Haeger Paola, Cuevas Rolando, Forray María Inés, Rojas Rolando, Daza Claudia, Rivadeneira Juan, Gysling Katia
Department of Cell and Molecular Biology, Faculty of Biological Sciences, Catholic University of Chile, Alameda 340, Santiago, Chile.
Brain Res Mol Brain Res. 2005 Sep 13;139(1):115-28. doi: 10.1016/j.molbrainres.2005.05.024.
Recently, it has been shown the endogenous expression of an antisense urocortin (Ucn) transcript in the rat brain and other tissues. In the present work, by means of two complementary techniques, specific-strand RT-PCR and in situ hybridization, we showed the natural expression of a second novel antisense Ucn RNA of higher size. Specific-strand RT-PCR of total RNA, cloning and sequence analysis together with the different subcellular localization observed for both antisense Ucn RNAs indicated that this novel antisense Ucn transcript corresponded to the immature form of the previously described antisense Ucn RNA. Sequence analysis indicated that this immature antisense Ucn transcript uses non-consensus CT-AC splice sites, exactly complementary to its sense counterpart. The mature antisense Ucn transcript was also amplified after specific-strand RT-PCR of poly(A)-RNA, suggesting that the mature antisense Ucn transcript is polyadenylated. We also proved that the region complementary to the promoter of sense Ucn RNA, including the TATA box, is part of the antisense Ucn RNA. Finally, we showed that the region complementary to the 3'-end of Ucn mRNA behaves as a functional promoter for the transcription of antisense Ucn RNA. Thus, the results indicate that the 3'-ends of both sense and antisense Ucn RNAs are the only non-complementary sequences between them. In conclusion, the present findings suggest that the Ucn gene locus naturally undergoes bidirectional transcription yielding a sense and an antisense RNA expanding the spectrum of antisense RNAs originated from the same genomic loci to antisense transcripts that are spliced using these non-consensus CT-AC splice sites.
最近，已证明大鼠脑和其他组织中存在反义促肾上腺皮质激素原（Ucn）转录本的内源性表达。在本研究中，我们通过特异性链RT-PCR和原位杂交这两种互补技术，展示了一种更大尺寸的新型反义Ucn RNA的天然表达。对总RNA进行特异性链RT-PCR、克隆和序列分析，以及观察到的两种反义Ucn RNA不同的亚细胞定位，表明这种新型反义Ucn转录本对应于先前描述的反义Ucn RNA的未成熟形式。序列分析表明，这种未成熟的反义Ucn转录本使用非保守的CT-AC剪接位点，与其正义对应物完全互补。对聚腺苷酸（poly(A)）RNA进行特异性链RT-PCR后，也扩增出了成熟的反义Ucn转录本，这表明成熟的反义Ucn转录本是多聚腺苷酸化的。我们还证明，与正义Ucn RNA启动子互补的区域，包括TATA盒，是反义Ucn RNA的一部分。最后，我们表明与Ucn mRNA 3'端互补的区域可作为反义Ucn RNA转录的功能性启动子。因此，结果表明正义和反义Ucn RNA的3'端是它们之间唯一的非互补序列。总之，本研究结果表明，Ucn基因位点自然地进行双向转录，产生正义和反义RNA，将源自同一基因组位点的反义RNA谱扩展到使用这些非保守CT-AC剪接位点进行剪接的反义转录本。
