In Vitro Expression Analysis Reveals HML6-c14 to Be an Attractive Research Target

Abstract

HML6-c14, a long terminal repeat (LTR)-type retrotransposon identified by expressed sequence tag (EST) database screening, was found to undergo RNA processing resembling that of placental tissue by in vitro expression analysis. Previous in situ hybridization studies using normal placental tissue showed that the transcript remained in the nucleus. However, among the transcripts forcedly expressed in cultured cells, the transcript that retained the 3.3 kb intron was observed in the nucleus, and a part of the spliced transcript was observed outside the nucleus. To verify whether this cytoplasmic transcript could be translated, we examined the coding potential of the open reading frame (ORF), consisting of 109 codons on the spliced transcript, along with two other putative ORFs detected in the intronic region. As a result, none of the ORF-derived products could be detected by Western blotting as fusion proteins tagged with the FLAG epitope, suggesting that HML6-c14 belongs to a group of long non-coding RNA (lncRNA) genes. Promoter analysis of the upstream 6.4 kb genomic region also suggested that the 5′-LTR itself potentially retains high promoter activity. Despite losing the ability to produce functional proteins, HML6-c14 continues to retain its transcriptional ability while converting to an lncRNA gene, which is an interesting subject for future research.