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)是在成熟的mRNA中选择性地包含或排除选择性外显子以产生多种不同功能的mRNA和蛋白质异构体的过程。近年来，深度测序技术使转录组的研究成为可能，并极大地促进了对选择性剪接的研究。有几条证据表明，这是调节干细胞的多能性网络的一个重要过程。然而，该领域缺乏关于体内干细胞中AS的转录组范围的观点。淡水浮游生物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--即所谓的新生细胞--构成了它们惊人的再生能力。新生细胞是多能的，可以大量分离，很容易通过功能丧失技术进行操作。拉杰斯基实验室最近的转录学研究发现，在脊椎动物和哺乳动物干细胞之间的多能性机制存在着深刻的保守性。因此，对于人类干细胞生物学来说，平面动物是一个信息量很大的体内模型。我们已经确定并实验验证了浮游生物干细胞特异性的外显子，并研究了可能的组织特异性因素(未发表，初步数据)。这些实验表明，只有两种AS因子-CELF和MBNL因子-发挥这种调节的大部分作用，并拮抗干细胞中调节AS的作用。CELF和MBNL因子通过以相反的方式影响替代外显子包涵体水平，分别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还只是粗略的描述。因此，这些观察指出了这样一种情况，即AS是调节哺乳动物和脊椎动物干细胞中干细胞的关键保守过程。为了扩大我们关于这种拮抗如何调节干细胞的知识，我想表征CELF和MBNL功能丧失在脊椎动物干细胞中的功能后果，并阐明它们直接的RNA结合靶标。我将描述CELF和MBNL RNAi敲除对干细胞维持或分化和脊椎动物再生的细胞效应，以及对选择性剪接的分子效应。我还致力于在活体内开发在浮游生物中的PAR-CLIP，这是一种允许在核苷酸分辨率下在转录组范围内识别RNA结合位置的方法。整合功能和RNA结合数据将揭示保守的CELF/MBNL拮抗作用对体内干性或多能性的功能后果，并表征其在mRNA调控中的作用机制。这项研究项目将深入了解转录后机制如何在体内调节干细胞，并提供关于这一过程的机制信息，以及它的进化保守。

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validated planarian stem cell-specific AS exons, and studied putative tissue specific AS factors in planarians (unpublished, preliminary data).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.