Linkage of Nonsense Codons and RNA Splicing

无义密码子与 RNA 剪接的联系

• 批准号: 0316793
• 负责人: Miles Wilkinson
• 金额: $ 64万
• 依托单位: University of Texas, M.D. Anderson Cancer Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0316793&HistoricalAwards=false
• 关键词: Linkage; Nonsense; Codons; RNA; Splicing

It is widely accepted that post-transcriptional events in eukaryotic cells are compartmentalized. Transcripts are spliced by spliceosomes in the nucleus and then translocated to the cytoplasm, where ribosomes and tRNAs collaborate to read the codons in these transcripts. This view has been cast into doubt, however, by the surprising observation that premature termination (nonsense) codons (PTCs) affect not only cytoplasmic events but also nuclear-associated events. Three distinct nonsense codon-induced responses have been characterized. First, many mRNAs containing PTCs are degraded in the nuclear fraction of mammalian cells. Although there is controversy as to whether this nonsense-mediated decay (NMD) mechanism occurs in the nucleus proper, several lines of evidence implicate nuclear events as being essential to trigger it. Second, the insertion of PTCs in some genes increases the levels of alternatively spliced transcripts that skip the introduced PTC. Because RNA splicing occurs in the nucleus, this nonsense-associated altered splicing (NAS) response clearly involves the nucleus. Third, the insertion of PTCs in some genes increases the levels of their precursor mRNAs at or near the site of transcription in the nucleus. To solve the paradox of how translation signals regulate nuclear events, the molecular mechanisms responsible for NAS and NMUP will be assessed. T-cell receptor-beta (TCRbeta) transcripts will be used for this research, as the TCRbeta gene frequently acquires PTCs during normal T-cell development, and therefore mechanisms that monitor acquisition of PTCs in this gene may be critical for normal immune cell function. Objective 1 is to determine the molecular mechanism responsible for NAS, including identifying the factors essential for it to occur and determining whether it occurs by a feedback mechanism. Objective 2 is to identify the molecular mechanism responsible for NMUP, including its factor requirements and its triggering mechanism, as well as to establish whether it is an early event coupled with transcription, whether it occurs as a result of inhibited splicing or precursor mRNA stabilization, whether it depends on translation, and what regulatory elements are essential for it to occur. This research should reveal whether nonsense codon-recognition occurs in the cytoplasm, triggering a signaling mechanism that regulates nuclear events (including RNA splicing), or whether instead nonsense codons are directly read in the nucleus as part of a nuclear scanning mechanism that proofreads nascent mRNAs, a controversial notion for which there is increasing evidence.

人们普遍认为，真核细胞中的转录后事件是区域化的。转录本由细胞核中的剪接体拼接，然后转移到细胞质，在那里核糖体和tRNAs协作阅读这些转录本中的密码子。然而，令人惊讶的是，过早终止(无意义)密码子不仅影响细胞质事件，还影响与核相关的事件，这一观点受到了质疑。三种不同的无意义密码子诱导的反应已经被表征。首先，许多含有PTCs的mRNAs在哺乳动物细胞的核部分中被降解。尽管对于这种无意义介导的衰变(NMD)机制是否发生在细胞核本身存在争议，但有几条证据表明，核事件是触发这种机制的关键。其次，在一些基因中插入PTC会增加跳过引入的PTC的选择性剪接转录本的水平。由于RNA剪接发生在细胞核中，这种无意义相关的改变剪接(NAS)反应显然涉及到细胞核。第三，在一些基因中插入PTCs会增加其前体mRNAs在核内转录位置或附近的水平。为了解决翻译信号如何调节核事件的悖论，将评估负责NAS和NMUP的分子机制。T细胞受体-β(TCRbeta)转录本将用于这项研究，因为TCRbeta基因在正常的T细胞发育过程中经常获得PTCs，因此监控该基因中PTCs的获取机制可能对正常免疫细胞功能至关重要。目标1是确定NAS的分子机制，包括确定NAS发生的必要因素，以及确定NAS是否通过反馈机制发生。目的2确定NMUP的分子机制，包括它的因子要求和触发机制，以及它是否是一个与转录结合的早期事件，它是由于剪接抑制还是前体mRNA稳定发生的结果，它是否依赖于翻译，以及什么调控元件是它发生所必需的。这项研究应该揭示无义密码子识别是发生在细胞质中，触发调节核事件(包括RNA剪接)的信号机制，还是作为校对新生mRNAs的核扫描机制的一部分，无义密码子直接在细胞核中读取，这是一个有争议的概念，证据越来越多。