Alternative splicing of the first intron of the steroid receptor RNA activator (SRA) participates in the generation of coding and noncoding RNA isoforms in breast cancer cell lines
Alternative splicing of the first intron of the steroid receptor RNA activator (SRA) participates in the generation of coding and noncoding RNA isoforms in breast cancer cell lines
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DOI：
10.1089/dna.2006.25.418
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发表时间：
2006-07-01
影响因子：
3.1
通讯作者：
Leygue, Etienne
中科院分区：
文献类型：
--
作者：
Hube, Florent;Guo, Jimin;Leygue, Etienne
类固醇受体RNA激活剂I(SRA1)最初被描述为一种非编码RNA，能特异性激活类固醇受体的转录活性。然而，我们已经在人类乳腺组织中发现了外显子1扩展的SRA1亚型；包含两个起始的AUG密码子，编码一种我们称为SRAP的蛋白质。我们最近报道，在过度表达SRAP的乳腺癌细胞中，雌激素受体活性降低，这表明SRA1RNA和SRAP发挥了拮抗作用。SRA1似乎是第一个在RNA和蛋白质水平上都活跃的分子。目前还没有关于编码和非编码功能SRA1 RNA的产生可能涉及的机制的数据。利用5‘-RACE技术，我们在第二个蛋氨酸密码子周围确定了几个可能的转录起始点，并用于产生编码的SRA1转录本。在这个过程中，我们还鉴定了一个选择性剪接的非编码SRA1转录本，其中仍然包含内含子-1序列。利用靶向RT-PCR方法，我们证实了乳腺癌细胞系中存在含有完整和部分内含子-1序列的SRA1 RNAs，并确定了这些RNAs的相对比例在乳腺癌细胞系中有所不同。使用“微基因”策略，我们还显示了含有SRA1内含子-1序列的人造RNA在乳腺癌细胞系中可以交替剪接。有趣的是，微型基因产物的剪接模式与内源SRA1转录本的剪接模式相似。总之，我们的数据表明，内含子1及其周围的初级基因组序列足以导致该内含子的差异剪接。我们认为，内含子-1的选择性剪接是乳腺癌细胞用来调节编码和功能非编码SRA1 RNA之间的平衡的一种机制。
The Steroid Receptor RNA Activator I (SRA1) has originally been described as a noncoding RNA specifically activating steroid receptor transcriptional activity. We have, however, identified, in human breast tissue, exon1 extended SRA1 isoforms; containing two initiating AUG codons and encoding a protein we called SRAP. We recently reported a decreased estrogen receptor activity in breast cancer cells overexpressing SRAP, suggesting antagonist roles played by SRA1 RNA and SRAP. SRA1 appears to be the first example of a molecule active both at the RNA and at the protein level. No data are currently available regarding the mechanisms possibly involved in the generation of coding and noncoding functional SRA1 RNAs. Using 5'-Rapid Amplification of cDNA Extremities (5'-RACE), we have herein identified several putative transcription initiation sites surrounding the second methionine codon and used to generate coding SRA1 transcripts. In the process, we also identified an alternatively spliced noncoding SRA1 transcript still containing an intron-1 sequence. Using targeted RT-PCR approaches, we confirmed the presence in breast cancer cell lines of SRA1 RNAs containing a full as well as a partial intron-1 sequence and established that the relative proportion of these RNAs varied within breast cancer cell lines. Using a "minigene" strategy, we also showed that artificial RNAs containing the SRA1 intron-1 sequence are alternatively spliced in breast cancer cell lines. Interestingly, the splicing pattern of the minigene products parallels the one of the endogenous SRA1 transcripts. Altogether, our data suggest that the primary genomic sequence in and around intron-1 is sufficient to lead to a differential splicing of this intron. We propose that alternative splicing of intron-1 is one mechanism used by breast cancer cells to regulate the balance between coding and functional noncoding SRA1 RNAs.