Novel Collagen II Alternative Transcripts and Mouse Skeletal Development

描述（由申请人提供）：在我们联合PI R21拨款申请的重新提交中，我们建议研究新发现的II型前胶原mRNA转录物（称为IIC）在骨骼发育中的作用。其他实验室先前的工作表明，II型前胶原基因（Col2a1）可以通过包含（IIA型）或排除（IIB型）外显子2（编码前胶原分子氨基前肽中富含半胱氨酸的结构域）来选择性剪接产生两种异构体。此外，这是一个发育调控的事件，其中软骨祖细胞主要合成IIA型mRNA，而分化的软骨细胞主要产生IIB型mRNA。我们最近在体外软骨形成试验中发现了两个额外的Col2a1亚型，我们将其命名为IIC和IID。IIC mRNA仅包含外显子2的前34个核苷酸，使用外显子2的替代剪接位点导致过早终止密码子。IID mRNA在外显子2的末端包含额外的三个核苷酸，编码色氨酸（人类）或精氨酸（小鼠），而不改变随后的蛋白质阅读框。我们假设IIC剪接事件是胚胎发育过程中软骨（可能还有其他组织）正确形成所需的基本机制。因此，我们提出(1)构建IIC剪接失活的重组敲入小鼠模型，(2)研究Col2a1 IIC mRNA在体外软骨形成过程中的调控和功能。这是一种优雅的方法，因为在体内和体外，我们将特异性地抑制一种mRNA异构体的产生，同时保留细胞产生其他三种Col2a1异构体的能力。我们使用迷你基因构建的初步体外研究表明，删除IIC剪接位点导致IIA/IID与IIB的比例发生显著变化。未翻译的IIC异构体的功能可能是调节其他翻译的Col2a1异构体的比例。因此，我们可以在敲入小鼠模型中预测IIC抑制对其他Col2a1亚型的产生以及对软骨发育的影响。我们认为IIC转录物仅在mRNA水平上起作用，并且在合成后很快就会迅速降解。我们计划研究IIC mRNA丰度是否受到一种称为无义介导的衰变（NMD）的过程的调节，该过程与其他具有类似调节作用的mRNA一起发生。使用胰岛素（ATDC5）处理后发生软骨分化的细胞系，我们将用反义寡核苷酸转染这些细胞，这些寡核苷酸将阻断IIC剪接位点的剪接。这应该对软骨形成产生与敲入小鼠相同的影响，敲入小鼠具有突变的IIC剪接位点。这个体外系统将使我们能够研究阻止IIC mRNA的产生对其他Col2a1亚型产生的影响。为了产生敲入小鼠而操纵剪接位点的创新方法，以及在体外阻断剪接位点的创新方法，将被证明是研究任何受选择性剪接影响的基因的有用策略。公共卫生相关性：该拨款提案将主要通过生成重组小鼠模型来研究新型II型胶原（Col2a1基因）替代转录本在骨骼发育中的意义。迄今为止，很少有小鼠模型用于研究选择性剪接位点的重要性。由于II型胶原mRNA的选择性剪接是骨骼形成的关键因素，因此开发敲入模型来研究II型胶原剪接在骨骼发育中的作用是结缔组织生物学领域的重要一步。近年来，选择性剪接在遗传多样性产生中的重要性已经变得明显，未来将需要许多其他基因的选择性剪接相关敲入小鼠。我们还预计，本研究中建立的小鼠模型将揭示与II型胶原蛋白表达有关的其他发育过程。IIA型前胶原mRNA在许多胚胎非软骨组织（如脊索、体裂体中胚层、胎儿脑、脊髓、发育中的眼睛、肌腱和韧带）的发育过程中表达水平相对较低。该项目的进一步工作将研究在分化和发育过程中调节每种可选剪接mRNA类型数量的机制。

DESCRIPTION (provided by applicant): In this resubmission of our joint PI R21 grant application, we propose to investigate the role of a newly-discovered type II procollagen mRNA transcripts, termed IIC, with respect to skeletal development. Previous work in other labs have shown that the type II procollagen gene (Col2a1) can be alternatively spliced to produce two isoforms by inclusion (type IIA) or exclusion (type IIB) of exon 2, which encodes a cysteine-rich domain within the amino propeptide of the procollagen molecule. In addition, this is a developmentally-regulated event where chondroprogenitor cells synthesize mainly type IIA mRNA, while differentiated chondrocytes produce mainly type IIB mRNA. We recently discovered two additional Col2a1 isoforms during in vitro chondrogenesis assays that we have named IIC and IID. IIC mRNA contains only the first 34 nucleotides of exon 2 by use of an alternative splice site in exon 2 resulting in a premature termination codon. IID mRNA contains an extra three nucleotides at the end of exon 2 that encodes either tryptophan (in human) or arginine (in mouse) without altering the subsequent protein reading frame. We hypothesize that the IIC splicing event is an essential mechanism required for proper formation of cartilage (and possibly other tissues) during embryonic development. We therefore propose to (1) generate a recombinant knock-in mouse model with an inactivated IIC splice site, and (2) To study the regulation and function of Col2a1 IIC mRNA during chondrogenesis in vitro. This is an elegant approach, as in vivo and in vitro we will specifically inhibit production of one mRNA isoform while retaining the cell's ability produce the other three Col2a1 isoforms. Our preliminary in vitro studies using a mini-gene construct has shown that deleting the IIC splice site results in a significant change in the ratio of IIA/IID to IIB. The function of the untranslated IIC isoform may be to regulate the proportions of the other translated alternatively spliced Col2a1 isoforms. Therefore, we can predict the effect of IIC inhibition upon production of other Col2a1 isoforms, and on cartilage development in the knock-in mouse model. We believe that the IIC transcript functions only at the level of mRNA and is rapidly degraded soon after it is synthesized. We plan to investigate whether IIC mRNA abundance is regulated by a process called nonsense-mediated decay (NMD), which occurs iwith other mRNAs that have similar regulatory roles. Using a cell line that undergoes chondrogenic differentiation when treated with insulin (ATDC5) we will transfect these cells with antisense oligonucleotides that will block splicing at the IIC splice site. This should have the same effect upon chondrogenesis as in the knock-in mouse, which has a mutated IIC splice site. This in vitro system will allow us to study the effect of preventing IIC mRNA production upon production of the other Col2a1 soforms. The innovative methods of manipulating a splice site in order to generate a knock-in mouse, and blocking a splice site in vitro, will prove to be a useful strategy to study any gene that is subject to alternative splicing. PUBLIC HEALTH RELEVANCE: This grant proposal will investigate the significance of novel type II collagen (Col2a1 gene) alternative transcripts within the context of skeletal development, primarily by generation of a recombinant mouse model. Very few mouse models to study the importance of alternative splicing sites have been developed to date. Since alternative splicing of collagen type II mRNA is a key element of skeletogenesis, the development of a knock-in model to study the role of collagen II splicing in skeletal development is an important step forward in the field of connective tissue biology. The importance of alternative splicing in the production of genetic diversity has become evident in recent years, and alternative splicing-related knock-in mice will be required for numerous other genes in the future. We also anticipate that the mouse model developed in this study will shed light upon other developmental processes that have been shown to involve expression of type II collagen. Type IIA procollagen mRNA is expressed at relatively low levels during development in a number of embryonic non-cartilage tissues such as the notochord, somitic mesoderm, fetal brain, spinal cord, developing eye, tendons and ligaments. Additional work on this project will investigate the mechanisms for regulating the amount of each alternatively spliced mRNA type during differentiation and development.