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.

13 C{2 H} rotational echo double resonance NMR has been used to provide the first evidence for the formation of quinone-derived cross-links in mussel byssal plaques. Labeling of byssus was achieved by allowing mussels to filter feed from seawater containing L-[phenol-4-13 C]tyrosine and L-[ring-d 4 ]tyrosine for 2 days. Plaques and threads were harvested from two groups of mussels over a period of 28 days. One group was maintained in stationary water while the other was exposed to turbulent flow at 20 cm/s. The flow-stressed byssal plaques exhibited significantly enhanced levels of 5, 5-di-dihydroxyphenylalanine cross-links. The average concentration of di-dihydroxyphenylalanine cross-links in byssal plaques is 1 per 1800 total protein amino acid residues.The attachment strategies of marine organisms that rely on DOPA-containing 1 adhesive proteins have recently come under much scrutiny. A number of these organisms, including tubebuilding polychaetes, black corals, ascidians, and mussels, cure their proteinaceous adhesives by a process called "quinone tanning" (1). Despite frequent references to in vitro studies postulating that quinone tanning involves nucleophilic addition of amine groups to quinones (2), nothing of substance is known about the actual cross-linking chemistry in these organisms. Indeed, previous efforts from our own laboratories seemed to rule out the existence of lysine-aromatic or aromaticaromatic coupling products in the mussel byssus (3, 4). In 1994, Dolmer and Svane (5) reported that individual threads in a mussel byssus behaved as "smart materials" in flow: by doubling the flow of seawater, the tensile strength of the threads could be doubled. This suggested that there were different degrees of quinone-tanning in a given material that relied in some way on maturation by imposed stresses. This paper reports on the continuation of work on the biosynthetic labeling of byssus with 13 C-and 2 H-containing analogs of tyrosine and the in situ analysis of this label-rich material by rotational echo double resonance (REDOR) NMR (6). We have repeated the byssus labeling under high flow conditions and compared directly REDOR spectra of byssal plaques collected under stressed (flow) and unstressed (stationary or minimal flow) conditions. The difference between these REDOR spectra reveal the preferential routing of tyrosine labels to diphenolics. The 13 C{ 2 H} REDOR dephasing is only consistent with the formation of 5, 5Ј-di-DOPA covalent crosslinks stabilizing the byssal plaques formed under stress. EXPERIMENTAL PROCEDURESMussel Labeling-170 Adult mussels (Mytilus edulis) were collected in the vicinity of Roosevelt Inlet at Lewes, Delaware. The mussels ranged in size from 3 to 10 cm. They were tethered to acrylic plates as described previously (4). The plates were put into two 140-liter marine aquarium tanks with salt water (no flow) at 14°C. The tanks were aerated. Labels H 4 ]tyrosine. The mussels were incubated with the labels for 48 h, after which control samples of plaques and threads were harv...