Symmetrical In Vivo Transcription of Mitochondrial DNA in HeLa Cells

Abstract

RNA DNA hybridization experiments utilizing separated strands of HeLa mitochondrial DNA and mit-RNA from HeLa cells exposed to short pulses of have shown that the labeled RNA hybridizes with both the light (L) and the heavy (H) strand, though to a different relative extent depending upon the labeling time. Thus, hybridization of pulse-labeled RNA is about equal with the two strands when the pulse is very short (1-5 min), and becomes more and more predominant with the H strand with increasing pulse length. Pulse-labeled fast-sedimenting mit-RNA forms RNase-resistant doublestranded structures up to more than 5 jAm long when selfannealed or annealed with an excess of unlabeled mit-RNA. These observations and the previous evidence of complete transcription of the H strand strongly suggest that mit-DNA is transcribed in HeLa cells symmetrically over a considerable portion of its length, with the transcript of the L strand being rapidly degraded or otherwise removed from the mitochondrial fraction.RNA -DNA hybridization experiments utilizing separated strands of HeLa mit-DNA and mit-RNA from HeLa cells uniformly labeled with [5-3H]uridine have shown that this RNA contains sequences complementary to the whole or almost whole length of the heavy (H) strand and to a short portion of the light (L) strand (1, 2). These results have been interpreted to indicate that mit-DNA in HeLa cells is completely or almost completely transcribed, and, furthermore, that this transcription occurs almost exclusively from the H strand, as previously reported for rat liver mit-DNA (3). However, these results did not exclude the possibility that the L strand is also transcribed to a considerable extent, if not completely, with the majority of this transcript being subsequently degraded or otherwise removed from the mitochondrial fraction. In the course of investigations on the synthesis and processing of mit-RNA, an analysis of the hybridization properties of RNA from HeLa cells labeled for a short term with [5-3H]uridine has unexpectedly revealed that the above mentioned possibility is indeed true. This paper describes these experiments. MATERIALS AND METHODS 1757and extraction of mitochondrion-associated RNA have been previously described (4) (the isopycnic centrifugation was performed here for 40 min at 25,000 rpm in the Spinco SW25.1 rotor).The RNA samples, fractionated on a sucrose gradient, to be used in RNA DNA or RNA RNA hybridizations were subjected to DNase digestion and phenol-sodium dodecylsulfate (SDS) extraction and either sedimented again through a sucrose gradient or fractionated on Sephadex G-100 (1).Hybridization of mit-RNA with H or L mit-DNA strands (which had been separated in an alkaline CsCl density gradi- for 4 hr; after rapid cooling, the mixtures were treated with 5 ,ug/ml of previously heated pancreatic RNase for 20 min at room temperature, then filtered through nitrocellulose membranes, which were washed with 100 ml of 2X SSC (SSC = 0.15 M NaCl-0.015 M Na citrate). Unless otherwise specified, the RNA pre...

The RNase P Associated with HeLa Cell Mitochondria Contains an Essential RNA Component Identical in Sequence to That of the Nuclear RNase P

“…It should be noted that the mtDNA light-strand transcripts, which contain the sequences of eight tRNAs, are synthesized at a rate at least 10 to 15 times higher than required to account for the steadystate levels of these tRNAs (7). Furthermore, it is known that these transcripts have a much shorter half-life than the heavystrand transcripts and do not accumulate to any significant extent (2,10). It is therefore a plausible hypothesis that the vast majority of these transcripts decay before the occurrence of any processing, which would lead to a maturation and stabilization of the tRNAs (7).…”

Synthesis of Complementary RNA Sequences During Productive Adenovirus Infection

“…Aloni and coworkers have reported that mitochrondrial DNA and DNA from papovaviruses are transcribed in a manner which leads to synthesis of complementary RNA sequences (19)(20)(21)(22). From studies on transcription of SV40 DNA Aloni (20) has proposed a model which postulates that both strands of SV40 DNA are completely transcribed.…”

Long noncoding RNAs are generated from the mitochondrial genome and regulated by nuclear-encoded proteins

“…Mitochondrial genes for proteins and tRNAs are located on both the heavy and light strands of the genome, which are transcribed as large polycistronic transcripts covering almost the entire length of each strand (Aloni and Attardi 1971;Murphy et al 1975;Montoya et al 1981;Mercer et al 2011). A third transcript covering the start of the heavy strand and the two rRNA genes is also produced (Christianson and Clayton 1988).…”