American Society of Human Genetics Meeting; October 19-23, 1999; San Francisco, CA.

CONSORTIUM TO CLONE AND MAP AND STUDY HUMAN MITOCHONDRIAL RIBOSOMAL PROTEIN GENES
James E. Sylvester¹, Hanns-Ruediger Graack², Jiguo Liu³, Edward B. Mougey¹, Beth A. Maguire¹, Nathan Fischel-Ghodsian⁴, Brigitte Wittmann-Liebold⁵ and Thomas W. O’Brien³. 
1) Nemours Children's Clinic, Jacksonville, FL; 2) Institute for Genetics, AG Kress, Free University of Berlin, Germany; 3) University of Florida, Gainesville, FL; 4) Cedars-Sinai Medical Center, Los Angeles, CA; 5) Max-Delbruck-Center for Molecular Medicine, Berlin, Germany.

Mammalian mitochondria have their own separate translation system comprised of components distinct from their cytoplasmic counterpart. Whereas mitochondrial DNA encodes tRNAs and the two rRNAs, the remaining genes for the translation system are found in nuclear DNA. Upward of 100 mitochondrial ribosomal proteins (MRPs) are imported into the mitochondria, assembled into ribosomes which are responsible for translating the 13 mRNAs for oxidative phosphorylation proteins. Since mutations in mitochondrial tRNA and rRNA can cause various pathological states, we hypothesize that mutations in MRP genes are also candidates for human disorders. A necessary first step, therefore, is to identify, clone, and map the gene for each human MRP. Our approach is to use N-terminal and internal amino acid sequence data obtained from purified bovine or rat MRPs (Goldschmidt-Reisin, et al., J.Biol.Chem. 273: 34828, 1998) to search EST databases. A representative I.M.A.G.E. clone (ATTC) is purchased and used to screen a human lambda genomic library. Chromosome map positions are ascertained by in situ hybridization (FISH) with genomic sequences and/or by using in silico methods to search Genbank and GeneMap through NCBI. At present, we have over 30 different human MRPs at various stages of characterization and have established a consortium of investigators to complete the project. We are currently investigating one MRP as a potential candidate for Russell-Silver Syndrome (RSS) (see abstract, Mougey, et al., this meeting). In addition to studying their clinical relevance, long term characterization of MRP genes should lead to important insights into mammalian evolution, coordinate regulation of nuclear and mitochondrial gene expression, and ribosome function.
 

American Society for Human Genetics Meeting
Philadelphia, October 3-8, 2000

HEART-SPECIFIC SPLICE-VARIANT OF HUMAN MITOCHONDRIAL RIBOSOMAL PROTEIN L5 (MRP-L5).
O. Spirina1, Y. Bykhovskaya1, A.V. Kajava2, T.W. O'Brien3, D.P. Nierlich4,
E.B. Mougey5, J.F. Sylvester5, H.R. Graak6, B. Wittman-Liebold7, N.
Fischel-Ghodsian1. 
1)Cedars-Sinai Medical Center and UCLA School of Medicine, Los Angeles, CA, 2)Center for Molecular Modeling, NIH, Bethesda, MD, 3)University of Florida, Gainesville, FL, 4) UCLA, Los Angeles, CA, 5)Nemours Children's Clinic, Jacksonville, FL, 6)Free University, Berlin, Germany, 7)Max-Delbruck Center, Berlin, Germany.
It has been proposed that splice variants of proteins involved in mitochondrial RNA processing and translation may be involved in the tissue specificity of mitochondrial DNA disease mutations (Mol Genet Metab 65:97, 1998). To identify and characterize the structural components of mitochondrial RNA processing and translation, the Mammalian Mitochondrial Ribosomal Consortium has been formed. The 338 a.a. long MRP-L5 was identified (J Biol Chem 274:36043, 1999), and its transcript was screened
for tissue specific splice variants. Screening of the EST databases revealed a single putative splice variant, due to the insertion of an exon consisting of 89 nucleotides prior to the last exon. Screening of multiple cDNA libraries revealed this inserted exon to be present only in heart tissue, in addition to the predominant MRP-L5 transcript. Sequencing of this region confirmed the EST sequence, and showed in the splice variant a termination triplet at the beginning of the last exon. Thus the inserted exon replaces the regular last exon, and creates a new 353 a.a. long protein (MRP-L5+) with a differnt C-terminus. Sequence analysis and 3-D modeling reveal similarity between MRP-L5 and threonyl-t-RNA synthetases, and likely RNA binding sites within MRP-L5, with the C-terminus in proximity to the RNA binding sites. Sequence analysis of MRP-L5+ also suggests a likely transmembrane domain at the C-terminus. Thus it is possible that the MRP-L5+ C-terminus could interfere with RNA binding and may have gained a
transmembrane domain. Further studies will be required to elucidate the functional significance of MRP-L5+.  This work was done as part of the Mammalian Mitochondrial Ribosomal Consortium and is supported by NIH/NIDCD grant RO1DC04092.
(Nota bene: MRPL5 is bovine-based nomenclature; the human alias for this protein is MRPL42 in the HGNC nomenclature)
 
 
 
 

1) Annual Meeting of the German Society for Cell Biology (Karlsruhe, March 26 - 30, 2000;

2) 2nd Colloquium on Mitochondria and Myopathies (Halle/Saale, March 31-April 2, 2000).

Mammalian Mitochondrial Ribosomal Proteins (MRPs) and their corresponding genes: Identification, Characterization and Genetics

Hanns-Rüdiger Graack1, James E. Sylvester2, Edward B. Mougey2, Nathan Fischel-Ghodsian3, Brigitte Wittmann-Liebold4 and Thomas W. O’Brien5. The Mammalian Mitochondrial Ribosomal Consortium: 1) Institute for Genetics, AG Kress, Free University of Berlin, Germany; 2) Nemours Children's Clinic, Jacksonville, Florida, USA; 3) Cedars-Sinai Medical Center, Los Angeles, California, USA; 4) Max-Delbrück-Center for Molecular Medicine, Berlin, Germany; 5) University of Florida, Gainesville, Florida, USA.

Mammalian mitochondria have their own separate translation system comprised of components distinct from their cytoplasmic counterpart. Whereas mitochondrial DNA encodes tRNAs and the two rRNAs, the remaining genes for the translation system are found in nuclear DNA. Upward of 100 mitochondrial ribosomal proteins (MRPs) are imported into the mitochondria, assembled into ribosomes which are responsible for translating the 13 mRNAs for oxidative phosphorylation proteins in humans. Since mutations in mitochondrial tRNA and rRNA can cause various pathological states, we hypothesize that mutations in MRP genes are also candidates for human disorders. A necessary first step, therefore, is to identify, clone, and map the gene for each human MRP. Our approach is to use N-terminal and internal amino acid sequence data obtained from purified bovine or rat MRPs (1, 2, 3) to search EST databases. A representative I.M.A.G.E. clone (ATTC) is purchased and used to screen a human lambda genomic library. Chromosome map positions are ascertained by radiation hybrid mapping or in situ hybridization (FISH) with genomic sequences and/or by using in silico methods to search Genbank and GeneMap through NCBI. At present, we have over 30 different human MRPs at various stages of characterization and have established a consortium of investigators to complete the project. In addition to studying their clinical relevance, long term characterization of MRP genes should lead to important insights into mammalian evolution, coordinate regulation of nuclear and mitochondrial gene expression, and ribosome function.
(1) Goldschmidt-Reisin, S. et al. (1998). J. Biol. Chem. 273, 34828-34836. (2) Graack, H.-R. et al. (1999) Biochemistry, 38, 16569-16577. (3) O’Brien, T. W. et al. (1999) J. Biol. Chem. 274, 36043-36051.
NFG and TWO gratefully acknowledge support by NIH/NIDCD grant R01 DC04092-01.
 

American Society for Human Genetics Meeting
San Diego, October 12-16, 2001

MAPPING GENES FOR HUMAN MITOCHONDRIAL RIBOSOMAL PROTEINS
Thomas W. O’Brien1, Hanns-Ruediger Graack2, Nathan Fischel-Ghodsian 3, Edward B. Mougey4, Beth A. Maguire4, Brigitte Wittmann-Liebold5, Donald P. Nierlich6, James E. Sylvester4.  1) University of Florida, Gainesville, FL; 2) Max Planck Institute for Infection Biology, Berlin, Germany; 3) Cedars-Sinai Medical Center, Los Angeles, CA; 4) Nemours Children's Clinic, Jacksonville, FL); 5) Max-Delbruck-Center for Molecular Medicine, Berlin, Germany; 6) UCLA, Los Angeles, CA.
Mitochondrial DNA encodes tRNAs and rRNAs, but the other genes for the mitochondrial translation system are found in nuclear DNA. Upward of 85 mammalian mitochondrial ribosomal proteins (MRPs) are imported into mitochondria where they assemble into ribosomes that are responsible for translating the 13 mRNAs for essential proteins of the oxidative phosphorylation system.  Since mutations in mitochondrial tRNA and rRNA can cause various pathological states, we hypothesize that mutations in MRP genes are also candidates for human disorders. Our approach is to use N-terminal and internal amino acid sequence data obtained from purified bovine MRPs (O'Brien, et al. (2000) J Biol Chem. 275: 18153) to search EST databases.  Chromosome map positions for the MRP genes are ascertained by in situ hybridization (FISH) with genomic sequences and/or by using in silico methods to search Genbank and GeneMap through NCBI.  At present, we have over 45 different human MRPs at various stages of characterization. We are currently investigating one MRP as a potential candidate for Russell-Silver Syndrome (RSS), a dwarfism characterized by low birth weight and lateral asymmetry, characteristics that are consistent with reduced mitochondrial function. DNA from RSS patients is being analyzed for possible mutations in MRPs.   In addition to studying their clinical relevance, long term characterization of MRP genes should lead to important insights into mammalian evolution, coordinate regulation of nuclear and mitochondrial gene expression, and ribosome function. This work is being done as part of the Mammalian Mitochondrial Ribosomal Consortium and is supported by NIH/NIDCD grant RO1DC04092 and the Nemours Research Program.