Robert Karwan

IMPAIRMENT OF TRNA PROCESSING BY POINT MUTATIONS IN MITOCHONDRIAL TRNALEU(UUR) ASSOCIATED WITH MITOCHONDRIAL DISEASES

Several point mutations in mitochondrial tRNA genes have been linked to distinct clinical subgroups of mitochondrial diseases. A particularly large number of different mutations is found in the tRNALeu(UUR) gene. We show that base substitutions at nucleotide position 3256, 3260, and 3271 of the mitochondrial genome, located in the D and anticodon stem of this tRNA, and mutation 3243 changing a base involved in a tertiary interaction, significantly impair the processing of the tRNA precursor in vitro. In correlation with other studies, our results suggest that inefficient processing of certain mutant variants of mitochondrial tRNALeu(UUR) is a primary molecular impairment leading to mitochondrial dysfunction and consequently to disease.

Definition of the Th/To ribonucleoprotein by RNase P and RNase MRP

We show that the Th/ To ribonucleoprotein is defined by (i) the co-immunoprecipitation of two RNAs, (ii) the co-immunoprecipitation of four major polypeptides and (iii) the quantitative immune recognition of both RNase P and RNase MRP. No serum was found that recognizes either one of these two enzymes exelusively. The specific co-immunoprecipitation of RNase MRP and RNase P by all Th/ To ribonucleoprotein autoantibodies indicates that the anti-Th/ To autoimmune response is directed against both enzymes in a quantitatively indistinguishable manner. Thus the Th/ To ribonucleoprotein is defined by RNase P and RNase MRP.

Three ribonucleases H and a reverse transcriptase from the yeast, Saccharomyces cerevisiae

From the yeast, Saccharomyces cerevisiae, three proteins exhibiting ribonuclease H activity were isolated. These proteins differ in molecular weights and enzymatic properties. The two smaller ones, RNAase H(55) and RNAase H(42) are immunologically and structurally related to each other. Neither reacts with antibodies against the largest one, RNAase H(70). Highly purified preparations of RNAase H(70) contain two polypeptides (Mr 70,000 and 160,000) and display reverse transcriptase activity. Deletion of part of the gene for the 160 kDa polypeptide results in mutants possessing about twice the amount of DNA as do wild-type cells. DNA polymerase stimulating activity resides in the 70,000 polypeptide. The processivity of yeast DNA polymerase A(I) does not change in presence of that protein. Possible functions of RNAases H are discussed.

Further characterization of human RNase MRP/RNase P and related autoantibodies.

We characterized a panel of human RNase MRP/RNase P autoantibodies by immunoprecipitation, immunodepletion, immunoaffinity purification and immunoblotting. We report on the protein spectrum that is recognized by RNase MRP/RNase P autoantibodies. We also describe another, related patient serum that based on these assays does not immunoprecipitate RNase P/MRP/Th40. This autoantibody ‘KC’, however, coimmunoprecipitates the RNase MRP/RNase P associated RNAs from HeLa and La9 cell extracts as shown by nuclease protection experiments.

A Ribonuclease H from Yeast Stimulates DNA Polymerase in Vitro

Recently we have purified a ribonuclease-H (RNase-H) activity from the yeast, Saccharomyces cerevisiae (1) which differs in many of its properties (see Table 1) from ribonucleases-H described earlier for this organism by others (2, 3). The enzyme specifically degrades the RNA part of a DNA-RNA hybrid in an endonucleolytic mode and is completely inactive with single-stranded PNA or native DNA (Table 2).