The Crithidia fasciculata RNH1 gene encodes both nuclear and mitochondrial isoforms of RNase H

The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA–DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the kinetoplast. These results suggest that the kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.     

Purification and characterization of Rpp25, an RNA-binding protein subunit of human ribonuclease P

In HeLa cells, ribonuclease P (RNase P), the tRNA processing enzyme consists of an RNA subunit (H1 RNA) associated with at least nine protein subunits, Rpp14, Rpp20, Rpp21, Rpp29 (hPop4), Rpp30, Rpp38, Rpp40, hPop1, and hPop5 (18.8 kDa). We report here the cloning and immuno-biochemical analysis of Rpp25, another protein subunit of RNase P. Polyclonal rabbit antibodies raised against recombinant Rpp25 recognize their corresponding antigens in RNase P-containing fractions purified from HeLa cells, and they also precipitate active holoenzyme. Furthermore, this protein has general RNA binding properties.     

Purification of Saccharomyces cerevisiae RNase H(70) and identification of the corresponding gene

We purified Saccharomyces cerevisiae RNase H(70) to homogeneity, using an optimized chromatographic purification procedure. Renaturation gel assay assigned RNase H activity to a 70 kDa polypeptide. Sequencing of tryptic peptides identified the open reading frame YGR276c on chromosome VII of the S. cerevisiae genome as the corresponding gene, which encodes a putative polypeptide of molecular mass of 62849. We therefore renamed this gene RNH70. Immunofluorescence microscopy using a RNH70-EGFP fusion construct indicates nuclear localization of RNase H(70). Deletion of RNH70 from the yeast genome did not result in any serious phenotype under the conditions tested. Homology searches revealed striking similarity with a number of eukaryotic proteins and open reading frames, among them the chimpanzee GOR protein, a homolog of a human autoimmune antigen, found to elicit autoimmune response in patients infected with hepatitis C virus.     

RNA-protein interactions in the human RNase MRP ribonucleoprotein complex

The eukaryotic nucleolus contains a large number of small RNA molecules that, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. One of the snoRNPs that has been shown to possess enzymatic activity is the RNase MRP. RNase MRP is an endoribonuclease involved in the formation of the 5' end of 5.8S rRNA. In this study the association of the hPop1 protein with the RNase MRP complex was investigated. The hPop1 protein seems not to be directly bound to the RNA component, but requires nt 1-86 and 116-176 of the MRP RNA to associate with the RNase MRP complex via protein-protein interactions. UV crosslinking followed by ribonuclease treatment and immunoprecipitation with anti-Th/To antibodies revealed three human proteins of about 20, 25, and 40 kDa that can associate with the RNase MRP complex. The 20- and 25-kDa proteins appear to bind to stem-loop I of the MRP RNA whereas the 40-kDa protein requires the central part of the MRP RNA (nt 86-176) for association with the RNase MRP complex. In addition, we show that the human RNase P proteins Rpp30 and Rpp38 are also associated with the RNase MRP complex. Expression of Vesicular Stomatitis Virus- (VSV) tagged versions of these proteins in HeLa cells followed by anti-VSV immunoprecipitation resulted in coprecipitation of both RNase P and RNase MRP complexes. Furthermore, UV crosslinking followed by anti-Th/To and anti-Rpp38 immunoprecipitation revealed that the 40-kDa protein we detected in UV crosslinking is probably identical to Rpp38.     

Eukaryotic RNAse H shares a conserved domain with caulimovirus proteins that facilitate translation of polycistronic RNA.

RNAse H (RNH1 protein) from the trypanosomatid Crithidia fasciculata has a functionally uncharacterized N-terminal domain dispensable for the RNAse H activity. Using computer methods for database search and multiple alignment, we show that the N-terminal domains of RNH1 and its homologue encoded by a cDNA from chicken lens are related to the conserved domain in caulimovirus ORF VI product that facilitates translation of polycistronic virus RNA in plant cells. We hypothesize that the N-terminal domain of eukaryotic RNAse H performs an as yet uncharacterized regulatory function, possibly in mRNA translation or turnover.     

Bovine Brain Ribonuclease Is the Functional Homolog of Human Ribonuclease 1

Mounting evidence suggests that human pancreatic ribonuclease (RNase 1) plays important roles in vivo, ranging from regulating blood clotting and inflammation to directly counteracting tumorigenic cells. Understanding these putative roles has been pursued with continual comparisons of human RNase 1 to bovine RNase A, an enzyme that appears to function primarily in the ruminant gut. Our results imply a different physiology for human RNase 1. We demonstrate distinct functional differences between human RNase 1 and bovine RNase A. Moreover, we characterize another RNase 1 homolog, bovine brain ribonuclease, and find pronounced similarities between that enzyme and human RNase 1. We report that human RNase 1 and bovine brain ribonuclease share high catalytic activity against double-stranded RNA substrates, a rare quality among ribonucleases. Both human RNase 1 and bovine brain RNase are readily endocytosed by mammalian cells, aided by tight interactions with cell surface glycans. Finally, we show that both human RNase 1 and bovine brain RNase are secreted from endothelial cells in a regulated manner, implying a potential role in vascular homeostasis. Our results suggest that brain ribonuclease, not RNase A, is the true bovine homolog of human RNase 1, and provide fundamental insight into the ancestral roles and functional adaptations of RNase 1 in mammals.     

Alterations in the intracellular level of a protein subunit of human RNase P affect processing of tRNA precursors

The human ribonucleoprotein ribonuclease P (RNase P), processing tRNA, has at least 10 distinct protein subunits. Many of these subunits, including the autoimmune antigen Rpp38, are shared by RNase MRP, a ribonucleoprotein enzyme required for processing of rRNA. We here show that constitutive expression of exogenous, tagged Rpp38 protein in HeLa cells affects processing of tRNA precursors. Alterations in the site-specific cleavage and in the steady-state level of 3′ sequences of the internal transcribed spacer 1 of rRNA are also observed. These processing defects are accompanied by selective shut-off of expression of Rpp38 and by low expression of the tagged protein. RNase P purified from these cells exhibits impaired activity in vitro. Moreover, inhibition of Rpp38 by the use of small interfering RNA causes accumulation of the initiator methionine tRNA precursor. Expression of other protein components, but not of the H1 RNA subunit, is coordinately inhibited. Our results reveal that normal expression of Rpp38 is required for the biosynthesis of intact RNase P and for the normal processing of stable RNA in human cells.     

Two ribonucleases H from cultured plant cells.

Two forms of enzyme with ribonuclease H (RNase H) [EC 3.1.4.34] activities, have been partially purified from cultured plant cells, strain GD-2, derived from carrot root. One is an Mn2+-dependent RNase H, and the second is an Mg2+-dependent RNase H. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at around 0.2 M and 0.4 M potassium chloride in phosphocellulose chromatography, and were further purified using blue Sepharose. Mg2+-dependent RNase H exhibits maximal activity at pH 9.0, and requires 10 to 15 mM Mg2+ for maximal activity, whereas the Mn2+-dependent enzyme is most active at pH 8.0, is maximally active at an Mn2+ concentration of 0.4 mM, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. The enzymes liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. The apparent molecular weight of the Mg2+-dependent RNase H was estimated to 18--20 X 10(4) and that of the Mn 2+- dependent RNase H was estimated to be 14 x 10(4) by gel filtration.     

Rpp14 and Rpp29, two protein subunits of human ribonuclease P

In HeLa cells, the tRNA processing enzyme ribonuclease P (RNase P) consists of an RNA molecule associated with at least eight protein subunits, hPop1, Rpp14, Rpp20, Rpp25, Rpp29, Rpp30, Rpp38, and Rpp40. Five of these proteins (hPop1p, Rpp20, Rpp30, Rpp38, and Rpp40) have been partially characterized. Here we report on the cDNA cloning and immunobiochemical analysis of Rpp14 and Rpp29. Polyclonal rabbit antibodies raised against recombinant Rpp14 and Rpp29 recognize their corresponding antigens in HeLa cells and precipitate catalytically active RNase P. Rpp29 shows 23% identity with Pop4p, a subunit of yeast nuclear RNase P and the ribosomal RNA processing enzyme RNase MRP. Rpp14, by contrast, exhibits no significant homology to any known yeast gene. Thus, human RNase P differs in the details of its protein composition, and perhaps in the functions of some of these proteins, from the yeast enzyme.     

The Crithidia fasciculata CRK gene encodes a novel cdc2-related protein containing large inserts between highly conserved domains.

A gene (CRK) encoding a cdc2-related protein has been identified in the trypanosomatid Crithidia fasciculata. CRK has a high degree of sequence identity with the human cdc2 gene and contains the sixteen amino acid PSTAIR motif, characteristic of p34cdc2 protein-serine/threonine kinases, with four amino acid substitutions in the motif. In addition, two inserts of more than sixty amino acids have been found between conserved domains of this putative protein-serine/threonine kinase. CRK is a single copy gene and is expressed on a 3.8 kb mRNA. Anti-CRK antibodies detect a 53kDa protein in extracts of C.fasciculata in agreement with the size predicted from the nucleotide sequence of the cloned gene. These antibodies also recognize proteins of 48 and 60 kDa in extracts of the trypanosomatid Leishmania tarentolae. Antibodies against the human PSTAIR peptide detect the p34cdc2 protein in human nuclear extracts but fail to detect a 34 kDa protein in C.fasciculata extracts. These results suggest that novel higher molecular weight forms of the cdc2 protein family may be involved in cell cycle control in trypanosomes.     

A structure-specific DNA endonuclease is enriched in kinetoplasts purified from Crithidia fasciculata.

The mitochondrial DNA (kinetoplast DNA) of the trypanosomatid Crithidia fasciculata consists of minicircles and maxicircles topologically interlocked in a single network per cell. Individual minicircles replicate unidirectionally from either of two replication origins located 180 degrees apart on the minicircle DNA. Initiation of minicircle leading-strand synthesis involves the synthesis of an RNA primer which is removed in the last stage of replication. We report here the purification to near homogeneity of a structure-specific DNA endo-nuclease based on the RNase H activity of the enzyme on a poly(rA).poly(dT) substrate. RNase H activity gel analysis of whole cell and kinetoplast extracts shows that the enzyme is enriched in kinetoplast fractions. The DNA endonuclease activity of the enzyme is specific for DNA primers annealed to a template strand and requires an unannealed 5' tail. The enzyme cleaves 3' of the first base paired nucleotide releasing the intact tail. The purified enzyme migrates as a 32 kDa protein on SDS gels and has a Stoke's radius of 21.5 A and a sedimentation coefficient of 3.7 s, indicating that the protein is a monomer in solution with a native molecular mass of 32.4 kDa. These results suggest that the enzyme may be involved in RNA primer removal during minicircle replication.     

A ribonuclease zymogen activated by the NS3 protease of the hepatitis C virus

Translating proteases as inactive precursors, or zymogens, protects cells from the potentially lethal action of unregulated proteolytic activity. Here, we impose this strategy on bovine pancreatic ribonuclease (RNase A) by creating a zymogen in which quiescent ribonucleolytic activity is activated by the NS3 protease of the hepatitis C virus. Connecting the N-terminus and C-terminus of RNase A with a 14-residue linker was found to diminish its ribonucleolytic activity by both occluding an RNA substrate and dislocating active-site residues, which are devices used by natural zymogens. After cleavage of the linker by the NS3 protease, the ribonucleolytic activity of the RNase A zymogen increased 105-fold. Both before and after activation, the RNase A zymogen displayed high conformational stability and evasion of the endogenous ribonuclease inhibitor protein of the mammalian cytosol. Thus, the creation of ribonuclease zymogens provides a means to control ribonucleolytic activity and has the potential to provide a new class of antiviral chemotherapeutic agents.     

RNase A及其链亲和素融合蛋白在pET系统中的表达

在大肠杆菌中用pET28a表达载体表达重组RNaseA。变性条件下,利用His-Resin亲和纯化,得到60mg／L电泳纯的RNaseA。纯化的RNaseA复性后,利用含大量RNA分子的碱法抽提质粒为底物,测定重组RNaseA活性,与商品化的RNaseA活性相当。同时在RNaseA活性测定体系中加入4mol／L尿素会使RNA分子切割效率提高10倍左右。在此基础上,成功表达RNaseA与链亲和素(streptavidjn)的融合蛋白,经纯化复性后,该融合蛋白同时具有核酸酶、biotin结合活性,在分子生物学中具有重要的应用价值。     

Selection and characterization of randomly produced mutants in the gene coding for M1 RNA

The gene for M1 RNA, the catalytic subunit of RNase P of Escherichia coli, was subjected to random chemical mutagenesis in vitro. Mutations were selected by electrophoresis in denaturing gradient gels. Twenty-seven different mutants of the gene for M1 RNA were selected, and in 24 cases the mutations were identified as single base substitutions. The mutant forms of M1 RNA were analyzed in vitro for catalytic activity in the absence and in the presence of the protein subunit of RNase P (C5 protein). The structure of mutant RNAs was probed by limited digestion with ribonuclease T1; a correlation between reduced catalytic activity of mutant M1 RNAs and perturbations in secondary and tertiary structure was noted in many cases. The results indicate the involvement of specific regions of the M1 RNA molecule in the catalytic function of RNase P, in the binding of the C5 protein, and in substrate binding.     

A common 40 amino acid motif in eukaryotic RNases H1 and caulimovirus ORF VI proteins binds to duplex RNAs.

Eukaryotic RNases H from Saccharomyces cerevisiae , Schizosaccharomyces pombe and Crithidia fasciculata , unlike the related Escherichia coli RNase HI, contain a non-RNase H domain with a common motif. Previously we showed that S.cerevisiae RNase H1 binds to duplex RNAs (either RNA-DNA hybrids or double-stranded RNA) through a region related to the double-stranded RNA binding motif. A very similar amino acid sequence is present in caulimovirus ORF VI proteins. The hallmark of the RNase H/caulimovirus nucleic acid binding motif is a stretch of 40 amino acids with 11 highly conserved residues, seven of which are aromatic. Point mutations, insertions and deletions indicated that integrity of the motif is important for binding. However, additional amino acids are required because a minimal peptide containing the motif was disordered in solution and failed to bind to duplex RNAs, whereas a longer protein bound well. Schizosaccharomyces pombe RNase H1 also bound to duplex RNAs, as did proteins in which the S.cerevisiae RNase H1 binding motif was replaced by either the C.fasciculata or by the cauliflower mosaic virus ORF VI sequence. The similarity between the RNase H and the caulimovirus domain suggest a common interaction with duplex RNAs of these two different groups of proteins.