Identification and characterisation of a novel antisense non-coding RNA from the RBM5 gene locus.
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.
Rintala-Maki
Nina D
ND
Tumour Biology Group, Regional Cancer Program, Sudbury Regional Hospital, Sudbury, Canada.
Sutherland
Leslie C
LC
D017398:Alternative Splicing / Q000235:genetics; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D016372:RNA, Antisense / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D022661:RNA, Untranslated / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C473448:RBM5 protein, human; D016372:RNA, Antisense; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins; D025521:Tumor Suppressor Proteins