K. Li et al., SUBCELLULAR PARTITIONING OF MRP RNA ASSESSED BY ULTRASTRUCTURAL AND BIOCHEMICAL-ANALYSIS, The Journal of cell biology, 124(6), 1994, pp. 871-882
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.