Alternative splicing in the regulation of planarian stem cells in vivo: a conserved CELF/MBNL antagonism regulates stem cell self-renewal and differentiation.

选择性剪接（AS）是在成熟mRNA中选择性地包括或排除选择性外显子以产生多种mRNA和蛋白质亚型的过程，通常具有不同的功能。近年来，深度测序技术使转录组研究成为可能，并大大促进了选择性剪接的研究。一些证据表明，AS是调节干细胞的多能性网络的重要过程。然而，该领域缺乏AS在干细胞体内的转录组全视角。淡水涡虫为体内干细胞研究提供了一个极好的平台。它们独特的干细胞——所谓的新生细胞——是它们惊人的再生能力的基础。新母细胞是多能性的，可以大量分离，并且很容易通过功能丧失技术进行操作。最近来自Rajewsky实验室的转录组学研究发现，在涡虫和哺乳动物干细胞之间，控制多能性的机制有着很深的保守性。因此，涡虫是人类干细胞生物学的体内模型。我们已经确定并实验验证了涡虫干细胞特异性AS外显子，并研究了涡虫中可能的组织特异性AS因子（未发表的初步数据）。这些实验表明，只有两种AS因子- celf和MBNL因子-在干细胞中发挥了大部分的调节和拮抗作用。CELF和MBNL因子分别通过以相反的方式影响备选外显子包含水平来促进和抑制干细胞特异性AS。此外，MBNL因子的作用最近被证明在人类干细胞中是保守的，而人类CELF因子在这一过程中的拮抗作用仅被概述。因此，这些观察结果表明，在哺乳动物和涡虫干细胞中，AS是调节干细胞的关键保守过程。为了扩大我们对这种拮抗剂如何调节干细胞的认识，我想描述CELF和MBNL在真肠干细胞中功能丧失的功能后果，并阐明它们的直接RNA结合靶点。我将描述CELF和MBNL RNAi敲除对干细胞维持或分化和原虫再生的细胞效应以及对选择性剪接的分子效应。我还致力于在涡虫体内开发PAR-CLIP，这是一种允许在核苷酸分辨率下转录组范围内识别RNA结合位点的方法。功能和RNA结合数据的整合将揭示保守的CELF/MBNL拮抗剂对体内干细胞性或多能性的功能影响，并表征其在mRNA调控中的作用机制。该研究项目将深入了解体内转录后机制如何调节干细胞，并提供有关该过程的机制信息，以及其进化保护。

Alternative splicing (AS) is the process by which alternative exons are selectively included or excluded in the mature mRNA to produce multiple mRNA and protein isoforms, often with different functions. In recent years, deep sequencing techniques have enabled transcriptome-wide studies and substantially boosted research of alternative splicing. Several lines of evidence show AS being an essential process for the pluripotency network that regulates stem cells. However, the field lacks a transcriptome-wide perspective of AS in stem cells in vivo. Freshwater planarians offer an excellent platform for in vivo stem cell research. Their unique stem cells -the so-called neoblasts- underlie their amazing power of regeneration. Neoblasts are pluripotent, can be isolated in large numbers and easily manipulated by loss of function techniques. Recent transcriptomic studies from the Rajewsky laboratory uncovered that there is a deep conservation of the mechanisms governing pluripotency between planarian and mammalian stem cells. Thus, planarians are an informative in vivo model for human stem cell biology. We have identified and experimentally validated planarian stem cell-specific AS exons, and studied putative tissue specific AS factors in planarians (unpublished, preliminary data). These experiments have revealed that only two kinds of AS factors -CELF and MBNL factors- exert most of this regulation and antagonize to regulate AS in stem cells. CELF and MBNL factors act promoting and repressing the stem cell-specific AS respectively, by affecting alternative exon inclusion levels in opposing ways. Furthermore, the role of MBNL factors has been recently shown to be conserved in human stem cells, while the antagonism of human CELF factors in this process has only been sketched. These observations therefore point to a scenario in which AS is a key conserved process for regulating stem cells, in both mammalian and planarian stem cells. To expand our knowledge on how this antagonism regulates stem cells I want to characterize the functional consequences of CELF and MBNL loss-of-function in planarian stem cells and to elucidate their direct RNA binding targets. I will characterize the cellular effects of CELF and MBNL RNAi knock down on stem cell maintenance or differentiation and planarian regeneration and the molecular effects on alternative splicing. I also aim at developing in vivo PAR-CLIP in planarians, a method that allows the transcriptome-wide identification of RNA binding sites at nucleotide resolution. Integration of functional and RNA binding data will reveal the functional consequences of the conserved CELF/MBNL antagonism for stemness or pluripotency in vivo and characterize its mechanism of action in mRNA regulation. This research project will generate insights into how post-transcriptional mechanisms regulate stem cells in vivo and provide mechanistic information on the process, as well as on its evolutionary conservation.