核RNA监视和剪接偶联机制

Splicing is an important mechanism to increase the proteome complexity of higher eukaryotes. Splicing is traditionally considered to be highly accurate and efficient as the spliceosome's specific recognition of the splicing sites. However, many evidences suggest that the splicing of many pre-mRNAs is suboptimal. Unspliced transcripts can be detected under various physiological conditions. The aberrant splicing would produce truncated proteins, which have been linked to many human diseases, including neurodegenerative diseases and cancers. So far, most studies are focusing on the recognition and degradation of the unspliced transcripts by nonsense-mediated mRNA decay. In fact, the key components of nonsense-mediated mRNA decay are located in the cytoplasm, but most of the unspliced transcripts are mainly trapped in nucleus. On the other hand, the level of unspliced transcripts increased dramatically with deletion of nuclear exosome/TRAMP subunits. According to the current model, substrates of the nuclear exosome can be recognized and then recruited to the nuclear exosome by its cofactor, TRAMP complex. Therefore the rapid decay of pre-mRNA in the nucleus very likely reflects the importance of nuclear exosome/TRAMP in the splicing process. However,so far, there is no experimental evidence to support the model that nuclear exosome/TRAMP are directly involved in the regulation of the splicing machinery. In this study, we will use budding yeast as a model organism, employing a combination of genetic and molecular biological methods, to elucidate the mechanism of the coupling of splicing and nuclear RNA surveillance. The mechanisms of when and how the unspliced transcripts are recognized and degraded by nuclear RNA surveillance system will be explored. The regulatory role of nuclear RNA surveillance system on splicing will also be addressed. This project will bring to light the new concept that nuclear RNA surveillance system not only play a key role to eliminate the aberrant unspliced transcripts, but also directly involve in the regulation of the splicing process.

剪接是增加高等生物蛋白质组复杂性的重要机制，传统上被认为是高精度的。然而，有证据表明剪接并非是完善的，在不同的生理条件下可发现未剪接的转录体；异常剪接可产生有缺陷的蛋白并和许多疾病有直接关系。目前关於未剪接mRNA 识别和降解机制的研究多集中在无义密码子介导的mRNA 降解路径NMD 上。然而未剪接的mRNA 主要在细胞核，而NMD 的关键部分均位于细胞质中。文献及我们的前期实验均显示未剪接转录体水平会因核外切体亚基的缺失而大幅增加，反映了核外切体在剪接过程中的重要性。目前尚未有实验证据来支持核外切体直接参与剪接过程。在本研究中，我们将结合遗传和分子生物方法来阐明剪接和核RNA 监视偶联机制，并探讨核RNA 监视于何时以及如何识别并降解未剪接的转录体，研究核RNA 监视在剪接机制中的调控作用，为阐明RNA监视与剪接调控机制提供实验依据。

TRAMP复合物通过增加核外切体活性和调节底物特异性两个方面广泛降解包括内含子在内核糖核酸（RNA）。然而，TRAMP调节底物特异性的详细机制仍不清楚。在这项研究中，我们证明TRAMP在RNA聚合酶Ⅱ介导的新生转录本剪接中发生了共转录，并且功能性的与RNA降解相耦联。（1）TRAMP是通过共转录途径募集到新生转录中，并富集在特定的内含子序列；（2）TRAMP组件缺失导致未剪接的前信使RNA进一步积累；（3）TRMP参与了剪接因子Ms15p的最佳募集过程。因此，我们提出了一个新的自动防故障机制，即通过耦联核RNA质量控制和前信使RNA的剪接，确保TRAMP在剪接之前或期间进行共转录募集，以准备后续的剪接内含子靶被核外切体快速降解。否则，潜在有害的内含子miRNA和环状RNA可能因此产生并干扰正常的基因表达。