Mutually exclusive synthetic pathways for sea urchin mitochondrial rRNA and mRNA.

Abstract

The structure and abundance of mitochondrial transcripts in sea urchin embryos were investigated by a combination of RNA blot-hybridization, SI mapping, and primer extension assays. Between the egg and blastula stages, the relative abundance of mitochondrial rRNAs declined slightly, while that of mitochondrial mRNAs increased up to 10-fold. Fine mapping of the termini of the rRNAs and of the adjacent transcripts indicated that, although they appeared to be butt-joined at their 5' ends to the upstream transcripts, tRNA-Phe 5' to the small subunit (12S) rRNA and NADH dehydrogenase subunit 2 mRNA 5' to the large subunit (16S) rRNA, respectively, their 3' ends were found to overlap the 5' ends of the downstream transcripts. 12S rRNA was found to extend 7 to 13 nucleotides into the sequence of tRNA-Glu; 16S rRNA was shown to terminate 3 to 5 nucleotides inside the coding region of cytochrome oxidase subunit I (COI) and 8 to 10 nucleotides from the mapped 5' end of COI mRNA. The rRNAs and the downstream transcripts must therefore be synthesized by distinct pathways, either by alternative processing of the same primary transcript(s) or by processing of different precursors. In either case, the events which select the ribosomal 3' ends preclude the production of functional transcripts of the downstream genes from the same precursor molecule. No developmental alterations in transcript structure were detected. We propose that mitochondrial RNA levels are regulated in early development by the selection of alternate and mutually exclusive RNA-processing pathways.Efficient protein synthesis in all cells and organelles depends on the maintenance of a large excess of rRNA over mRNA. In mammalian mitochondria, rRNA levels are up to 50 to 100 times those of individual mRNAs (2). This appears to be achieved by differential rates of synthesis of the two classes of transcript, with control exercised at the level of transcriptional termination (2, 9, 11, 13, 24). Transcription of the major coding strand of mammalian mitochondrial DNA (mtDNA) commences at the gene for tRNA-Phe (23, 24) just upstream of the effectively contiguous rRNA genes, which are separated only by tRNA-Val. Early termination of transcription at the 3' end of the large subunit (16S) rRNA gene results in the production of a subgenomic transcript which acts as a precursor to both rRNAs and probably to the two tRNAs. Relatively infrequent readthrough of this terminator is believed to result in the production of a genome-length transcript which is processed to the remaining mRNAs and tRNAs encoded on this strand.In sea urchins (Fig. 1) represented in the RNA populations of eggs and embryos (7,28), and at least one mRNA, encoding cytochrome oxidase subunit I (COI), accumulates during embryogenesis (28), whereas the level of the large subunit (16S) rRNA remains approximately constant. The ratio of mitochondrial mRNA to rRNA would therefore appear to be under developmental regulation.In order to gain insight into the mechanism by which this ratio is determined and modula...

Transcript Mapping and Genome Annotation of Ascidian mtDNA Using EST Data

“…This motif is well conserved near the 3Ј end of 16S rRNA in a wide range of organisms, from protozoa to metazoa, independent of the identity of the adjacent gene (Valverde et al 1994). In sea urchin, the rRNA termination box is located exactly where the 16S rRNA transcript terminates in vivo (Elliott and Jacobs 1989), at the same relative position inferred for Halocynthia from EST data. The rRNA transcription termination box has been found also in Ciona mtDNA within the tRNA-Ile sequence (C. Gissi, F. Iannelli, and G. Pesole, in prep.…”

“…Large mt genomes with relaxed packaging of genetic information are transcribed into multiple small transcripts from individual promoters scattered over the genome (i.e., the yeast Saccharomyces cerevisiae and plants) (Costanzo and Fox 1990;Binder et al 1996). The details of transcription have been investigated widely only in mammals (human and rodents), whereas partial data on mapping and processing of mt transcripts are available for a few other animals, such as sea urchin Paracentrotus lividus (Elliott and Jacobs 1989;Cantatore et al 1990), and Drosophila (Berthier et al 1986). In mammals, the promoters for transcription of the mtDNA heavy (H) and light (L) strands are located in the D-loop region and lead to the synthesis, for each strand, of a long primary transcript covering the whole or almost the whole genome.…”

Cloning and characterisation of mtDBP, a DNA-binding protein which binds two distinct regions of sea urchin mitochondrial DNA

“…Moreover, mtDBP displays a significant sequence homology with the human mitochondrial transcription termination factor mTERF (15), a protein that also binds DNA as a monomer and contains three leucine zippers (16). These data, together with the above reported features of the sea urchin mitochondrial genetic system (1)(2)(3)(6)(7)(8)(9)(10)(11)(12)(13)(14), point to a dual role of mtDBP in regulating both mtDNA replication and transcription.…”

“…The L-strand replication origin has not formally been mapped; the lagging strand probably initiates from multiple points, one of which appears to be located near the main H-strand replication pause site, at the junction between the genes for ATPase 6 and COIII (7). The sea urchin mitochondrial transcription mechanism appears to differ from that existing in vertebrates: studies in Paracentrotus lividus and S.purpuratus supported a mechanism based on multiple and probably overlapping transcription units in which post-transcriptional processing events play a relevant role (8,9). In particular, it is intriguing that the 3′-ends of 12S and 16S rRNAs were both located a few bases inside the adjacent downstream gene.…”

A conserved heptamer motif for ribosomal RNA transcription termination in animal mitochondria.

“…Instead, the gene encoding the COI maps in this region ofthe mtDNA. By using S1 nuclease-mapping analysis (18), the 16S rRNA transcription has been reported to terminate 3-5 nt inside the COI gene. Fig.…”