We reported the presence in human cells of a noncoding mitochondrial RNA that contains an inverted repeat (IR) of 815 nucleotides (nt) covalently linked to the 5 end of the mitochondrial 16S RNA (16S mtrRNA). The transcript contains a stem-loop structure and is expressed in human proliferating cells but not in resting cells. Here, we demonstrate that, in addition to this transcript, normal human proliferating cells in culture express 2 antisense mitochondrial transcripts. These transcripts also contain stem-loop structures but strikingly they are down-regulated in tumor cell lines and tumor cells present in 17 different tumor types. The differential expression of these transcripts distinguishes normal from tumor cells and might contribute a unique vision on cancer biology and diagnostics.differential expression in cancer ͉ RNAs with stem-loop structures R ecently, we described a novel human mitochondrial transcript of 2,374 nt that contains a long inverted repeat (IR) linked to the 5Ј end of the 16S mitochondrial rRNA (16S mtrRNA) (1, 2), which we designated noncoding mitochondrial RNA or ncmtRNA (2). The IR generates a stem-loop structure with an 820-bp doublestranded region and a 40-nt loop (2). In situ hybridization (ISH) showed that the ncmtRNA is overexpressed in several tumor cell lines but not in nondividing cells, suggesting that the ncmtRNA may play a role in cell proliferation (2).Because the results described before were obtained using tumor cell lines (2), we asked whether the ncmtRNA (from now on, sense ncmtRNA, SncmtRNA) is expressed in normal proliferating cells. Here we show that ISH of human umbilical vein endothelial cells (HUVEC), foreskin keratinocytes (HFK) (3), and human tonsil endothelial cells (HUTEC) (4) also express the SncmtRNA. However, and in striking contrast with tumor cell lines, ISH of normal proliferating cells revealed expression of antisense transcripts. These molecules were identified as 2 unique transcripts containing IRs linked to the 5Ј region of the antisense 16S mtRNA transcribed from the L-strand of the mtDNA (1, 2). We named these transcripts antisense ncmtRNA-1 (ASncmtRNA-1) and antisense ncmtRNA-2 (ASncmtRNA-2). Finally, we show that the antisense transcripts are also expressed in proliferating cells present in normal human tissues but are down-regulated in cells present in human tumors of different types and patients.

Previously, we reported the presence in mouse cells of a mitochondrial RNA which contains an inverted repeat (IR) of 121 nucleotides (nt) covalently linked to the 5′ end of the mitochondrial 16S RNA (16S mtrRNA). Here, we report the structure of an equivalent transcript of 2374 nt which is over-expressed in human proliferating cells but not in resting cells. The transcript contains a hairpin structure comprising an IR of 815 nt linked to the 5′ end of the 16S mtrRNA and forming a long double-stranded structure or stem and a loop of 40 nt. The stem is resistant to RNase A and can be detected and isolated after digestion with the enzyme. This novel transcript is a non-coding RNA (ncRNA) and several evidences suggest that the transcript is synthesized in mitochondria. The expression of this transcript can be induced in resting lymphocytes stimulated with phytohaemagglutinin (PHA). Moreover, aphidicolin treatment of DU145 cells reversibly blocks proliferation and expression of the transcript. If the drug is removed, the cells re-assume proliferation and over-express the ncmtRNA. These results suggest that the expression of the ncmtRNA correlates with the replicative state of the cell and it may play a role in cell proliferation.

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