Long Open Reading Frame Transcripts Escape Nonsense-Mediated mRNA Decay in Yeast

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Telomere Length Regulation and Telomeric Chromatin Require the Nonsense-Mediated mRNA Decay Pathway

Rap1p localization factor 4 (RLF4) is a Saccharomyces cerevisiae gene that was identified in a screen for mutants that affect telomere function and alter the localization of the telomere binding protein Rap1p. In rlf4 mutants, telomeric silencing is reduced and telomere DNA tracts are shorter, indicating that RLF4 is required for both the establishment and/or maintenance of telomeric chromatin and for the control of telomere length. In this paper, we demonstrate that RLF4 is allelic to NMD2/UPF2, a gene required for the nonsense-mediated mRNA decay (NMD) pathway (Y. Cui, K. W. Hagan, S. Zhang, and S. W. Peltz, Mol. Cell. Biol. 9:423–436, 1995, and F. He and A. Jacobson, Genes Dev. 9:437–454, 1995). The NMD pathway, which requires Nmd2p/Rlf4p together with two other proteins, (Upf1p and Upf3p), targets nonsense messages for degradation in the cytoplasm by the exoribonuclease Xrn1p. Deletion of UPF1 and UPF3 caused telomere-associated defects like those caused by rlf4 mutations, implying that the NMD pathway, rather than an NMD-independent function of Nmd2p/Rlf4p, is required for telomere functions. In addition, telomere length regulation required Xrn1p but not Rat1p, a nuclear exoribonuclease with functional similarity to Xrn1p (A. W. Johnson, Mol. Cell. Biol. 17:6122–6130, 1997). In contrast, telomere-associated defects were not observed in pan2, pan3, or pan2 pan3 strains, which are defective in the intrinsic deadenylation-dependent decay of normal (as opposed to nonsense) mRNAs. Thus, loss of the NMD pathway specifically causes defects at telomeres, demonstrating a physiological requirement for the NMD pathway in normal cell functions. We propose a model in which the NMD pathway regulates the levels of specific mRNAs that are important for telomere functions.     

基于RNA-Seq数据识别果蝇剪接位点和可变剪接事件

完整基因结构的预测是当前生命科学研究的一个重要基础课题,其中一个关键环节是剪接位点和各种可变剪接事件的精确识别.基于转录组测序（RNA-seq）数据,识别剪接位点和可变剪接事件是近几年随着新一代测序技术发展起来的新技术策略和方法.本工作基于黑腹果蝇睾丸RNA-seq数据,使用TopHat软件成功识别出39718个果蝇剪接位点,其中有10584个新剪接位点.同时,基于剪接位点的不同组合,针对各类型可变剪接特征开发出计算识别算法,成功识别了8477个可变剪接事件（其中新识别的可变剪接事件3922个）,包括可变供体位点、可变受体位点、内含子保留和外显子缺失4种类型.RT-PCR实验验证了2个果蝇基因上新识别的可变剪接事件,发现了全新的剪接异构体.进一步表明,RNA-seq数据可有效应用于识别剪接位点和可变剪接事件,为深入揭示剪接机制及可变剪接生物学功能提供新思路和新手段.     

The Hierarchy of Exon-Junction Complex Assembly by the Spliceosome Explains Key Features of Mammalian Nonsense-Mediated mRNA Decay

Exon junction complexes (EJCs) link nuclear splicing to key features of mRNA function including mRNA stability, translation, and localization. We analyzed the formation of EJCs by the spliceosome, the physiological EJC assembly machinery. We studied a comprehensive set of eIF4A3, MAGOH, and BTZ mutants in complete or C-complex–arrested splicing reactions and identified essential interactions of EJC proteins during and after EJC assembly. These data establish that EJC deposition proceeds through a defined intermediate, the pre-EJC, as an ordered, sequential process that is coordinated by splicing. The pre-EJC consists of eIF4A3 and MAGOH-Y14, is formed before exon ligation, and provides a binding platform for peripheral EJC components that join after release from the spliceosome and connect the core structure with function. Specifically, we identified BTZ to bridge the EJC to the nonsense-mediated messenger RNA (mRNA) decay protein UPF1, uncovering a critical link between mRNP architecture and mRNA stability. Based on this systematic analysis of EJC assembly by the spliceosome, we propose a model of how a functional EJC is assembled in a strictly sequential and hierarchical fashion, including nuclear splicing-dependent and cytoplasmic steps.     

一种新的Srrm1/SRm160可变剪接体经历无义突变介导的mRNA降解

目的:研究基因Srrm1/SRm160的可变剪接。方法:应用RT-PCR研究Srrm1/SRm160的可变剪接,通过蛋白质的翻译抑制和RNA干扰研究剪接异构体是否经历无义突变介导的mRNA降解(NMD)过程。结果:获得Srrm1/SRm160新的可变剪接异构体,该异构体产生提前终止密码子,翻译抑制和RNA干扰证实含有提前终止密码子的剪接体经过NMD而降解。结论:Srrm1/SRm160通过可变剪接和NMD调节自身的表达水平,作为剪接因子进一步调节其他基因的可变剪接。     

Nonsense-Mediated Decay Enables Intron Gain in Drosophila

Intron number varies considerably among genomes, but despite their fundamental importance, the mutational mechanisms and evolutionary processes underlying the expansion of intron number remain unknown. Here we show that Drosophila, in contrast to most eukaryotic lineages, is still undergoing a dramatic rate of intron gain. These novel introns carry significantly weaker splice sites that may impede their identification by the spliceosome. Novel introns are more likely to encode a premature termination codon (PTC), indicating that nonsense-mediated decay (NMD) functions as a backup for weak splicing of new introns. Our data suggest that new introns originate when genomic insertions with weak splice sites are hidden from selection by NMD. This mechanism reduces the sequence requirement imposed on novel introns and implies that the capacity of the spliceosome to recognize weak splice sites was a prerequisite for intron gain during eukaryotic evolution.