果蝇发育中的U12型剪接调控

In eukaryotes pre-mRNA splicing is an essential step in gene expression and plays important regulatory roles in the development of metazoan. Splicing reaction is catalyzed by the highly dynamic complex, spliceosome, under the regulation of cis-elements in pre-mRNA and trans-factors. The major spliceosome，also called U2-type spliceosome, is composed of 5 snRNA(U1, U2, U4, U5 and U6) and more than 100 proteins factors. Additionally, splicing of a small number of introns is catalyzed by minor spliceosome, also called U12-type spliceosome, which is composed of U11, U12, U4atac, U6ata, U5 snRNA and U12-type specific protein factors, in addition to the common protein factors shared with U2-type spliceosome. Although called ‘minor’splicing, U12-type splicing plays essential roles for the viability and development of metazoan. Mutations in genes encoding U12-type splicing factors have been found in patients of many human diseases. In contrast to the comprehensive understanding of major splicing, the minor splicing is far less investigated. Even in model system Drosophila melanogaster, the U12-type specific protein factors were not fully recovered due to their poor conservation between fly and human. Only two of them were predicted in Drosophila. Most of the U12-type introns were predicted according to the consensus sequences, proposed based on the experimentally proved U12-type intron sequences across several other species. This may lead to real U12-type introns being omitted. In order to identify the possibility using Drosophila as an effective model to study U12-type splicing related human diseases, we design three set of experiments to comprehensively investigate the U12-type splicing in Drosophila. First, we are going to use multi-step purification, mass-spec technologies to identify the U12-type specific spliceosomal protein factors in Drosophila and using Drosophila to dissect how SNN mutation is linked to the defective assembly of snRNP complex in U12-type spliceosome as displayed in SMA patients. Second, to identify accurately the U12-type introns in Drosophila, we plan to use the newly developed CRISPR/Cas9 technique to generate U11 and U12 snRNA deficient fly strains and to compare RNA-seq data between fly samples by bioinformatics analysis, and finally to provide Drosophila consensus sequences of U12-type introns based on experimental confirmation. Lastly, we will investigate the regulatory roles of U12-type splicing in fly development through mutants phenotype analysis. Collectively, these results will provide comprehensive knowledge of U12-type splicing in Drosophila, and shed light on how to apply Drosophila as an effective model system in the study of U12-type splicing related human diseases.

RNA剪接对真核生物基因表达及个体发育起到重要调控作用。剪接有两种类型：U2型剪接，负责基因组中绝大多数内含子的去除，也称主要剪接；另一类为U12型剪接，也称次要剪接，负责剪接的内含子数量虽少，但对于生物个体正常发育是必需的。U12型剪接障碍与多种人类疾病的发生有关。然而与U2型剪接相比，U12型剪接的研究还相对薄弱。在经常用于人类疾病模型的果蝇中，由于保守性较低，U12型剪接体的特有蛋白不能完全通过同源性分析预测；基因组中U12型的内含子多是预测得到，难免有失全面。因此本课题将结合利用有效的生化手段、新的Cas9基因编辑术、高通量测序、生物信息分析及遗传表形分析等方法，从剪接体组分、内含子特征界定及发育中的剪接调控等多个方面深入研究果蝇个体发育中的U12型剪接，为果蝇疾病模型在人类U12型剪接相关疾病的研究中的合理利用提供更明确的研究基础。

RNA剪接对真核生物基因表达及个体发育起到重要调控作用。其中的U12型剪接，也称次要剪接，在高等真核生物中非常保守。次要剪接体负责催化的内含子数量相对较少，但这些内含子的剪接对于生物个体的正常发育是必需的。因此 U12型剪接障碍会导致多种人类疾病的发生发展。本课题利用多种手段，开展了一系列工作以深入研究次要剪接的机制，阐明其在高等生物个体发育、人类疾病中的作用。我们利用CRISPR/Cas9介导的基因编辑技术，构建了次要剪接体特异的U12 and U6atac snRNA基因突变果蝇品系。突变表型分析表明次要剪接缺陷导致果蝇出现类似于脊肌萎缩症（SMA）的表型。利用这些突变品系进行RNA高通量测序及本课题新开发的生物信息学方法，我们发现了大量对次要剪接缺陷敏感的剪接事件和内含子。我们进一步对与神经系统功能有关的、含有次要剪接敏感内含子的基因进行了分析，并最终通过遗传回补cDNA的实验鉴定出三个神经系统相关基因与SMA疾病表型的产生直接相关。另外，本研究的结果还表明次要剪接敏感内含子中的许多剪接位点能够被次要和主要剪接体识别，但是只有当5’和3’SS同时被次要或主要剪接体识别的内含子才能发生有效的剪接。这揭示了一种通过次要和主要剪接体之间的竞争从而进行剪接调控的新模式。综上所述，我们发现了次要剪接内含子的新特征，在原有基础上增加了次要剪接内含子的数量；提出了次要和主要剪接竞争的剪接调控新模式；还进一步确定了与SMA疾病发生有直接关系的、含有次要剪接内含子的基因。研究结果加深了对次要剪接机制的理解、扩展了对次要剪接相关基因功能的认识，并进一步明晰了次要剪接缺陷和SMA病症的关系，开避了一些新的研究方向。