 5-10 years is the CRISPR system. CRISPRs are DNA sequences found in the genomes of many bacteria and archaea. They consist of short repeats flanking "spacers" that are often derived from viral sequences. The CRISPR region of the genome is transcribed to make an RNA copy, which is then chopped up to generate individual CRISPR RNA molecules, each with the potential to match the sequence of an invading virus. CRISPR-mediated viral defence (or Interference) is mediated by two large, complex molecular machines, CASCADE and CMR, targetting viral DNA and RNA, respectively. This project is focused on the CMR complex, which is less well understood. We have shown that CMR uses CRISPR RNA to degrade viral RNA targets in a sequence specific reaction. We now wish to understand the molecular basis for this reaction, which is probably unique in prokaryotes. Studies will focus on the active sites of the enzyme that degrades the RNA and the details of the interactions between RNA and protein. Further, we aim to harness the CMR complex to develop sequence dependent RNA silencing in prokaryotes. This technology is not available in a robust form for prokaryotes, unlike in eukaryotes where the RNAi system is used to silence specific genes. If we can develop this technology it will open the door to new possibilities in the manipulation of gene expression in bacteria and archaea with many downstream applications.

病毒和细胞之间的战斗是残酷的，经常被比作“军备竞赛”。它是最古老的战争--和生命本身一样古老。病毒感染每年在全球造成数百万人伤亡。病毒对细胞施加的极端进化压力，反之亦然，一直是并仍然是进化的关键驱动力。对人类来说，显然有必要探索病毒介导的细胞杀死致病细菌的可能性，反过来，利用自然界中的细菌病毒免疫系统来促进粮食生产。可以说，在过去的5-10年里，原核分子生物学中最令人兴奋的发现是CRISPR系统。CRISPR是在许多细菌和古细菌的基因组中发现的DNA序列。它们由侧接“间隔区”的短重复序列组成，所述间隔区通常来源于病毒序列。基因组的CRISPR区域被转录以产生RNA拷贝，然后将其切碎以产生单独的CRISPR RNA分子，每个分子都有可能匹配入侵病毒的序列。CRISPR介导的病毒防御（或干扰）由两个大型复杂的分子机器介导，CASCADE和CMR，分别靶向病毒DNA和RNA。该项目的重点是CMR复合体，这是不太好理解。我们已经证明CMR使用CRISPR RNA在序列特异性反应中降解病毒RNA靶标。我们现在希望了解这种反应的分子基础，这在原核生物中可能是独一无二的。研究将集中在降解RNA的酶的活性位点以及RNA和蛋白质之间相互作用的细节上。此外，我们的目标是利用CMR复合物来开发原核生物中的序列依赖性RNA沉默。这种技术对于原核生物来说并不强大，不像真核生物，RNAi系统用于沉默特定的基因。如果我们能够开发出这项技术，它将为操纵细菌和古细菌中的基因表达提供新的可能性，并具有许多下游应用。

项目成果

Expression and Purification of the CMR (Type III-B) Complex in Sulfolobus solfataricus.

• DOI: 10.1007/978-1-4939-2687-9_12
• 发表时间: 2015
• 期刊: Methods in molecular biology
• 作者: Jing Zhang;M. F. White

CRISPR interference: a structural perspective.

• DOI: 10.1042/bj20130316
• 发表时间: 2013-07-15
• 期刊: The Biochemical journal
• 作者: Reeks J;Naismith JH;White MF
• 通讯作者: White MF

Hot and crispy: CRISPR-Cas systems in the hyperthermophile Sulfolobus solfataricus.

又热又脆：超嗜热微生物硫磺硫化叶菌中的 CRISPR-Cas 系统。

• DOI: 10.1042/bst20130031
• 发表时间: 2013
• 期刊: Biochemical Society transactions
• 影响因子: 3.9
• 作者: Zhang J

CRISPR-mediated targeted mRNA degradation in the archaeon Sulfolobus solfataricus.

• DOI: 10.1093/nar/gku161
• 发表时间: 2014-04
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Zebec Z;Manica A;Zhang J;White MF;Schleper C
• 通讯作者: Schleper C

Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site for CRISPR RNA processing.

• DOI: 10.1042/bj20130269
• 发表时间: 2013-06-01
• 期刊: The Biochemical journal
• 作者: Reeks J;Sokolowski RD;Graham S;Liu H;Naismith JH;White MF
• 通讯作者: White MF