snRNA maturation in Saccharomyces cerevisie

剪接体从初级转录物中去除内含子序列以产生开放的阅读框架，使得蛋白质能够正确表达。为此，剪接体每次都在具有内含子序列的mRNA上新组装。它们由几种蛋白质和五种snRNAs组成：U1，U2，U4，U 5和U6。除了由RNAP III合成的U6外，所有snRNA都由RNAP II转录。随后，在成熟snRNA被掺入剪接体之前，发生几个成熟步骤。很长一段时间以来，人们认为面包酵母的snRNAs不会穿梭出细胞核，但我们现在可以证明情况并非如此。与人类细胞中的snRNA一样，酵母snRNA也会离开细胞核。不同的成熟步骤，如Sm-环组装，防止降解，发生在细胞质中。在我们最近的出版物中，我们已经表明酵母在进化中也不例外，但核snRNA输出在所有真核生物中都是必要的。我们更进一步，用这种优秀的模式生物来说明为什么穿梭是必不可少的。通过用输出突变体阻断核snRNA输出，我们已经表明，在这种情况下，核保留的未成熟snRNA被并入装配剪接体。它们的组装导致非功能性剪接体和全基因组剪接缺陷。这表明未成熟snRNAs的直接核输出代表了完整剪接所必需的质量保证机制。我们的研究揭示了几个新的开放性问题，我们希望在本研究项目中解决这些问题。我们想更仔细地看看核进出口进程。例如，不清楚RNA输出因子Mex 67（人TAP）是否直接接触snRNA或是否需要衔接蛋白。此外，我们可以证明保卫蛋白Np 13、Gbp 2和Hrb 1与snRNA结合。在后续的实验中，我们想知道保卫蛋白是否真的也在保卫snRNA的成熟，例如它们是否有一个合适的5 '端。其他的质量控制机制可能是snRNA结合蛋白的正确结合顺序。此外，我们已经证明U6也穿过细胞质。我们怀疑U4/U6 di-snRNP在细胞质中形成，并因此被转运到细胞核中。与其他snRNA类似，U6也是首先合成为较长的前体。我们不仅将分析snRNP的共转录加载，而且还调查，加工是否发生在出口和再进口之前或之后，总之，这项研究将导致更好地了解snRNA成熟和随后的mRNA剪接和基因表达，它将有助于识别这些过程中可能导致神经退行性疾病和癌症的潜在缺陷。

Spliceosomes remove intron sequences from primary transcripts to generate an open reading frame that enables correct protein expression. For this purpose, the spliceosomes assemble every time newly on mRNAs with intron sequences. They are composed of several proteins and five snRNAs: U1, U2, U4, U5 and U6. With the exception of U6, which is synthesized from RNAP III, all snRNAs are transcribed from RNAP II. Subsequently, several maturation steps occur, before the mature snRNAs are incorporated into the spliceosome. For a long time it was thought that snRNAs from bakers yeast do not shuttle out of the nucleus, but we could now show that this is not the case. Like the snRNA from human cells, yeast snRNAs leave the nucleus. Different maturation steps such as the Sm-ring assembly, which protects from degradation, occur in the cytoplasm. In our recent publication we have shown that yeast is no exception in evolution, but that nuclear snRNA export is necessary in all eukaryotes. We went further and used this excellent model organism to show why shuttling is essential. By blocking nuclear snRNA export with export mutants, we have shown that the nuclear retained immature snRNAs are in this situation incorporated into the assembling spliceosome. Their assembly leads to non-functional spliceosomes and genome-wide splicing defects. This shows that the immediate nuclear export of immature snRNAs represents a quality assurance mechanism that is essential for intact splicing.Our study revealed several new open questions, which we would like to address in this research project. We would like to take a closer look at the nuclear export and import processes. It is for example unclear if the RNA export factor Mex67 (human TAP) contacts the snRNA directly or if an adapter protein is required. Furthermore we could show that the guard proteinsNpl3, Gbp2 and Hrb1 bind to the snRNA. In continuative experiments we would like to find out, whether the guard proteins indeed are also guarding the snRNA maturation, e.g. if they have a proper 5’-end. Other quality control mechanisms might be the correct order in association of snRNA binding proteins.Moreover we have shown that U6 against the current view also traverses through the cytoplasm. We suspect that the U4/U6 di-snRNP is formed in the cytoplasm and as such is transported into the nucleus. Similar to the other snRNAs, also U6 is first synthesized as a longer precursor. We will not only analyze the co-transcriptional loading of the snRNP, but also investigate, whether processing occurs before or after the export and re-import.In summary, this study will lead to a better understanding of snRNA maturation and subsequent mRNA splicing and gene expression and it will help to recognize potential defects in these processes that can lead to neurodegenerative diseases and cancer.