Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis

Kang Li, Cynthia S. Smagula, William J. Parsons, James A. Richardson, Mark Gonzalez, Herbert K. Hagler, R. Sanders Williams

Research output: Contribution to journalArticle

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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 nucleo-mitochondrial 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 subcellular 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.

Original languageEnglish (US)
Pages (from-to)871-882
Number of pages12
JournalJournal of Cell Biology
Volume124
Issue number6
StatePublished - Mar 1994

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RNA
Mitochondria
Genes
RNA Probes
Replication Origin
Endonucleases
Ribonucleases
Biotin
DNA Replication
Mitochondrial DNA
Cardiac Myocytes
Nucleic Acids
In Situ Hybridization
Cytoplasm
Nucleotides

ASJC Scopus subject areas

  • Cell Biology

Cite this

Li, K., Smagula, C. S., Parsons, W. J., Richardson, J. A., Gonzalez, M., Hagler, H. K., & Williams, R. S. (1994). Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis. Journal of Cell Biology, 124(6), 871-882.

Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis. / Li, Kang; Smagula, Cynthia S.; Parsons, William J.; Richardson, James A.; Gonzalez, Mark; Hagler, Herbert K.; Williams, R. Sanders.

In: Journal of Cell Biology, Vol. 124, No. 6, 03.1994, p. 871-882.

Research output: Contribution to journalArticle

Li, K, Smagula, CS, Parsons, WJ, Richardson, JA, Gonzalez, M, Hagler, HK & Williams, RS 1994, 'Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis', Journal of Cell Biology, vol. 124, no. 6, pp. 871-882.
Li K, Smagula CS, Parsons WJ, Richardson JA, Gonzalez M, Hagler HK et al. Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis. Journal of Cell Biology. 1994 Mar;124(6):871-882.
Li, Kang ; Smagula, Cynthia S. ; Parsons, William J. ; Richardson, James A. ; Gonzalez, Mark ; Hagler, Herbert K. ; Williams, R. Sanders. / Subcellular partitioning of MRP RNA assessed by ultrastructural and biochemical analysis. In: Journal of Cell Biology. 1994 ; Vol. 124, No. 6. pp. 871-882.
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