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PubMed1998

Structural analysis and molecular model of a self-incompatibility RNase from wild tomato.

Self-incompatibility RNases (S-RNases) are an allelic series of style glycoproteins associated with rejection of self-pollen in solanaceous plants. The nucleotide sequences of S-RNase alleles from several genera have been determined, but the structure of the gene products has only been described for those from Nicotiana alata. We report on the N-glycan structures and the disulfide bonding of the S3-RNase from wild tomato (Lycopersicon peruvianum) and use this and other information to construct a model of this molecule. The S3-RNase has a single N-glycosylation site (Asn-28) to which one of three N-glycans is attached. S3-RNase has seven Cys residues; six are involved in disulfide linkages (Cys-16-Cys-21, Cys-46-Cys-91, and Cys-166-Cys-177), and one has a free thiol group (Cys-150). The disulfide-bonding pattern is consistent with that observed in RNase Rh, a related RNase for which radiographic-crystallographic information is available. A molecular model of the S3-RNase shows that four of the most variable regions of the S-RNases are clustered on one surface of the molecule. This is discussed in the context of recent experiments that set out to determine the regions of the S-RNase important for recognition during the self-incompatibility response.

Amino Acid Sequence↗
PubMed1991

Three RNases in Senescent and Nonsenescent Wheat Leaves : Characterization by Activity Staining in Sodium Dodecyl Sulfate-Polyacrylamide Gels.

We have described three RNases in wheat leaves (Triticum aestivum L. cv Chinese Spring) and developed assays for measuring each RNase individually in crude leaf extracts. We initially used activity staining in sodium dodecyl sulfate-polyacrylamide gels to characterize RNases in extracts of primary and flag leaves. We thus identified acid RNase (EC 3.1.27.1, here designated RNase WL(A)), and two apparently novel enzymes, designated RNases WL(B) and WL(C). RNase WL(B) activity displays a distinctive isozyme pattern, a molecular mass of 26 kilodaltons (major species), a broad pH range with an optimum near neutrality, insensitivity to EDTA, and stimulation by moderate concentrations of KCl and by MgCl(2). RNase WL(C) activity exhibits a molecular mass of 27 kilodaltons, a neutral pH optimum, insensitivity to EDTA, and inhibition by KCl, MgCl(2), and tri-(hydroxymethyl)aminomethane. Based on distinctive catalytic properties established in gels, we designed conventional solution assays for selective quantitation of each RNase activity. We used the assays to monitor the individual RNases after gel filtration chromatography and native gel electrophoresis of extracts. In accompanying work, we used the assays to monitor RNases WL(A), WL(B), and WL(C), which are present in senescent and nonsenescent leaves, during the course of leaf senescence.

Journal Article↗
PubMed1980

Effects of purified macrophage RNase on granuloma fibroblasts with reference to silicosis.

Two alkaline RNases, designated RNase 1 and RNase 2, were isolated from the culture media of silica-treated and non-treated macrophages. The yield of RNase from the medium of silica-treated macrophages was 30% of that from the non-treated control. The effects of these RNases on cultured granuloma fibroblasts and on granulation-tissue nuclei were studied. RNase 1 inhibited thymidine incorporation into fibroblasts except at low concentrations, where it was observed to be stimulatory. RNase 1 also inhibited the protein synthesis of fibroblasts. The incorporation of cytidine into RNA in cultured fibroblasts was not affected by RNase 1, but the incorporation into isolated nuclei was decreased. In pulse chase experiments RNase 1 increased the release of cytidine, but not that of thymidine, from the cells. RNase 2 had no effect on the protein or nucleic acid metabolism of the fibroblasts or on the RNA metabolism of isolated nuclei, perhaps because of impermeability. These experiments confirm that macrophage RNase activity is able to regulate the metabolism of granulation-tissue fibroblasts by increasing RNA degradation. Through this action it also regulates DNA and protein synthesis and other metabolic functions of those cells.

Animals↗
PubMed1971

Erythroid cell RNase: activation by urea and localization to the cell membrane.

Assays of ribonuclease activity in components of mature and immature mammalian erythroid cells indicate that RNase activity is present both in the membrane-free hemolysate and the washed membranes. Erythroid cell RNase exists in an active and latent form. The majority of total cell RNase activity is in the latent state, and is localized to the erythroid cell membrane. Both total and latent RNase activity decline as the cell matures. The latent RNase is released from its relatively firm attachment to the cell membrane and activated by centrifugation or, optimally, by exposure to 4 M urea. The active sites of membrane-associated RNase are apparently oriented toward the inner side of the cell membrane. The properties of the latent membrane-bound RNase which is activated by urea, including K(m), pH optimum, inhibition of enzyme activity by cations, and response to metabolic inhibitors, do not differ significantly from those of the soluble RNase in the membrane-free hemolysate, suggesting that there is only one type of RNase in the erythroid cell. Binding of Rnase to the erythroid cell membrane stabilized the enzyme against inactivation during incubation at 37 degrees C, and the findings suggest that membrane-bound RNase may play a particular part in degrading ribosomes. The findings indicate that the cell membrane has a major role in RNA metabolism in the maturing mammalian erythroid cell.

Animals↗
PubMed1985

Independent regulation of ppp(A2'p)nA-dependent RNase in NIH 3T3, clone 1 cells by growth arrest and interferon treatment.

The regulation of ppp(A2'p)nA-(2-5A)-dependent RNase (RNase L or RNase F) was investigated in NIH 3T3, clone 1 cells using 2-5A-binding and nuclease activity assays. Minimal levels of 2-5A-dependent RNase were detected in actively dividing clone 1 cells; these levels were independently induced by growth arrest or interferon treatment. Accordingly, levels of the RNase were enhanced during growth arrest by confluency regardless of the presence or absence of interferon or antibody to interferon in the media. Measurement of 2-5A-dependent RNase was unaffected by the addition of any of six different proteinase inhibitors to the cells prior to extraction. The expression of 2-5A-dependent RNase in growth-arrested, interferon-treated cells was still relatively low (about one-third to one-half of that found in similarly treated murine Ehrlich ascites tumor cells). Although this amount of 2-5A-dependent RNase could not be detected by 2-5A-mediated ribosomal RNA cleavage, the activity was identified using a more sensitive novel assay for 2-5A-dependent RNase. In addition, introduction of 2-5A or poly(I) X poly(C) into growth-arrested, interferon-treated cells resulted in some inhibition of protein synthesis. The results indicated that the expression of 2-5A-dependent RNase in NIH 3T3, clone 1 cells is regulated under different physiological conditions and that low levels of 2-5A-dependent RNase were insufficient to significantly inhibit encephalomyocarditis virus replication.

Adenine Nucleotides↗
PubMed2003

Evaluation of bacterial RNase P RNA as a drug target.

RNA has gained increasing importance as a therapeutic target. However, so far mRNAs rather than stable cellular RNAs have been considered in such studies. In bacteria, the tRNA-processing enzyme RNase P has a catalytic RNA subunit. Fundamental differences in structure and function between bacterial and eukaryotic RNase P, and its indispensability for cell viability make the bacterial enzyme an attractive drug target candidate. Herein we describe two approaches utilized to evaluate whether the catalytic RNA subunit of bacterial RNase P is amenable to inactivation by antisense-based strategies. In the first approach, we rationally designed RNA hairpin oligonucleotides targeted at the tRNA 3'-CCA binding site (P15 loop region) of bacterial RNase P RNA by attempting to include principles derived from the natural CopA-CopT antisense system. Substantial inactivation of RNase P RNA was observed for Type A RNase P RNA (such as that in Escherichia coli) but not for Type B (as in Mycoplasma hyopneumoniae). Moreover, only an RNA oligonucleotide (Eco 3') complementary to the CCA binding site and its 3' flanking sequences was shown to be an efficient inhibitor. Mutation of Eco 3' and analysis of other natural RNase P RNAs with sequence deviations in the P15 loop region showed that inhibition is due to interaction of Eco 3' with this region and occurs in a highly sequence-specific manner. A DNA version of Eco 3' was a less potent inhibitor. The potential of Eco 3' to form an initial kissing complex with the P15 loop did not prove advantageous. In a second approach, we tested a set of oligonucleotides against E. coli RNase P RNA which were designed by algorithms developed for the selection of suitable mRNA targets. This approach identified the P10/11-J11/12 region of bacterial RNase P RNA as another accessible region. In conclusion, both the P15 loop and P10/11-J11/12 regions of Type A RNase P RNAs seem to be promising antisense target sites since they are easily accessible and sufficiently interspersed with nonhelical sequence elements, and oligonucleotide binding directly interferes with substrate docking to these two regions.

Algorithms↗
PubMed1994

Residues 36-42 of liver RNase PL3 contribute to its uridine-preferring substrate specificity. Cloning of the cDNA and site-directed mutagenesis studies.

Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative members of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a secreted RNase. Expression of the cDNA in Escherichia coli yielded 1.5 mg of purified protein/liter of culture. The recombinant enzyme was indistinguishable from the enzyme isolated from porcine liver based on the following criteria: amino acid analysis, N-terminal amino acid sequence, molecular weight, specific activity toward yeast RNA, and kinetic parameters for the hydrolysis of uridylyl(3',5')adenosine and cytidylyl(3',5')adenosine. Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compared to pancreatic RNase A. Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most members of the RNase superfamily, strongly prefers uridine over cytidine on the 5' side of the scissile bond. Replacement, by site-directed mutagenesis, of residues 36-42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase in the catalytic efficiency for cytidine-containing substrates. This suggests that this region of the molecule contains some of the elements that determine substrate specificity.

Amino Acid Sequence↗
PubMed1995

Kinetics and thermodynamics of the RNase P RNA cleavage reaction: analysis of tRNA 3'-end variants.

We have studied the interaction of 3'-end variants of a (pre-)tRNAGly with ribonuclease P (RNase P) RNAs from Escherichia coli and Thermus thermophilus. To dissect the thermodynamics of tRNA binding from the overall catalytic reaction, specific binding of mature tRNAGly variants to RNase P RNAs was studied by gel retardation. A newly developed assay, based on the reduction of Pb(2+)-hydrolysis at the CCA end due to complex formation of tRNA and RNase P RNA, was utilized to confirm the dissociation constants. The binding data were supplemented by single and multiple turnover kinetic analyses of the corresponding pre-tRNAGly variants. For E. coli RNase P RNA the following results were obtained. Extensions of CCA by pCp or three nucleotides (AUA) stabilized gel-resolved tRNAGly binding by 1 to 1.5 kcal/mol. Changing the first C in CCA to A, G or U resulted in a more than 100-fold reduction in binding affinity, which corresponds to a loss of 3.5 to 4.5 kcal/mol of binding energy. However, single turnover rate constants were only slightly affected, indicating that a disruption or loss of the tRNA 3'-end-mediated interaction with RNase P RNA does not preferentially destabilize the transition state. Our data suggest another kinetic step following initial substrate binding to E. coli RNase P RNA (possibly a conformational rearrangement). For T. thermophilus RNase P RNA, product release of wild-type tRNAGly CCAAUA was not rate-limiting in the multiple turnover reaction. However, the effects of CCA mutations were similar to those attained with E. coli RNase P RNA. This supports the notion that a high-affinity binding site for the tRNA 3'-end is a ubiquitous feature of eubacterial P RNAs. Finally, the results obtained here provide further evidence that the gel retardation assay is suitable for binding interference studies to identify the structural elements of RNase P RNAs and tRNAs that are crucial for the formation of a specific RNase P RNA-tRNA complex.

Base Sequence↗
PubMed2000

Effects of phosphorothioate modifications on precursor tRNA processing by eukaryotic RNase P enzymes.

The cleavage mechanism has been studied for nuclear RNase P from Saccharomyces cerevisiae, Homo sapiens sapiens and Dictyostelium discoideum, representing distantly related branches of the Eukarya. This was accomplished by using precursor tRNAs (ptRNAs) carrying a single Rp or Sp-phosphorothioate modification at the normal RNase P cleavage site (position -1/+1). All three eukaryotic RNase P enzymes cleaved the Sp-diastereomeric ptRNA exclusively one nucleotide upstream (position -2/-1) of the modified canonical cleavage site. Rp-diastereomeric ptRNA was cleaved with low efficiency at the modified -1/+1 site by human RNase P, at both the -2/-1 and -1/+1 site by yeast RNase P, and exclusively at the -2/-1 site by D. discoideum RNase P. The presence of Mn(2+ )and particularly Cd(2+) inhibited the activity of all three enzymes. Nevertheless, a Mn(2+ )rescue of cleavage at the modified -1/+1 site was observed with yeast RNase P and the Rp-diastereomeric ptRNA, consistent with direct metal ion coordination to the (pro)-Rp substituent during catalysis as observed for bacterial RNase P enzymes. In summary, our results have revealed common active-site constraints for eukaryotic and bacterial RNase P enzymes. In all cases, an Rp as well as an Sp-phosphorothioate modification at the RNase P cleavage site strongly interfered with the catalytic process, whereas substantial functional interference is essentially restricted to one of the two diastereomers in other RNA and protein-catalyzed hydrolysis reactions, such as those catalyzed by the Tetrahymena ribozyme and nuclease P1.

Animals↗
PubMed1977

Nuclear and cytoplasmic RNase-activity in regenerating mouse liver.

Nuclear and cytoplasmic RNase activities at pH 5.0 and 7.6 were analyzed in regenerating mouse liver at 6, 12, 24, 48, and 72 h after partial hepatectomy. Two different nucleus-isolation methods were used, one in a EDTA-spermidine medium free from divalent cations, and one in a sucrose medium containing these ions. During regeneration, the cytoplasmic alkaline RNase activity in the sucrose medium was unchanged, but in the spermidine medium showed an increase toward the end of the period. Also the cytoplasmic acid RNase activity was unchanged in sucrose medium, whereas in the spermidine it slightly increased during regeneration. The nuclear alkaline RNase activity showed a notable peak 6 h after the operation and later decreased. Also the nuclear acid RNase activity displayed a similar marked peak 6 h after operation, then decreased, but remained high throughout the period. The nuclear RNase activities were about 1% of the corresponding cytoplasmic RNase activities. The absolute activities varied greatly according to the nucleus-isolation methods. In the controls, the absolute activity of nuclear alkaline RNase was slightly above (1.2 times) that of the corresponding acid activity after the spermidine method. After the sucrose method the nuclear alkaline activity was 2.7 times that of the acid activity. The absoluted activity of cytoplasmic alkaline RNase was slightly above (1.2 times) the acid activity after the spermidine method but after the sucrose method it was only 0.25 times that of the acid activity. In sham-operated animals, cytoplasmic acid and alkaline RNase activities generally were fairly similar to the normal value, but corresponding nuclear activities showed marked variations indicating an influence by anesthesia.

Animals↗
PubMed2004

RNase activity requires formation of disulfide bonds and is regulated by the redox state.

The activity of many RNases requires the formation of one or more disulfide bonds which can contribute to their stability. In this study, we show that RNase activity and, to a much lesser extent, nuclease activity, are redox regulated. Intracellular RNase activity was altered in vitro by changes in the glutathione redox state. Moreover, RNase activity was abolished following exposure to reducing agents such as beta-ME or DTT. Following reduction with glutathione (GSH), RNase activity could be fully reactivated with oxidized glutathione (GSSG). In contrast, RNase activity could not be reactivated when reduced with DTT. Decreasing the level of glutathione in vivo in wheat increased RNase activity. Tobacco engineered to have an increased glutathione redox state exhibited substantially lower RNase activity during dark-induced senescence. These results suggest that RNase activity requires the presence of one or more disulfide bonds that are regulated by glutathione and demonstrate for the first time that RNase activity can be altered with an alteration in cellular redox state.

Darkness↗
PubMed2004

Cytotoxicity of RNases is increased by cationization and counteracted by K(Ca) channels.

K(Ca) channels are involved in control of cell proliferation and differentiation. Here we have revealed their role in overcoming the RNase-induced cytotoxicity. Toxic effects of Streptomyces aureofaciens RNases Sa, Sa2, Sa3, and of RNase Sa charge reversal mutants on the human embryonic kidney cell lines differing only by the presence of K(Ca) channels were characterized. In contrast to other RNases, a basic variant of RNase Sa and RNase Sa3 exhibit significant cytotoxic activity of the same order of magnitude as onconase. Our data indicate the absence of a correlation between catalytic activity and stability of RNases and cytotoxicity. On the other hand, cationization enhances toxic effect of an RNase indicating the major role of a positive charge. Essentially lower sensitivity to cytotoxic microbial RNases of cells expressing K(Ca) channels was found. These results suggest that cells without the K(Ca) channel activity cannot counteract toxic effect of RNases.

Calcium Channels↗
PubMed2004

Expression of interferon-inducible recombinant human RNase L causes RNA degradation and inhibition of cell growth in Escherichia coli.

Interferon-inducible ribonuclease L (RNase L) is a unique ankyrin-repeat containing endoribonuclease activated by 2',5'-oligoadenylate (2-5A) cofactor leading to RNA degradation and apoptosis during antiviral response in mammalian cells. We report that expression of recombinant human RNase L (1-741 a.a.) caused RNA degradation and inhibition of cell growth in Escherichia coli in absence of exogenous 2-5A. On the contrary, expression of a homologous but dominant negative form of murine RNase L (1-656 a.a.), lacking the RNA binding and ribonuclease domain, did not show RNA degradation, rather it stimulated cell growth. Upon computational analysis by pBLAST search, a putative transcription factor (yahD, F64758, and NP_414852) from the E. coli genome showed highest homology (E value=1e(-17)) with 90-259 a.a. region of human RNase L due to ankyrin repeats with conserved GKT motifs. Ankyrin repeats 6-9 of RNase L are involved in 2-5A binding, dimerization, and activation of the ribonuclease. Thus, a biochemically active human RNase L in E. coli strongly suggests for a prokaryotic cell growth-inhibitory mechanism possibly through ankyrin-ankyrin interaction of YahD and RNase L leading to RNA degradation. The mammalian interferon-inducible RNase L and E. coli yahD protein may have common origin for the ankyrin repeats with 2-5A binding sites. Thus, RNA degradation and cell growth inhibition by recombinant human RNase L biochemically reconstituted mammalian cellular response to interferon in E. coli. RNase L has prokaryotic evolutionary history, it is not only an antiviral but also an antibacterial gene.

Amino Acid Sequence↗
PubMed2006

The role of endoribonucleases in the regulation of RNase R.

RNase R is an important exoribonuclease involved in the maturation and degradation of RNA. RNase R is co-transcribed with other genes in the same operon. In this report, we show that under physiological conditions maturation of these co-transcripts and the levels of RNase R are mainly dependent on the endoribonuclease RNase E. The presence of the full-length RNase E is necessary for the decay of intermediary products that arise from the maturation of transcripts from the rnr operon. RNase G and RNase III do not seem to have a primary role in the processing of the rnr transcripts. However, the accumulation of intermediary transcripts in an rng mutant suggests that RNase G may act in the degradation of the transcripts already cleaved by RNase E. These results demonstrated that other ribonucleases can act as an additional level of regulation in the control of the expression of RNase R.

Endoribonucleases↗
PubMed2005

Targeting HIV-1 integrase with aptamers selected against the purified RNase H domain of HIV-1 RT.

Several in vitro strategies have been developed to selectively screen for nucleic acid sequences that bind to specific proteins. We previously used the SELEX procedure to search for aptamers against HIV-1 RNase H activity associated with reverse transcriptase (RT) and human RNase H1. Aptamers containing G-rich sequences were selected in both cases. To investigate whether the interaction with G-rich oligonucleotides (ODNs) was a characteristic of these enzymes, a second in vitro selection was performed with an isolated RNase H domain of HIV-1 RT (p15) as a target and a new DNA library. In this work we found that the second SELEX led again to the isolation of G-rich aptamers. But in contrast to the first selection, these latter ODNs were not able to inhibit the RNase H activity of either the p15 domain or the RNase H embedded in the complete RT. On the other hand, the aptamers from the first SELEX that were inhibitors of the RT-associated RNase H did not inhibit the activity of the isolated p15 domain. This suggests that the active conformation of both RNase H domains is different according to the presence or absence of the DNA polymerase domain. HIV-1 RNase H and integrase both belong to the phosphotransferase family and share structural similarities. An interesting result was obtained when the DNA aptamers initially raised against p15 RNase H were assayed against HIV-1 integrase. In contrast to RNase H, the HIV-1 integrase was inhibited by these aptamers. Our results point out that prototype structures can be exploited to develop inhibitors of two related enzymes.

Aptamers, Nucleotide↗
PubMed2001

Crystallographic and modeling studies of RNase III suggest a mechanism for double-stranded RNA cleavage.

BACKGROUND: Aquifex aeolicus Ribonuclease III (Aa-RNase III) belongs to the family of Mg(2+)-dependent endonucleases that show specificity for double-stranded RNA (dsRNA). RNase III is conserved in all known bacteria and eukaryotes and has 1-2 copies of a 9-residue consensus sequence, known as the RNase III signature motif. The bacterial RNase III proteins are the simplest, consisting of two domains: an N-terminal endonuclease domain, followed by a double-stranded RNA binding domain (dsRBD). The three-dimensional structure of the dsRBD in Escherichia coli RNase III has been elucidated; no structural information is available for the endonuclease domain of any RNase III. RESULTS: We present the crystal structures of the Aa-RNase III endonuclease domain in its ligand-free form and in complex with Mn(2+). The structures reveal a novel protein fold and suggest a mechanism for dsRNA cleavage. On the basis of structural, genetic, and biological data, we have constructed a hypothetical model of Aa-RNase III in complex with dsRNA and Mg(2+) ion, which provides the first glimpse of RNase III in action. CONCLUSIONS: The functional Aa-RNase III dimer is formed via mainly hydrophobic interactions, including a "ball-and-socket" junction that ensures accurate alignment of the two monomers. The fold of the polypeptide chain and its dimerization create a valley with two compound active centers at each end of the valley. The valley can accommodate a dsRNA substrate. Mn(2+) binding has significant impact on crystal packing, intermolecular interactions, thermal stability, and the formation of two RNA-cutting sites within each compound active center.

Amino Acid Sequence↗
PubMed2002

Phylogenetic analysis of the structure of RNase MRP RNA in yeasts.

RNase MRP is a ribonucleoprotein enzyme involved in processing precursor rRNA in eukaryotes. To facilitate our structure-function analysis of RNase MRP from Saccharomyces cerevisiae, we have determined the likely secondary structure of the RNA component by a phylogenetic approach in which we sequenced all or part of the RNase MRP RNAs from 17 additional species of the Saccharomycetaceae family. The structure deduced from these sequences contains the helices previously suggested to be common to the RNA subunit of RNase MRP and the related RNA subunit of RNase P, an enzyme cleaving tRNA precursors. However, outside this common region, the structure of RNase MRP RNA determined here differs from a previously proposed universal structure for RNase MRPs. Chemical and enzymatic structure probing analyses were consistent with our revised secondary structure. Comparison of all known RNase MRP RNA sequences revealed three regions with highly conserved nucleotides. Two of these regions are part of a helix implicated in RNA catalysis in RNase P, suggesting that RNase MRP may cleave rRNA using a similar catalytic mechanism.

Base Sequence↗
PubMed1999

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.

Apoptosis Regulatory Proteins↗