Detection and cDNA cloning of H-strand mitochondrial regulatory region RNAs in cultured human cells and human tissues

摘要

In the mitochondrion, essential genetic elements for replication and transcription are mostly housed within a short segment of its DNA located between tRNA(Phe) and tRNA(Pro) genes, which is called mitochondrial regulatory region (mrr). RNAs are known to be transcribed from mrr, the structures and the functions of which are yet to be fully characterized. We detected ca. 1.3 kb H-strand transcripts of mrr (mrrH-RNAs), and 0.2 kb L-strand transcripts of mrr (mrrL-RNAs) in various human cultured cells and tissues using double stranded mrrDNA probes. The steady state levels of mrrL-RNAs were generally high in cultured cells, while they varied among tissues. On the other hand, the levels of mrrH-RNAs varied among tissues and among cultured cells. A tendency was observed in these cells and tissues that a high level of mrrL-RNA is associated with cell proliferation, and a high level of mrrH-RNA with differentiation. Several cDNA clones to 1.3 kb mrrH-RNA were obtained from human skeletal muscle polyadenylated RNAs. The 5' terminus of the 1.3 kb RNA was determined to be at nucleotide position 15953 which is immediately downstream of tRNA(T)hr sequence. Polyadenylation site for most of the clones was demonstrated to be at nucleotide position 576 which is immediately upstream of tRNA(Phe) sequence. The longest cDNA insert obtained was 1177 bps long spanning from nucleotide positions 15969 to 576 which could code for a peptide of 76 amino acids. The cDNAs isolated here are the first cDNA clones reported to human mrrH-RNAs. These results, together with previous results, further substantiate that polyadenylated mrrH- and mrrL-RNAs are commonly present at varying levels among human tissues and cells. The 3' end sequences of the cloned mrrH-cDNA provides with insights into the mechanisms of transcription termination. The cDNA clones will provide tools to further the study of the function of mrr RNAs. References: 41