Identification and characterisation of a novel antisense non-coding RNA from the RBM5 gene locus.
Identification and characterisation of a novel antisense non-coding RNA from the RBM5 gene locus.
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DOI：
10.1016/j.gene.2009.06.009
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发表时间：
2009-09-15
期刊：
影响因子：
3.5
通讯作者：
Sutherland, Leslie C
中科院分区：
文献类型：
--
作者：
Rintala-Maki, Nina D;Sutherland, Leslie C
我们实验室之前的工作鉴定了一个326碱基对（bp）的cDNA，称为Je2，它映射到假定的肿瘤抑制基因RBM5/LUCA-15/H37的内含子6的反义链，并作为细胞凋亡抑制因子。本文所描述的工作的目的是确定Je2是否是较大转录本的一部分，克隆该转录本并检查其调节RBM5表达的能力。Northern blot分析结合链特异性反转录和PCR发现了两个新的转录本，一个是反义的，一个是正的，包括Je2和RBM5内含子4序列。利用cDNA末端快速扩增技术（RACE）克隆了一个包含Je2和内含子4的1.4 kb新产物。在体外转录/翻译没有导致产生任何蛋白质产物，从任何一条链。基因组DNA分析显示存在一个假定的启动子区5'至Je2，这表明克隆的1.4 kb RACE产物是一个反义转录物，在RBM5基因的内含子6内起始，在内含子4内终止。这种新的反义非编码RNA被称为LUST，用于luca -15特异性转录物。LUST的异位过表达与全长RBM5+5+6备选RBM5 RNA剪接变体的表达升高一致，而截断的细胞毒性RBM5+5+6t/克隆26备选RBM5 RNA剪接变体的表达降低。本文提出了一个模型，通过LUST共同转录来掩盖RBM5+5+6和RBM5+5+6t/克隆26转录本共有的义链调控序列，当被揭开时导致RBM5+5+6的过早终止，从而产生细胞毒性截断产物RBM5+5+6t/克隆26。这些结果表明，LUST是一种新的、功能性的非编码RNA，通过调节RBM5剪接变异体的表达，在决定细胞凋亡命运中发挥作用。
Previous work from our lab identified a 326 base-pair (bp) cDNA, termed Je2, which mapped to the antisense strand of intron 6 of the putative tumour suppressor gene RBM5/LUCA-15/H37, and functioned as an apoptosis suppressor. The purpose of the work described herein was to determine if Je2 is part of a larger transcript, to clone that transcript and to examine its ability to modulate RBM5 expression. Northern blot analyses in conjunction with strand-specific reverse transcription and PCR revealed two novel transcripts, one antisense and one sense, that included Je2 as well as RBM5 intron 4 sequence. Using rapid amplification of cDNA ends (RACE), a novel 1.4 kb product including Je2 and intron 4 was cloned. In vitro transcription/translation did not result in the production of any protein product, from either strand. Genomic DNA analysis revealed the presence of a putative promoter region 5' to Je2, suggesting that the cloned 1.4 kb RACE product represents an antisense transcript that initiates within intron 6 and terminates within intron 4 of the RBM5 gene. This novel antisense, non-coding RNA was termed LUST, for LUCA-15-specific transcript. Ectopic overexpression of LUST coincided with elevated expression of the full-length RBM5+5+6 alternative RBM5 RNA splice variant, and reduced expression of the truncated, cytotoxic RBM5+5+6t/Clone 26 alternative RBM5 RNA splice variant. A model is proposed whereby LUST functions co-transcriptionally to mask a sense-strand regulatory sequence, common to both RBM5+5+6 and RBM5+5+6t/Clone 26 transcripts, that when unmasked results in premature termination of RBM5+5+6, thereby generating the cytotoxic truncated product, RBM5+5+6t/Clone 26. These results suggest that LUST is a novel, functional, non-coding RNA that plays a role in determining the apoptotic fate of a cell by regulating the expression of RBM5 splice variants.