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

涡虫干细胞体内调节中的选择性剪接：保守的CELF/MBNL拮抗作用调节干细胞的自我更新和分化。

• 批准号: 256258154
• 负责人: Dr. Jordi Solana García
• 金额: --
• 依托单位: Department of Biological and Medical Sciences
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/256258154?language=en
• 关键词: Alternative; splicing; regulation; planarian; stem

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validated planarian stem cell-specific AS exons, and studied putative tissue specific AS factors in planarians (unpublished, preliminary data).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

选择性剪接（Alternative splicing，AS）是指选择性地将成熟mRNA中的外显子插入或排除，从而产生多种mRNA和蛋白质异构体的过程，这些异构体通常具有不同的功能。近年来，深度测序技术使转录组范围的研究成为可能，并大大促进了可变剪接的研究。一些证据表明AS是调节干细胞的多能性网络的一个重要过程。然而，该领域缺乏AS在体内干细胞中的转录组范围的观点。淡水真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。新成细胞是多能的，可以大量分离并且容易通过功能丧失技术操作。最近的转录组学研究从Raublsky实验室发现，有一个深刻的保护机制之间的多能性的涡虫和哺乳动物干细胞。因此，真涡虫是人类干细胞生物学的一个信息丰富的体内模型。我们已经确定并实验验证了涡虫干细胞特异性AS外显子，并研究了推定的组织特异性AS因素在涡虫（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-发挥大部分这种调节作用，并拮抗调节干细胞中的AS。CELF和MBNL因子通过以相反的方式影响选择性外显子包含水平，分别促进和抑制干细胞特异性AS。此外，MBNL因子的作用最近已被证明在人干细胞中是保守的，而人CELF因子在该过程中的拮抗作用仅被概述。因此，这些观察结果指出，AS是一个关键的保守过程，调节干细胞，在哺乳动物和涡虫干细胞的情况。为了扩大我们的知识，这种拮抗作用如何调节干细胞，我想表征的功能性后果的CELF和MBNL的功能丧失的涡虫干细胞，并阐明其直接的RNA结合的目标。我将描述CELF和MBNL RNAi敲低对干细胞维持或分化和真涡虫再生的细胞效应以及对选择性剪接的分子效应。我的目标还在于开发体内的PAR-CLIP在涡虫，一种方法，允许在核苷酸分辨率的RNA结合位点的转录组范围内的识别。功能和RNA结合数据的整合将揭示保守的CELF/MBNL拮抗作用在体内对干细胞性或多能性的功能后果，并表征其在mRNA调控中的作用机制。该研究项目将深入了解转录后机制如何在体内调节干细胞，并提供有关该过程及其进化保护的机制信息。