Linkage of Nonsense Codons and RNA Splicing

人们普遍认为真核细胞的转录后事件是区隔化的。转录本在细胞核内由剪接体剪接，然后转移到细胞质中，核糖体和trna合作读取这些转录本中的密码子。然而，令人惊讶的观察结果表明，过早终止（无义）密码子（ptc）不仅影响细胞质事件，而且影响核相关事件，这一观点受到了质疑。三种不同的无义密码子诱导反应已被表征。首先，许多含有ptc的mrna在哺乳动物细胞的核部分中被降解。尽管关于这种无义介导的衰变（NMD）机制是否发生在原子核本身存在争议，但有几条证据表明，核事件是触发它的必要条件。其次，PTC在某些基因中的插入增加了跳过引入的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，这种无义相关的剪接改变（NAS）反应显然涉及细胞核。第三，ptc在某些基因中的插入增加了其前体mrna在细胞核转录位点或附近的水平。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)转录本将用于本研究，因为TCRbeta基因在正常t细胞发育过程中经常获得ptc，因此监测该基因中ptc获取的机制可能对正常免疫细胞功能至关重要。目的1是确定NAS发生的分子机制，包括确定NAS发生的必要因素，确定NAS是否通过反馈机制发生。目的2是确定NMUP的分子机制，包括其因子需求和触发机制，并确定它是否是与转录耦合的早期事件，是否由于剪接抑制或前体mRNA稳定而发生，是否依赖于翻译，以及哪些调控元件是其发生所必需的。这项研究将揭示无义密码子识别是否发生在细胞质中，触发调节核事件（包括RNA剪接）的信号机制，或者是否在细胞核中直接读取无义密码子，作为核扫描机制的一部分，以校对新兴mrna，这是一个有争议的概念，有越来越多的证据。

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.