Identification of a 100‐kDa protein associated with nuclear ribonuclease P activity in <i>Schizosaccharomyces pombe</i>

Zimmerly, Steven; Drainas, Denis; Sylvers, Lee A.; Söll, Dieter

doi:10.1111/j.1432-1033.1993.tb18270.x

Cited by 29 publications

(24 citation statements)

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“…The fact that wheat germ RNase P is not inactivated by micrococcal nuclease suggests that its overall structure might be similar to that of other eukaryotic and also archaebacterial enzymes, with a relatively large protein subunit protecting the RNA from nuclease attack and contributing to the low density of the holoenzyme. Indeed, eukaryotic and archaebacterial RNases P tend to possess much larger protein subunits than the bacterial enzymes, as estimated from the size of the holoenzymes (Kmpp et al, 1986;Lee and Engelke, 1989;Bartkiewicz et al, 1989;Morales et al, 1989;Darr et al, 1990) and also after direct identification of the protein component (Morales et al, 1992;Dang and Martin, 1993;Zimmerly et a]., 1993).…”

Section: Discussionmentioning

confidence: 99%

Partial Purification and Characterization of Nuclear Ribonuclease P from Wheat

Arends

Schön

1997

European Journal of Biochemistry

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Ribonuclease P (RNase P) from wheat nuclei has been purified over 1000-fold, using wheat germ extract as starting material and a combination of poly(ethylenglyco1) precipitation and column chromatography. The enzyme was shown to be of nuclear origin by its characteristic ionic requirements; for optimum activity it requires 0.5-1.5 mM Mg", which can be partly replaced by MnZ+. With about 100 kDa, wheat nuclear RNase P has the lowest molecular mass reported so far for a eukaryotic RNase P. The enzyme has an isoelectric point of 5.0 and a buoyant density of 1.34 g/ml in CsCl, suggesting the presence of a nucleic acid component; it is, however, insensitive against treatment with micrococcal nuclease. Wheat germ RNase P requires an intact tertiary structure of the pre-tRNA substrate; its cleavage efficiency is also influenced by the presence of an intron, and by the nature of the 3' terminus of the substrate. The apparent K,,, and V,,, for an intronless plant pre-tRNATYr are 10.3 nM and 1.12 fmol/min, respectively.Keywords: enzyme purification; nuclear ribonuclease P; pre-tRNA processing ; substrate specificity ; Triticum aestivum.RNase P is the ubiquitous endonuclease required for the maturation of the 5' end of tRNAs. In all cases where the composition of this enzyme has been investigated in detail, it was found to consist of both protein and RNA subunits (Kmpp et al., 1986;Bartkiewicz et al., 1989;Lee and Engelke, 1989;Morales et al., 1989;Doria et al., 1991;Nieuwlandt et al., 1991 ; LaGrandeur et al., 1993;Marchfelder and Brennicke, 1994;Baum et al., 1996; Lee et al., 1996a,b; for the bacterial enzymes, see reviews by Altman et al., 1993a;Pace and Brown, 1995). RNase P RNAs from bacteria show enzymatic activity in vitro without the protein subunit; however, RNase P from archaebacteria or eukaryotes is only active as a holoenzyme. In contrast to our current knowledge on RNase P from bacteria and most eukaryotic cellular compartments, no detailed information is available on cleavage mechanism, substrate requirements, or subunit composition of RNase P from plant nuclei. Previous studies performed with a complete processing system from wheat germ did not allow unambigous determination of the order of cleavage events involved in end maturation of tRNAs (e.g. Stange and Beier, 1987). Similarly, although investigation of a variety of pre-tRNAs indicated that mutations disrupting the tRNA structure severely impair overall processing in this plant in vitro system (Stange et al., 1991 ;Fuchs et al., 1992;Teichmann et al., 1994), it is not clear at present which of the enzymes involved is most sensitive to these changes. To approach these questions, and to gain insight into the molecular composition and substrate requirements of nuclear RNase P from higher (EC 3.1.31.1).plants, we have purified and characterized this enzyme from wheat germ. Although an RNA component could not yet be identified in wheat germ RNase P, several properties of the enzyme indicate that it might be a ribonucleoprotein. The availability of a high...

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Section: Discussionmentioning

confidence: 99%

Partial Purification and Characterization of Nuclear Ribonuclease P from Wheat

Arends

Schön

1997

European Journal of Biochemistry

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“…A temperature-sensitive mutant of rprl accumulates pre-tRNAs that are spliced but 5' and 3' unprocessed (Lee et al 1991). Analysis of the protein components of the nuclear RNase P from S. cerevisiae has not been reported; however, highly purified RNase P from Schizosaccharomyces pombe contains a single protein component of 100 kD estimated molecular mass (Zimmerly et al 1993). RNase P from vertebrates can be immunoprecipitated by human Th autoim-Cold Spring Harbor Laboratory Press on May 12, 2018 -Published by genesdev.cshlp.org Downloaded from mune sera (also called To sera) .…”

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confidence: 99%

The POP1 gene encodes a protein component common to the RNase MRP and RNase P ribonucleoproteins.

Lygerou¹,

Mitchell²,

Petfalski³

et al. 1994

Genes Dev.

198

214

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Two forms of the yeast 5.8S rRNA are generated from a large precursor by distinct processing pathways. Cleavage at site A3 is required for synthesis of the major, short form, designated 5.8S(S), but not for synthesis of the long form, 5.8S(L). To identify components required for A3 cleavage, a bank of temperature-sensitive lethal mutants was screened for those with a reduced ratio of 5.8S(S):5.8S(L). The pop1-1 mutation (for processing of precursor RNAs) shows this phenotype and also inhibits A3 cleavage. The pre-rRNA processing defect of pop1-1 strains is similar to that reported for mutations in the RNA component of RNase MRP; we show that a mutation in the RNase MRP RNA also inhibits cleavage at site A3. This is the first site shown to require RNase MRP for cleavage in vivo. The pop1-1 mutation also leads to a block in the processing of pre-tRNA that is identical to that reported for mutations in the RNA component of RNase P. The RNA components of both RNase MRP and RNase P are underaccumulated in pop1-1 strains at the nonpermissive temperature, and immunoprecipitation demonstrates that POPlp is a component of both ribonucleoproteins.The POP1 gene encodes a protein with a predicted molecular mass of 100.5 kD and is essential for viability.POPlp is the first protein component of the nuclear RNase P or RNase MRP for which the gene has been cloned.[Key Words: POP1 gene; RNase MRP; RNase P; S. cerevisiae; RNA processing] Received April 4, 1994; revised version accepted May 9, 1994.Two ribonucleoprotein (RNP) complexes are known to have endoribonuclease (RNase) activity. These are the RNase P and RNase mitochondrial RNA processing (MRP) RNPs.All pre-tRNAs are transcribed with 5' and 3' extensions, and some contain intervening sequences that are removed to produce the mature tRNAs. RNase P is responsible for the 5' processing of pre-tRNAs in eubacteria, archaebacteria, and eukaryotes (for review, see Altman 1989;Dart et al. 1992;Altman et al. 1993). RNase P also processes the precursor of the 4.5S RNA in Escherichia coli (for review, see by Altman et al. 1993) but has not been reported to have substrates other than pretRNAs in archaebacteria or eukaryotes. The RNA component of eubacterial RNase P has catalytic activity in vitro in the absence of protein cofactors. In contrast, the RNA components of archaebacterial (Nieuwlandt et al. 1991;LaGrandeur et al. 1993) and eukaryotic RNase P have not been found to be catalytic in the absence of proteins, despite their considerable structural homology to the eubacterial RNAs. E. coli RNase P contains a single protein, designated C5, which is small (119 amino acids, 13.8 kD) and highly basic. This protein cofactor is required for RNase P activity in vivo and greatly stimulates activity in vitro. The protein plays important roles in the release of the product, the mature tRNA, from the enzyme, which is the rate-limiting step for the RNA catalyzed reaction in vitro (Reich et al. 1988;Tallsj6 and Kirsebom 1993), and in the binding of suboptimal substrates, including the 4.5S RN...

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“…polypeptide because other known eukaryotic nuclear RNase P are made up of a single large polypeptide (Zimmerly et al, 1993). The following evidence confirmed that nuclear RNase P Characterization of the RNA component of nuclear RNase P. To determine whether the RNA component is an essential was composed of a seven-polypeptide complex.…”

Section: Discussionmentioning

confidence: 76%

“…This means that eukaryotic RNase P is more proteinaceous lus species) is about 150 kDa (Darr et al, 1992) and the buoyant than its bacterial counterpart. Second, the molecular mass of density of RNase P of E. coli (Lawrence et al, 1987) and Haloeukaryotic RNase P is greater than that of its bacterial counterferax volcanii (Nieuwlandt et al, 1987) in a cesium sulfate grapart [for example, the molecular mass of RNase P of Saccharodient are 1.55 g/cm 3 and 1.61 g/cm 3 , respectively. The length of myces pombe is about 450 kDa (Kline et al, 1981; Krupp et al, the RNA component of bacterial RNase P is 340Ϫ400 nucleo-1986).…”

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Purification and characterization of the nuclear ribonuclease P of Aspergillus nidulans

Han¹,

Lee

Kang³

1998

European Journal of Biochemistry

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In Aspergillus nidulans, the nuclear ribonuclease P was separated from its mitochondrial counterpart by Q-Sepharose chromatography, and a precursor-tRNA His processing assay system was used to discriminate nuclear ribonuclease P activity from the mitochondrial counterpart. The nuclear ribonuclease P was purified to near homogeneity from whole-cell extracts. A 2150-fold purification with a yield of 2.3 % was achieved by five types of chromatography including tRNA affinity chromatography and glycerol gradient velocity sedimentation. This enzyme, which had a molecular mass of 580 kDa determined by both glycerol-gradient sedimentation analysis and gel-permeation chromatography, appeared to be composed of seven polypeptides and an RNA molecule. Seven polypeptides, with masses of 125, 85, 45, 33, 30, 21, 19 kDa, were consistently copurified with nuclear ribonuclease P activity through MonoS and tRNA affinity chromatography and in a glycerol gradient. As judged by a micrococcal-nuclease-sensitivity assay, nuclear ribonuclease P required an RNA component for its activity, as do other ribonuclease Ps. Analysis of the radiolabeled 5′ end of RNAs copurified with nuclear ribonuclease P implied that RNA molecules in the purified nuclear ribonuclease P originated from a common RNA molecule, the putative RNA molecule of nuclear ribonuclease P. Comparison of the two ribonuclease Ps in A. nidulans showed that the protein and RNA components of the nuclear ribonuclease P were different from those of the mitochondrial counterpart.Keywords : Aspergillus nidulans ; nuclear ribonuclease P; precursor-tRNA His processing ; copurifying RNA; protein component of nuclear ribonuclease P.Ribonuclease P (RNase P), an endonuclease involved in structures of the RNA components (cage-shape structure) are very similar to each other (Forster and Altman, 1990). 5′-end maturation of precursor-tRNA His (pre-tRNA), is a ribonucleoprotein. On the basis of micrococcal nuclease and proteAlthough RNase P in spinach chloroplast has physical properties inconsistent with the presence of any RNA (Thomas et al., ase-sensitivity assays, both RNA and protein components are essential for bacterial RNase P activity in vivo. The protein com-1995), RNase P in the nuclei and mitochondria of eukaryotes studied so far are also ribonucleoprotein complexes like their ponent of bacterial RNase P [such as C5 protein in Escherichia coli (Kole and Altman, 1981;Hansen et al., 1985) and P protein bacterial counterparts (Darr et al., 1992). Even if the RNase P functions in vivo in prokaryotes and eukaryotes are the same, in Bacillus subtilis (Ogasawara et al., 1985)] is a 14-kDa small basic protein. The function of the protein component in bacterial two properties of eukaryotic RNase Ps are different from the bacterial counterparts. First, the buoyant density in a cesium RNase P activity is not well known, but it may play a role as sulfate gradient of eukaryotic RNase P is less than that of bactean electrostatic screen allowing the negatively charged RNArial RNase P ; the buoya...

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Identification of a 100‐kDa protein associated with nuclear ribonuclease P activity in Schizosaccharomyces pombe

Cited by 29 publications

References 34 publications

Partial Purification and Characterization of Nuclear Ribonuclease P from Wheat

Partial Purification and Characterization of Nuclear Ribonuclease P from Wheat

The POP1 gene encodes a protein component common to the RNase MRP and RNase P ribonucleoproteins.

Purification and characterization of the nuclear ribonuclease P of Aspergillus nidulans

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