Analysis on how RNA splicing factors change global gene expression patterns and regulate male fertility.

分析RNA剪接因子如何改变全局基因表达模式并调节男性生育能力。

• 批准号: 2882792
• 金额: --
• 依托单位: Newcastle University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2882792
• 关键词: Analysis; how; RNA; splicing; factors

Infertility affects many couples, but the reasons causing it are usually unknown, as are the mechanisms required for normal fertility. This project will analyse RNA binding proteins implicated in male infertility, to understand their role in normal spermatogenesis. We will take advantage of existing mouse models and make cell lines that we can manipulate in vitro. This project will include world class training in both molecular biology and bioinformatics which will place the student in a strong place in the jobs market. Background: Recently the Elliott lab found that an RNA binding protein called RBMXL2 is essential for normal spermatogenesis, but why is not fully clear (PMID: 34927545). In the Veltman lab, heterozygous de novo mutations affecting RNA binding proteins have been identified that prevent normal healthy development of the testis (for example, mis-sense mutations in the RBM5 gene, PMID: 35013161). What gene expression pathways are affected by these RNA binding proteins, and why they affect normal healthy development of sperm is unknown. Both RBMXL2 and RBM54 are involved in controlling splicing. Splicing is critical since most human genes are split between exons and introns. A macromolecular machine called the spliceosome recognises exon sequences and splices them into mRNAs. Most genes can be spliced in more than one way, and changes in splicing patterns can alter gene function. Splicing factor proteins control whether exons are recognised or not, and this can change gene function in important ways. But these RNA binding proteins could also have additional unexpected roles in normal healthy germ cells.Hypothesis and aims: We hypothesise that mutations occurring in the human RBM5 gene and the mouse RBMXL2 gene will change impact normal healthy development of the testis. Our aim is to use mouse tissue and human cell lines to investigate this. Methodology: The student will analyse patterns of gene expression and cell biology in RBMXL2 knockout mice and wild type mice to identify defects caused by loss of RBMXL2 within an animal model. These will include splicing and gene expression changes, and corresponding changes in cellular processes important for spermatogenesis. The student will use genome engineering of human cultured cells to investigate the function of RBM5 and how this is changed by dominant negative mutations. A pool of predicted differentially spliced genes will be validated using RT-PCR and capillary gel electrophoresis. The student will interrogate mechanisms how normal patterns of gene expression of key genes is achieved, using minigenes made by genomic amplification and transfected into cells with the wild type and mutated versions of RBM5 and RBMXL2. As a backup to analysing endogenous targets the student will also characterise the effects of de novo mutations on RBM5 and RBMXL2 function on candidate minigenes that are already available. Potential impact: Up to 7% of men suffer from infertility. Despite this, the mechanisms of male infertility are very poorly understood. This project will follow on from recent work in our collaborating groups to start to address what global pathways of gene expression pathways are mis-regulated as a result of these de novo mutations. If successful, we can expand on this work by performing similar studies in other splicing genes found to be mutated in male infertility. This work will contribute to improving the genetic diagnosis in infertile men, which will provide more insight into the potential success of assisted reproductive technologies, the chance of passing on infertility with these approaches and co-morbidities associated with the infertility.

不孕症影响许多夫妇，但导致它的原因通常是未知的，因为是正常生育所需的机制。该项目将分析与男性不育有关的RNA结合蛋白，以了解它们在正常精子发生中的作用。我们将利用现有的小鼠模型，制造可以在体外操作的细胞系。该项目将包括分子生物学和生物信息学方面的世界级培训，这将使学生在就业市场上处于有利地位。背景资料：最近，Elliott实验室发现一种称为RBMXL 2的RNA结合蛋白对正常精子发生至关重要，但原因尚不完全清楚（PMID：34927545）。在Veltman实验室中，已经确定了影响RNA结合蛋白的杂合从头突变，这些突变会阻止睾丸的正常健康发育（例如，RBM 5基因中的错义突变，PMID：35013161）。这些RNA结合蛋白影响哪些基因表达途径，以及它们为什么影响精子的正常健康发育尚不清楚。RBMXL 2和RBM 54都参与控制剪接。剪接是至关重要的，因为大多数人类基因在外显子和内含子之间分裂。一种叫做剪接体的大分子机器识别外显子序列并将它们剪接成mRNA。大多数基因可以以多种方式剪接，剪接模式的变化可以改变基因功能。剪接因子蛋白控制外显子是否被识别，这可以以重要的方式改变基因功能。但是这些RNA结合蛋白在正常健康的生殖细胞中也可能具有额外的意想不到的作用。假设和目的：我们假设人类RBM 5基因和小鼠RBMXL 2基因中发生的突变会影响睾丸的正常健康发育。我们的目标是使用小鼠组织和人类细胞系来研究这一点。方法学：学生将分析RBMXL 2基因敲除小鼠和野生型小鼠的基因表达和细胞生物学模式，以确定动物模型中RBMXL 2缺失引起的缺陷。这些将包括剪接和基因表达的变化，以及对精子发生重要的细胞过程的相应变化。学生将使用人类培养细胞的基因组工程来研究RBM 5的功能以及显性负突变如何改变RBM 5的功能。将使用RT-PCR和毛细管凝胶电泳对预测的差异剪接基因库进行验证。学生将询问机制如何实现关键基因的基因表达的正常模式，使用通过基因组扩增制成的小基因，并转染到具有RBM 5和RBMXL 2的野生型和突变版本的细胞中。作为分析内源性靶点的备份，学生还将研究新生突变对RBM 5和RBMXL 2功能的影响，这些功能对已经可用的候选小基因有影响。潜在影响：高达7%的男性患有不孕症。尽管如此，男性不育的机制仍然知之甚少。该项目将继续我们合作小组最近的工作，开始解决由于这些从头突变而导致的基因表达途径的全球途径的错误调节。如果成功，我们可以通过在男性不育症中发现突变的其他剪接基因中进行类似的研究来扩展这项工作。这项工作将有助于改善不育男性的遗传诊断，这将为辅助生殖技术的潜在成功提供更多的见解，通过这些方法传递不育的机会以及与不育相关的并发症。