The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation.
The splicing regulator PTBP1 controls the activity of the transcription factor Pbx1 during neuronal differentiation.
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DOI：
10.7554/elife.09268
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发表时间：
2015-12-24
期刊：
影响因子：
7.7
通讯作者：
Black DL
中科院分区：
文献类型：
--
作者：
Linares AJ;Lin CH;Damianov A;Adams KL;Novitch BG;Black DL
RNA结合蛋白PTBP1和PTBP2控制神经元发育过程中的选择性剪接程序。在神经前体细胞分化为早期神经元的过程中，发现PTBP2能维持许多突触和细胞骨架蛋白的胚胎剪接模式。然而，早期的PTBP1计划在胚胎干细胞(ESCs)和神经前体细胞中的作用尚不清楚。我们发现PTBP1控制神经基因表达的程序，其中包括转录因子Pbx1。当小鼠胚胎干细胞分化为神经前体细胞时，我们发现了由PTBP1而不是PTBP2特异性调控的外显子。我们发现PTBP1抑制胚胎干细胞中Pbx1外显子7和神经元Pbx1a亚型的表达。使用CRISPR-Cas9删除外显子7的调控元件，我们诱导了ESCs中Pbx1a的表达，发现这激活了神经元基因的转录。因此，PTBP1在诱导鼻咽癌发育之前控制Pbx1的活性以抑制其神经转录程序。DOI：http://dx.doi.org/10.7554/eLife.09268.001是在神经系统周围传递信息的神经元，经历了几个阶段的发育。胚胎干细胞专门形成神经前体细胞，然后再发育成神经元。这些细胞类型具有不同的特征，部分原因是它们产生不同的蛋白质或相同蛋白质的不同版本。为了制造蛋白质，基因的DNA序列被用来构建核糖核酸(RNA)分子，作为蛋白质的模板。然而，并不是所有的序列都编码蛋白质。非编码区必须从RNA中移除，其余的“外显子”连接在一起形成最终的“信使核糖核酸”模板。并不是所有的外显子都必须包含在最终的信使核糖核酸分子中。通过将不同的外显子组合结合在一起，一个基因可以产生几个不同版本的蛋白质。这一过程被称为替代剪接。控制选择性剪接的一种方法是通过与RNA结合的蛋白质来决定最终的mRNA分子中包含或排除哪些外显子。PTBP1是一种RNA结合蛋白，控制胚胎干细胞和神经前体细胞的选择性剪接。胚胎干细胞具有发育成人体所有细胞的能力。相比之下，神经前体细胞在发育过程中受到限制，只能产生神经系统的特化细胞。PTBP1在这些特性中的作用尚不清楚。Linares等人。现在使用了一系列技术来研究这两种细胞类型中产生的RNA分子，以及当PTBP1被移除时这些RNA如何变化。这发现了许多剪接受PTBP1调控的RNA，包括产生一种名为Pbx1的蛋白质的基因的mRNAs，Pbx1是神经元发育的重要调节因素。进一步的研究表明，PTBP1阻止了特定的外显子被包括在Pbx1的mRNA模板中。这创造了一种不影响神经基因的胚胎干细胞形式的Pbx1。PTBP1的去除允许Pbx1外显子的剪接，并产生在神经前体细胞中发现的Pbx1版本，该版本可启动神经元基因。因此，通过对Pbx1的作用，PTBP1的一个作用是使干细胞保持其非神经元特性，并防止它们过早发育为神经元前体细胞。Pbx1基因只是PTBP1在剪接水平上控制的众多基因中的一个。未来的一个挑战将是了解这些基因如何在一个共同的程序中协同工作，该程序决定了干细胞的特性。另一个问题涉及干细胞和神经前体细胞中不同的Pbx1蛋白如何在制造它们的细胞中发挥不同的作用。DOI：http://dx.doi.org/10.7554/eLife.09268.002
The RNA-binding proteins PTBP1 and PTBP2 control programs of alternative splicing during neuronal development. PTBP2 was found to maintain embryonic splicing patterns of many synaptic and cytoskeletal proteins during differentiation of neuronal progenitor cells (NPCs) into early neurons. However, the role of the earlier PTBP1 program in embryonic stem cells (ESCs) and NPCs was not clear. We show that PTBP1 controls a program of neuronal gene expression that includes the transcription factor Pbx1. We identify exons specifically regulated by PTBP1 and not PTBP2 as mouse ESCs differentiate into NPCs. We find that PTBP1 represses Pbx1 exon 7 and the expression of the neuronal Pbx1a isoform in ESCs. Using CRISPR-Cas9 to delete regulatory elements for exon 7, we induce Pbx1a expression in ESCs, finding that this activates transcription of neuronal genes. Thus, PTBP1 controls the activity of Pbx1 to suppress its neuronal transcriptional program prior to induction of NPC development. DOI: http://dx.doi.org/10.7554/eLife.09268.001 The neurons that transmit information around the nervous system develop in several stages. Embryonic stem cells specialize to form neuronal progenitor cells, which then develop into neurons. These cell types have different characteristics, in part because they make different proteins or different versions of the same proteins. To make a protein, the DNA sequence of a gene is used to build a molecule of ribonucleic acid (RNA) that acts as a template for the protein. However, not all of this sequence codes for the protein. The non-coding regions must be removed from the RNA, and the remaining “exons” joined together to form the final “mRNA” template. Not all of the exons are necessarily included in the final mRNA molecule. By joining together different combinations of exons, several different versions of a protein can be produced from a single gene. This process is known as alternative splicing. One way that alternative splicing is controlled is through proteins that bind to RNA and determine which exons are included or excluded from the final mRNA molecule. PTBP1 is an RNA-binding protein that controls alternative splicing in embryonic stem cells and neuronal progenitor cells. Embryonic stem cells have the ability to develop into all the cells of the body. In contrast, neuronal progenitor cells are restricted in their development and only give rise to specialized cells of the nervous system. The role of PTBP1 in these properties was not clear. Linares et al. have now used a range of techniques to study the RNA molecules produced in these two cell types and how these RNAs change when PTBP1 is removed. This identified many RNAs whose splicing is regulated by PTBP1, including mRNAs of the gene that produces a protein called Pbx1, which is an important regulator of neuronal development. Further investigation revealed that PTBP1 prevents a particular exon being included in the mRNA template for Pbx1. This creates an embryonic stem cell form of Pbx1 that does not affect neuronal genes. Removal of PTBP1 allows splicing of the Pbx1 exon and produces a version of Pbx1 that is found in neuronal progenitor cells and which turns on neuronal genes. Thus, through its action on Pbx1, one role of PTBP1 is to enable stem cells to maintain their non-neuronal properties and prevent their premature development into neuronal progenitor cells. The gene for Pbx1 is only one of many genes controlled by PTBP1 at the level of splicing. One challenge for the future will be to understand how these genes work together in a common program that determines the properties of stem cells. Another question regards how the different Pbx1 proteins in stem cells and in neuronal progenitors can exert different effects in the cells where they are made. DOI: http://dx.doi.org/10.7554/eLife.09268.002