How do a conserved family of RNA binding proteins protect the transcriptome from aberrant processing?

研究目的：基因表达需要通过RNA加工正确产生成熟的蛋白质编码RNA（mRNA）。这包括剪接，它连接基因的一部分称为“外显子”，并删除其他称为“内含子”。RNA加工由短RNA序列指导。该项目旨在确定RBMXL2和RBMX蛋白如何抑制使用“神秘”RNA加工序列的机制，否则这些序列会破坏生产性基因表达。该项目解决了目前的三个知识差距。首先，在mRNA成熟过程中有效识别多长外显子目前知之甚少，但对于关键基因的表达至关重要，包括小鼠Meioc（减数分裂前期所需）和人类BRCA 2（基因组稳定性所需的肿瘤抑制因子）。特别是，隐藏的RNA加工事件如何在长外显子内被抑制的了解非常少，但可能会导致与RBMX家族蛋白缺乏相关的问题（男性不育，基因组不稳定性和Shashi智力障碍综合征）。第三，RBMX蛋白如何抑制剪接位点是未知的。由于抑制的RNA加工位点是我们在初步筛选中鉴定的大多数靶点，这表明抑制是RBMXL2和RBMX蛋白的主要作用机制。时间性和金钱价值：该项目是及时的，因为（1）我们已经制作并进行了RBMXL2敲除小鼠的初步表征。这只小鼠是雄性不育的，并且已经使我们能够鉴定出一些被RBMXL2抑制的隐蔽剪接位点。(2)通过与位于爱丁堡的两个世界领先的研究小组（Ian亚当斯教授和Donal O'Carroll教授）的合作，我们将能够纯化在我们的RBMXL 2敲除小鼠中停滞的确切细胞类型，并将其用于RNA测序分析。(3)提供物有所值，奥卡罗尔集团将为我们提供一个小鼠线，表达GFP在减数分裂期间在不收取运费以外。该细胞系将能够从RBMXL2敲除小鼠中纯化约99%的减数分裂细胞，然后我们可以将其纯化。(4)提供额外的价值，该项目将利用毛细管凝胶电泳系统，这是最近购买的赠款从病理学会和匹配的资金从纽卡斯尔大学。(5)我们已经确定了体细胞中被RBMX抑制的新型隐蔽RNA加工位点，并进行了实验以全面确定人类细胞中的RBMX结合位点（目前在我们基因组学核心设施的测序队列中）。结果：我们期望该项目的结果将在理解剪接和多聚腺苷酸化机制如何使长外显子的适当RNA加工，并避免包括隐藏的外显子方面具有重要意义。我们希望确定在男性减数分裂过程中依赖RBMXL2的基因表达模式，从而提高对男性不育原因的理解。由于我们的初步研究已经在癌细胞中检测到RBMX靶基因，这些基因对细胞如何对遗传毒性药物作出反应很重要，我们的研究可能会导致增加化疗疗效的新策略。这项工作的主要受益者将是将接受培训的科学家和学生，对基因表达感兴趣的其他科学家，我们将参与的公众成员，以及可能接受遗传毒性药物治疗的长期患者。

PURPOSE OF RESEARCH: Gene expression requires correct production of mature protein-coding RNAs (mRNAs) by RNA processing. This includes splicing, which joins parts of the gene called "exons" and removes others called "introns". RNA processing is guided by short RNA sequences. This project seeks identify the mechanism how RBMXL2 and RBMX proteins suppress use of "cryptic" RNA processing sequences that would otherwise corrupt productive gene expression. This project addresses three current knowledge gaps. Firstly, how long exons are efficiently recognised during mRNA maturation is currently poorly understood, yet critically important for expression of key genes, including mouse Meioc (required for meiotic prophase) and human BRCA2 (a tumour suppressor needed for genome stability). In particular, how cryptic RNA processing events are repressed within long exons is very poorly understood, yet may underpin problems associated with deficiency of RBMX family proteins (male infertility, genome instability and Shashi intellectual disability syndrome). Thirdly, how RBMX proteins repress splice sites is unknown. Since repressed RNA processing sites were the majority of targets we identified in our preliminary screens, this suggests that repression is the primary mechanism of action of RBMXL2 and RBMX proteins.TIMELINESS AND VALUE FOR MONEY: This project is timely since (1) We have already made and carried out initial characterisation of an RBMXL2 knockout mouse. This mouse is male infertile and has already enabled us to identify some cryptic splice sites repressed by RBMXL2. (2) Through collaboration with two world leading groups based in Edinburgh (Prof Ian Adams and Prof Donal O'Carroll) we will be able to purify the exact cell types that arrest in our RBMXL2 knockout mouse and use these for RNA sequencing analyses. (3) Providing value for money, the O'Carroll group will provide us with a mouse line that expresses GFP during meiosis at no charge other than shipping. This line will enable ~99% purification of cells in meiosis from RBMXL2 knockout mice that we can then characterise. (4) Providing extra value for money, this project will make use of a capillary gel electrophoresis system that was recently purchased by a grant from the Pathological Society and matching funds from Newcastle University. (5) We have already identified novel cryptic RNA processing sites that are repressed by RBMX in somatic cells and carried out experiments to globally identify RBMX binding sites in human cells (currently in the sequencing queue of our Genomics Core Facility). OUTCOMES: We expect that the results of this project will be significant in understanding how the splicing and polyadenylation machineries enable proper RNA processing of long exons, and avoid including cryptic exons. We expect to identify patterns of gene expression that depend on RBMXL2 during male meiosis, improving understanding of the causes of male infertility. Since our preliminary study has already detected RBMX target genes in cancer cells that are important for how cells respond to genotoxic drugs, our research may lead to new strategies to increase the efficacy of chemotherapy. The main beneficiaries from this work will be scientists and students who will be trained, other scientists interested in gene expression, members of the public that we will engage, and possibly in the longer term patients who are treated with genotoxic drugs.