SUBCELLULAR PARTITIONING OF MRP RNA ASSESSED BY ULTRASTRUCTURAL AND BIOCHEMICAL-ANALYSIS

A small RNA encoded within the nucleus is an essential subunit of a RN A processing endonuclease (RNase MRP) hypothesized to generate primers for mitochondrial DNA replication from the heavy strand origin of rep lication. Controversy has arisen, however, concerning the authenticity of an intramitochondrial pool of MRP RNA, and has called into questio n the existence of pathways for nucleomitochondrial transport of nucle ic acids in animal cells. In an effort to resolve this controversy, we combined ultrastructural in situ hybridization and biochemical techni ques to assess the subcellular partitioning of MRP RNA, Cryosections o f mouse cardiomyocytes were hybridized with biotin-labeled RNA probes complementary to different regions of MRP RNA and varying in length fr om 115 to 230 nucleotides, followed by immunogold labeling. In additio n, we transfected mouse C2C12 myogenic cells with constructs bearing m utated forms of the mouse MRP RNA gene and compared the relative abund ance 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 c omparison to the nucleoplasm and cytoplasm. In the latter series of st udies we observed that wild-type MRP RNA partitions to the mitochondri al fraction by comparison to other RNA transcripts that are localized to the extramitochondrial cytoplasmic space (28S rRNA) or to the nucle oplasm (U1 snRNA). Deletions within 5' or 3' regions of the MRP RNA ge ne produced transcripts that remain competent for mitochondrial target ing. 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 p artition to the mitochondrial fraction. We conclude that an authentic intramitochondrial pool of MRP RNA is present in these actively respir ing cells, and that specific structural determinants within the MRP RN A molecule permit it to be partitioned to mitochondria.