TCR RNA Surveillance

细胞的存活取决于检测基因表达错误的质量控制机制。这项提议的主题是无义介导的衰变（NMD），这是一种RNA监视途径，可以识别并降解具有提前终止（无义）密码子的异常mRNA。T细胞受体（TCR）和免疫球蛋白（IG）转录物是NMD的常见底物，因为它们通常由于淋巴细胞发育期间发生的程序性基因重排事件而获得无义密码子，以增加淋巴细胞上的受体库。其他NMD底物是从随机产生的突变基因（具有无义突变和移码）转录的异常mRNA 突变）和由于生物合成错误（转录和RNA剪接期间的错误）而产生的那些。奇怪的是，异常TCR和IG转录物的下调发生在细胞的核部分中，然而直到最近才知道触发NMD（无义密码子）的主要信号被细胞质中的翻译装置识别。细胞核在这种下调反应中发挥作用的进一步证据是，无义密码子下游的内含子作为触发NMD的第二个信号。TCR转录物似乎具有该第二信号或对该第二信号的反应不同，因为它们与所有其他已知转录物的不同之处在于无义密码子与下游密码子之间的最小距离。 需要内含子来引发NMD。此外，TCR转录物在结合无义密码子下游以触发该第二信号的一些因子方面不同。TCR在无义密码子的上游也具有独特的调控元件，其引起比来自非重排基因的所有已知转录物更强烈的下调（超过20倍）。本申请的具体目的是（1）表征和理解作用于无义密码子上游以引起TCR转录物的稳健下调的新调控元件的机制，（2）鉴定和表征作用于无义密码子下游调控元件的因子（外显子-外显子连接或内含子）以递送TCR NMD所必需的第二信号，和（3）通过产生和测试在NMD基因UPF 1中具有无效突变的淋巴细胞系来评估NMD的生理学意义。了解NMD的生理功能和机制是重要的，因为这种RNA监视途径在各种人类遗传疾病中被触发，并且可以保护个体免受由于无义和移码突变而产生的潜在有害的截短蛋白质的影响。

The survival of cells depends on quality-control mechanisms that detect mistakes in gene expression. The subject of this proposal is nonsense-mediated decay (NMD), an RNA surveillance pathway that recognizes and degrades aberrant mRNAs that possess premature termination (nonsense) codons. T-cell receptor (TCR) and immunoglobutin (Ig) transcripts are frequent substrates of NMD, as they commonly acquire nonsense codons as a result of the programmed gene rearrangement events that occur during lymphocyte development to increase the repertoire of receptors on lymphocytes. Other NMD substrates are aberrant mRNAs transcribed from randomly generated mutant gen_s (with nonsense mutations and frameshift mutations) and those generated as a result of biosynthetic errors (mistakes during transcription and RNA splicing). Paradoxically, the downregulation of aberrant TCR and Ig transcripts occurs in the nuclearfraction of cells, yet the primary signal that triggers NMD (a nonsense codon) was until recently only known to be recognized by the translation apparatus in the cytoplasm. Further evidence that the nucleus has a role in this downregulatory response is that an intron downstream of a nonsense codon serves as the second signal to trigger NMD. TCR transcripts appear to have or to respond differently to this second signal, as they differ from all other known transcripts in the minimum distance between the nonsense codon and the downstream intron required to elicit NMD. In addition, TCR transcripts differ in some of the factors that bind downstream of a nonsense codon to trigger this second signal. TCR also has unique regulatory elements upstream of the nonsense codon that elicit much more robust downregulation (more than 20-fold) than all known transcripts from non-rearranging genes. The Specific Aims of this application are (1) to characterize and understand the mechanism of the novel regulatory elements that act upstream of nonsense codons to cause robust downregulation of TCR transcripts, (2) to identify and characterize factors that act on the regulatory elements downstream of nonsense codons (exon-exon junctions or introns) to deliver the second signal essential for TCR NMD, and (3) to assess the physiological significance of NMD by generating and testing lymphocyte cell lines with a null mutation in the NMD gene UPF1. Understanding the physiological functions and mechanisms of NMD is important, as this RNA surveillance pathway is triggered in a variety of human genetic diseases and may protect individuals from the potentially deleterious truncated proteins generated as a result of nonsense and frameshift mutations.