Impairment of tRNA processing by point mutations in mitochondrial tRNA(Leu)(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 tRNA(Leu)(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 tRNA(Leu)(UUR) is a primary molecular impairment leading to mitochondrial dysfunction and consequently to disease.

Characterization of human mitochondrial RNase P: novel aspects in tRNA processing.

Human mitochondrial RNase P does not distinguish itself from other RNase P enzymes by most of its basic properties. 5' phosphates on tRNA products, strict dependence on a divalent cation, independence of ATP or other cofactors, and sensitivity to puromycin are generally characteristic for RNase P. Slow sedimentation of human mitochondrial RNase P in glycerol gradients suggests a molecular weight considerably lower than that of bacterial or nuclear RNase P. In contrast to fungi, all putative components of mammalian mitochondrial RNase P are encoded by the nucleus. Intriguingly, no indication of the involvement of a trans-acting RNA was found in mammalian mitochondrial tRNA processing. Mitochondrial RNase P is resistant to rigorous treatments with nucleases and exhibits a protein-like density in Cs2SO4 gradients. Moreover, an analysis of copurifying RNAs revealed no putative RNase P RNA candidates. These data suggest that mammalian mitochondrial RNase P, unlike its nuclear counterpart or its bacterial relatives, is not a ribonucleoprotein but a protein enzyme.

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.

Expression of mouse RNase MRP RNA in human embryonic kidney 293 cells.

We report on the expression of mouse RNase MRP RNA in human embryonic kidney 293 cells upon DNA transfection. Stable cell lines were selected by cotransfection with a neor gene. Transcription of wild-type and deletion mutants of MRP RNA and ribonucleoprotein formation were assessed by RNase protection and immunoprecipitation experiments. Mouse MRP RNA as expressed in 293 cells readily associates with human proteins to form a chimeric Th ribonucleoprotein. 5' truncated MRP RNAs, however, failed to associate with Th antigen(s) and deletion of the 3' sequences of MRP RNA greatly reduced the expression in stable as well as in transient transfectants.

Human mitochondrial tRNA processing.

tRNA processing is a central event in mammalian mitochondrial gene expression. We have identified key enzymatic activities (ribonuclease P, precursor tRNA 3'-endonuclease, and ATP(CTP)-tRNA-specific nucleotidyltransferase) that are involved in HeLa cell mitochondrial tRNA maturation. Different mitochondrial tRNA precursors are cleaved precisely at the tRNA 5'- and 3'-ends in a homologous mitochondrial in vitro processing system. The cleavage at the 5'-end precedes that at the 3'-end, and the tRNAs are substrates for the specific CCA addition in the same in vitro system. Using a comparative enzymatic approach as well as biochemical and immunological techniques, we furthermore demonstrate that human cells contain two distinct enzymes that remove 5'-extensions from tRNA precursors, the previously characterized nuclear and the newly identified mitochondrial ribonuclease P. These two cellular isoenzymes have different substrate specificities that seem to be well adapted to their structurally disparate mitochondrial and nuclear tRNA substrates. This kind of approach may also help to understand the structural diversities and commonalities of tRNAs.

RNase mitochondrial RNA processing cleaves RNA from the rat mitochondrial displacement loop at the origin of heavy-strand DNA replication.

Ribonuclease mitochondrial RNA processing cleaves RNAs from the mammalian mitochondrial main non-coding regulatory region, called the displacement loop. Our data demonstrate that rat cells contain a site-specific ribonuclease mitochondrial RNA processing activity. We found that this enzyme processes the rat mitochondrial displacement-loop RNA substrate at the level of the conserved sequence block 1, a result which is different from that for mouse. This finding correlates with the in-vivo transcriptional analysis of the rat displacement-loop region. Processing by homologous and heterologous ribonuclease mitochondrial RNA enzymes occurs in the same manner, suggesting a conserved mode of substrate recognition.

RNase MRP and RNase P share a common substrate.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that processes RNA from the mammalian mitochondrial displacement loop containing region. RNase P is a site-specific ribonucleoprotein endoribonuclease that processes pre-tRNAs to generate their mature 5'-ends. A similar structure for the RNase P and RNase MRP RNAs and a common cleavage mechanism for RNase MRP and RNase P enzymes have been proposed. Experiments with protein synthesis antibiotics have shown that both RNase MRP and RNase P are inhibited by puromycin. We also show that E. coli RNase P cleaves the RNase MRP substrate, mouse mitochondrial primer RNA, exactly at a site that is cleaved by RNase MRP.

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 exclusively. 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.

RNase MRP/RNase P: a structure-function relation conserved in evolution?

RNase P and RNase MRP are related ribonucleoproteins. RNase MRP processes mitochondrial precursor- (primer) RNAs, whereas RNase P cleaves precursor-tRNAs to produce their mature 5'-ends. Both RNase P and RNase MRP are associated with the Th/To ribonucleoprotein suggesting possible interrelated pathways and/or functions. All known RNase P and RNase MRP RNAs contain conserved structural elements possibly involved in catalysis/substrate binding, but these elements do not predict all cellular functions of the RNPs.

Nuclear RNase MRP processes RNA at multiple discrete sites: interaction with an upstream G box is required for subsequent downstream cleavages.

RNase MRP is a site-specific endoribonuclease that processes primer RNA from the leading-strand origin of mammalian mitochondrial DNA replication. It is present in active form as isolated from the nucleus, suggesting a bipartite cellular location and function. The relatively high abundance of nucleus-localized RNase MRP has permitted its purification to near homogeneity and, in turn, has led to the identification of protein components of this ribonucleoprotein. Analysis of the mode of RNA cleavage by nuclear RNase MRP revealed the surprising and unprecedented ability of the endonuclease to process RNA at multiple discrete locations. Substrate cleavage is dependent on the presence of a previously described G-rich sequence element adjacent to the primary site of RNA processing. Downstream cleavage occur in a distance- and sequence-specific manner.

Ribonuclease H(70) is a component of the yeast nuclear scaffold.

We have used monospecific antibodies against three ribonuclease H enzymes of Saccharomyces cerevisiae to investigate their intracellular localization. Fractionation experiments, as well as immunocytochemical staining, revealed a predominantly cytoplasmic localization of the RNase H proteins of 42,000 and 70,000 Mr, whereas that of 55,000 Mr showed equal distribution between nuclei and cytoplasm. The nuclear moiety of ribonuclease H(70) was found to be a part of the yeast nuclear scaffold, as investigated by immunoblotting and antibody inhibition experiments. The 42,000 and 55,000 Mr enzymes, on the other hand, are not scaffold-associated. We conclude that RNase H(70) is part of the nuclear substructure of yeast that was previously found to maintain specific interactions with yeast chromosomal origins of replication (ARS elements).

Identification of a yeast ribonuclease H as an Sm antigen.

We have isolated a 55-kDa enzyme from Saccharomyces cerevisiae on the basis of its ability to hydrolyze specifically the RNA moiety of RNA/DNA hybrids [RNase H(55)]. Remarkably, monospecific anti-[RNase H(55)] antibodies revealed that the protein associates with several small RNAs, including some of the essential yeast spliceosomal snRNAs. Moreover, immunoprecipitation as well as immunoblotting experiments demonstrated that the yeast enzyme reacts (a) with human anti-Sm autoantisera, (b) with a monoclonal antibody specific for the human snRNP proteins B/B', but (c) not with U1-ribonucleoprotein-specific autoantibodies. These results disclosed a hitherto unexpected degree of evolutionary conservation in snRNP protein structure between yeast and man. Additionally, our findings suggested a re-evaluation of the enzymatic mechanism of RNases H which recognize both RNA and RNA/DNA hybrids.

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.

In addition to RNase H(70) two other proteins of Saccharomyces cerevisiae exhibit ribonuclease H activity.

Two ribonucleases H (RNases H) were purified to apparent homogeneity from the yeast Saccharomyces cerevisiae. The enzymes were separated from the previously described yeast ribonuclease H (RNase H(70), Karwan, R., Blutsch, H., and Wintersberger, U. (1983) Biochemistry 22, 5500-5507) by chromatography on Mono Q and blue-Sepharose columns and from each other on a Mono S column. The two proteins, RNase H(55) of molecular weight around 55,000 and RNase H(42) of molecular weight around 42,000, exhibit distinct enzymatic properties: RNase H(55) acts as a 5'-exonuclease of low specific activity and produces predominantly monoribonucleotides from the synthetic hybrid poly(rA)-poly(dT). RNase H(42) efficiently releases oligoribonucleotides from the same substrate. Polyclonal antibodies against these proteins do not cross-react with RNase H(70), and thus, these two RNases H probably do not represent proteolytic breakdown products of RNase H(70). Peptide maps obtained by total digestion of RNase H(55) and RNase H(42) with trypsin reveal several common peptides and, therefore, suggest that the two enzymes are related to each other. We tentatively conclude that RNase H(55) is proteolytically processed to RNase H(42) in vivo.

Yeast ribonuclease H(70) cleaves RNA-DNA junctions.

A specific substrate, M13 DNA:RNA-[32P]DNA, was synthesized to investigate the mode of cleavage of enzymes with RNase H activity. RNase H(70) from Saccharomyces cerevisiae hydrolyzes the phosphodiester bond at the RNA-DNA junction of this substrate, thereby producing a 5'-monophosphate-terminated polydeoxyribonucleotide and 3'-hydroxyl-terminated oligoribonucleotides.

Ribonuclease H(70) from Saccharomyces cerevisiae possesses cryptic reverse transcriptase activity.

Yeast cells contain a protein of molecular size 70 kDa that possesses RNase H activity. A polyclonal antibody against it reacts in addition with proteins of molecular sizes 160 kDa from yeast extracts. All these immunologically related proteins exhibit reverse transcriptase activity and in this respect they resemble the products of retroviral pol genes, relatives of which reside in Ty elements and mitochondrial introns of yeast. Experimental evidence, however, indicates that the protein described here that combines RNase H and reverse transcriptase activity is not coded for by a known element of the retrotransposon family. It may originate from a cellular gene distantly related to retrotransposon sequences.