Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis

Abstract

Abstract. A small RNA encoded within the nucleus is an essential subunit of a RNA processing endonuclease (RNase MRP) hypothesized to generate primers for mitochondrial DNA replication from the heavy strand origin of replication. Controversy has arisen, however, concerning the authenticity of an intramitochondrial pool of MRP RNA, and has called into question the existence of pathways for nucleomitochondrial transport of nucleic acids in animal cells. In an effort to resolve this controversy, we combined ultrastructural in situ hybridization and biochemical techniques to assess the subeellular partitioning of MRP RNA. Cryosections of mouse cardiomyocytes were hybridized with biotin-labeled RNA probes complementary to different regions of MRP RNA and varying in length from 115 to 230 nucleotides, followed by immunogold labeling. In addition, we transfected mouse C2C12 myogenic cells with constructs bearing mutated forms of the mouse MRP RNA gene and compared the relative abundance of the resulting transcripts to that of control RNAs within whole cell and mitochondrial fractions. In the former analysis we observed preferential localization of MRP RNA to nucleoli and mitochondria in comparison to the nucleoplasm and cytoplasm. In the latter series of studies we observed that wild-type MRP RNA partitions to the mitochondrial fraction by comparison to other RNA transcripts that are localized to the extramitochondrial cytoplasmic space (28S rRNA) or to the nucleoplasm (U1 snRNA). Deletions within 5' or 3' regions of the MRP RNA gene produced transcripts that remain competent for mitochondrial targeting. In contrast, deletion of the midportion of the coding region (nt 118 to 175) of the MRP RNA gene resulted in transcripts that fail to partition to the mitochondrial fraction. We conclude that an authentic intramitochondrial pool of MRP RNA is present in these actively respiring cells, and that specific structural determinants within the MRP RNA molecule permit it to be partitioned to mitochondria.

Evidence for the Presence of 5S rRNA in Mammalian Mitochondria

“…Furthermore, only one of three tRNA Gln isoforms is imported into Tetrahymena mitochondria (Rusconi and Cech, 1996), and only tRNA Lys , and no other tRNA, is imported into yeast mitochondria , most likely on the basis of sequence-specific determinants (Entelis et al, 1998). Similarly, deletion experiments imply that mouse MRP RNA also contains importation determinants (Li et al, 1994). Thus, it is reasonable to assume that a similar importation specificity applies to mammalian 5S rRNA.…”

“…are encoded by nuclear DNA (nDNA), synthesized in the cytosol, and imported into the organelle (Schatz and Dobberstein, 1996;Neupert, 1997). The same is true for all enzymes involved in the myriad metabolic pathways that take place in the mitochondrial environment.Interestingly, at least two mitochondrial enzymes, RNase MRP (a site-specific endoribonuclease involved in primer RNA metabolism in mammalian mitochondria [Chang and Clayton, 1987;Topper and Clayton, 1990;Li et al, 1994]) and RNase P (an endoribonuclease involved in tRNA processing [Doerson et al, 1985]), are ribonucleoproteins that contain an RNA moiety that is encoded by nDNA and is imported into the organelle. However, unlike the mechanisms for protein import into mitochondria, the mechanisms of RNA import into mitochondria are poorly understood.…”

Functional equivalence of hairpins in the RNA subunits of RNase MRP and RNase P in Saccharomyces cerevisiae

“…RNase MRP (mitochondrial RNA processing) was first identified as a ribonucleoprotein enzyme capable of in vitro formation of primers for mitochondrial DNA replication (Chang & Clayton, 1987;Schmitt & Clayton, 1992;)+ However, most of the enzyme is localized to the nucleolus (Reddy et al+, 1981;Reimer et al+, 1988;Kiss & Filipowicz, 1992), with only minute amounts found in the mitochondria (Topper et al+, 1992;Li et al+, 1994)+ Its cellular location suggested that the primary function of RNase MRP is in ribosome biogenesis+ Indeed, recent genetic evidence has established that RNase MRP is required for normal processing of 5+8S rRNA in Saccharomyces cerevisiae (Schmitt & Clayton, 1993b;Chu et al+, 1994;Lygerou et al+, 1994Lygerou et al+, , 1996+ The 5+8S rRNA of S. cerevisiae exists in short (5+8S S ) and long (5+8S L ) versions, differing by 6-7 nt at their 59 ends (Rubin, 1974;Lindahl et al+, 1992)+ The ratio of 5+8S S :5+8S L in wild-type cells is 5-10:1+ However, a single base change in the RNA subunit of RNase MRP results in a substantially decreased ratio of 5+8S S to 5+8S L , as well as accumulation of a very long version of 5+8S rRNA with 149 bases of the ITS1 sequence attached at the 59 end (Shuai & Warner, 1991;Lindahl et al+, 1992;Chu et al+, 1994)+ These observations together with other experiments (Henry et al+, 1994) have led to the conclusion that RNase MRP is responsible for initiating the processing of the short form of 5+8S rRNA by cleaving the pre-rRNA in the transcribed spacer between the 18S and 5+8S moieties+ RNase MRP is structurally related to RNase P, the endonucleolytic enzyme that creates the mature 59 end of tRNAs+ Both enzymes have one RNA subunit (here called MRP RNA and P RNA) that share secondarystructure features (Forster & Altman, 1990;Schmitt & Clayton, 1993a)+ Furthermore, RNases MRP and P of S. cerevisiae contain eight common protein subunits (Lygerou et al+, 1994;Chu et al+, 1997;Dichtl & Tollervey, 1997;Stolc & Altman, 1997;Chamberlain et al+, 1998;Stolc et al+, 1998); only one protein specific to each of the two nucleases has been identified (Schmitt & Clayton, 1994;…”

Peptide nucleic acid delivery to human mitochondria

“…25,26 Schon and colleagues have recently reported that a 5S ribosomal RNA may be imported, but its function has yet to be determined. 27 It is therefore possible that mammalian mitochondria do not possess an efficient import mechanism for nucleic acids.…”