Nuclear Ribonuclease Research Articles

Promoter-dependent nuclear RNA degradation ensures cell cycle-specific gene expression

Cell cycle progression depends on phase-specific gene expression. Here we show that the nuclear RNA degradation machinery plays a lead role in promoting cell cycle-dependent gene expression by triggering promoter-dependent co-transcriptional RNA degradation. Single molecule quantification of RNA abundance in different phases of the cell cycle indicates that relative curtailment of gene expression in certain phases is attained even when transcription is not completely inhibited. When nuclear ribonucleases are deleted, transcription of the Saccharomyces cerevisiae G1-specific axial budding gene AXL2 is detected throughout the cell cycle and its phase-specific expression is lost. Promoter replacement abolished cell cycle-dependent RNA degradation and rendered the RNA insensitive to the deletion of nuclear ribonucleases. Together the data reveal a model of gene regulation whereby RNA abundance is controlled by promoter-dependent induction of RNA degradation.

The rice thermo-sensitive genic male sterility gene tms9: pollen abortion and gene isolation

AbstractThe development of two-line hybrid rice has done great contribution to the food security. It is imperative to study the male sterility mechanism of rice photo-thermo sensitive genic male sterile (PTGMS) line which is the core component of two-line hybrid rice. Zhu1S is a rice thermo-sensitive genic male sterile line used frequently as female parent in two-line hybrid seed production. A cytological examination of the development of the Zhu1S anther wall and the microspores showed that tms9 encoded male sterility is caused by the failure of the tapetum to degenerate normally, thereby starving the microspores and finally leading to pollen abortion. A fine-scale genetic map based on a large F2 population allowed tms9 to be located within a 30.2 Kb segment of chromosome 2 harboring seven open reading frames. A comparison between Zhu1S under sterile temperature condition (high temperature, male sterile) and fertile temperature condition (low temperature, fertile) showed that only one of the seven genes, LOC_Os02g12290, was differentially transcribed, with its transcript abundance being much lower under the sterile temperature growing condition. LOC_Os02g12290 encodes a nuclear ribonuclease Z. Re-sequencing demonstrated that the Zhu1S LOC_Os02g12290 allele differed from that present in non tms9 carrier alleles by two contiguous nucleotides in the first exon, inducing a truncated mRNA.

MicroRNAs as Modulators of the Platelet Proteome

MicroRNAs are short 21- to 24-nucleotide (nt) RNA species that act as key regulators of gene expression. Known primarily to modulate mRNA translation through recognition of specific binding sites located in the 3untranslated region (UTR) of messenger RNA (mRNA) targets, microRNAs may regulate between 30% to 92% of the genes in human, thereby controlling a plethora of biological processes. Although devoid of a nucleus and lacking genomic DNA, platelets may be no exception, as recent experimental evidences indicate that they contain all the protein and RNA components and features required for microRNA-regulated mRNA translation: (i) the platelet transcriptome is astonishingly diverse, representing between 15 and 32% of all human genes, (ii) platelet mRNAs can be translated into proteins, (iii) platelets contain an abundant and diverse array of microRNAs, and (iv) the host Dicer and Argonaute 2 (Ago2) complexes. The latter ones are functional in microRNA biogenesis and function, respectively. In this review article, we will summarize and discuss the experimental evidences as well as the most recent advances supporting a role for microRNAs as modulators of the platelet proteome. Expected to play a central role in health and disease, a dysfunctional microRNA-based regulation of gene expression in platelets may represent an important etiologic factor underlying platelet-related and cardiovascular diseases. Keywords: Cardiovascular diseases, gene regulation, messenger RNA, microRNA, RNA silencing, platelet, biogenesis, nuclear ribonuclease, mutation, neurological diseases

Expression of the ribonucleases Drosha, Dicer, and Ago2 in colorectal carcinomas

The pathogenesis of colorectal carcinoma (CRC) is a complex process that involves the recruitment of both genetic and epigenetic mechanisms. Recent studies underline the cardinal role of small, noncoding RNA molecules, called microRNAs (miRs), in the pathobiology of numerous physiological and pathological processes, including oncogenesis. MiR biogenesis and maturation is mainly regulated by the nuclear ribonuclease Drosha and the cytoplasmic ribonucleases Dicer and Ago2. In the present study, we investigated the expression and distribution of these molecules in three colon cancer cell lines and in human CRC samples. Drosha, Dicer, and Ago2 mRNA and protein expression was assessed with real-time PCR, western blotting, and immunofluorescence. Our experiments showed that Drosha, Dicer, and Ago2 were expressed in all the cell lines and in the majority of the CRC samples examined. The mRNA levels of Dicer were significantly augmented in stage III compared to stage II tumors. Our results suggest that Drosha, Dicer, and Ago2 are possibly implicated in CRC pathobiology and that Dicer might have a role in the progression of these tumors to advanced stages.

Fine mapping and candidate gene analysis of ptgms2-1, the photoperiod-thermo-sensitive genic male sterile gene in rice (Oryza sativa L.)

Photoperiod-thermo-sensitive genic male sterile (PTGMS) rice exhibits a number of desirable traits for hybrid rice production. The cloning genes responsible for PTGMS and those elucidating male sterility mechanisms and reversibility to fertility would be of great significance to provide a foundation to develop new male sterile lines. Guangzhan63S, a PTGMS line, is one of the most widely used indica two-line hybrid rice breeding systems in China. In this study, genetic analysis based on F(2) and BC(1)F(2) populations derived from a cross between Guangzhan63S and 1587, determined a single recessive gene controls male sterility in Guangzhan63S. Molecular marker techniques combined with bulked-segregant analysis (BSA) were used and located the target gene (named ptgms2-1) between two SSR markers RM12521 and RM12823. Fine mapping of the ptgms2-1 locus was conducted with 45 new Insertion-Deletion (InDel) markers developed between the RM12521 and RM12823 region, using 634 sterile individuals from F(2) and BC(1)F(2) populations. Ptgms2-1 was further mapped to a 50.4 kb DNA fragment between two InDel markers, S2-40 and S2-44, with genetic distances of 0.08 and 0.16 cM, respectively, which cosegregated with S2-43 located on the AP004039 BAC clone. Ten genes were identified in this region based on annotation results from the RiceGAAS system. A nuclear ribonuclease Z gene was identified as the candidate for the ptgms2-1 gene. This result will facilitate cloning the ptgms2-1 gene. The tightly linked markers for the ptgms2-1 gene locus will further provide a useful tool for marker-assisted selection of this gene in rice breeding programs.

Of proteins and RNA: The RNase P/MRP family

Nuclear ribonuclease (RNase) P is a ubiquitous essential ribonucleoprotein complex, one of only two known RNA-based enzymes found in all three domains of life. The RNA component is the catalytic moiety of RNases P across all phylogenetic domains; it contains a well-conserved core, whereas peripheral structural elements are diverse. RNA components of eukaryotic RNases P tend to be less complex than their bacterial counterparts, a simplification that is accompanied by a dramatic reduction of their catalytic ability in the absence of protein. The size and complexity of the protein moieties increase dramatically from bacterial to archaeal to eukaryotic enzymes, apparently reflecting the delegation of some structural functions from RNA to proteins and, perhaps, in response to the increased complexity of the cellular environment in the more evolutionarily advanced organisms; the reasons for the increased dependence on proteins are not clear. We review current information on RNase P and the closely related universal eukaryotic enzyme RNase MRP, focusing on their functions and structural organization.

Regulating the Regulator

MicroRNAs (miRNAs) have emerged as posttranscriptional regulators of gene expression that appear to have important roles in complex processes like development and the development of cancer. Although the abundance of miRNAs increases dramatically during development and is decreased in some cancer cells, how the production of miRNAs is regulated has not been known. Although transcription of miRNAs is one obvious control point, the processing steps mediated by the nuclear ribonuclease III (RNAse III) enzymes known as Drosha and Dicer might also contribute. Thomson et al. therefore monitored expression of the Let-7 family of miRNAs during mouse development. The amount of mature Let-7g increased several thousand-fold during mouse embryogenesis but was not correlated with production of the primary transcript. Rather, the change in abundance appeared more likely to reflect a block in processing of a specific group of miRNAs by Drosha. A set of genes was also identified encoding miRNAs that decreased in abundance in published tumor expression data. Again for this set of genes, there was no correlation between abundance of the primary transcript and the reduced expression of mature miRNA. The authors propose that the Drosha-mediated processing step is an important control point for modulating the abundance of miRNAs in normal and cancer cells. J. M. Thomson, M. Newman, J. S. Parker, E. M. Morin-Kensicki, T. Wright, S. M. Hammond, Extensive post-transcriptional regulation of microRNAs and its implications for cancer. Genes Dev. 20 , 2202-2207 (2006). [Abstract] [Full Text]

An active precursor in assembly of yeast nuclear ribonuclease P.

The RNA-protein subunit assembly of nuclear RNase P was investigated by specific isolation and characterization of the precursor and mature forms of RNase P using an RNA affinity ligand. Pre-RNase P was as active in pre-tRNA cleavage as mature RNase P, although it contained only seven of the nine proteins found in mature RNase P. Pop3p and Rpr2p were not required for maturation of the RPR1 RNA subunit and virtually absent from pre-RNase P, implying that they are dispensable for pre-tRNA substrate recognition and cleavage. The RNase P subunit assembly is likely to occur in the nucleolus, where both precursor and mature forms of RNase P RNA are primarily localized. The results provide insight into assembly of nuclear RNase P, and suggest pre-tRNA substrate recognition is largely determined by the RNA subunit.

Human ribonuclease P: subunits, function, and intranuclear localization.

Catalytic complexes of nuclear ribonuclease P (RNase P) ribonucleoproteins are composed of several protein subunits that appear to have specific roles in enzyme function in tRNA processing. This review describes recent progress made in the characterization of human RNase P, its relationship with the ribosomal RNA processing ribonucleoprotein RNase MRP, and the unexpected evolutionary conservation of its subunits. A new model for the biosynthesis of human RNase P is presented, in which this process is dynamic, transcription-dependent, and implicates functionally distinct nuclear compartments in tRNA biogenesis.

Function and subnuclear distribution of Rpp21, a protein subunit of the human ribonucleoprotein ribonuclease P.

Rpp21, a protein subunit of human nuclear ribonuclease P (RNase P) was cloned by virtue of its homology with Rpr2p, an essential subunit of Saccharomyces cerevisiae nuclear RNase P. Rpp21 is encoded by a gene that resides in the class I gene cluster of the major histocompatibility complex, is associated with highly purified RNase P, and binds precursor tRNA. Rpp21 is predominantly localized in the nucleoplasm but is also observed in nucleoli and Cajal bodies when expressed at high levels. Intron retention and splice-site selection in Rpp21 precursor mRNA regulate the intranuclear distribution of the protein products and their association with the RNase P holoenzyme. Our study reveals that dynamic nuclear structures that include nucleoli, the perinucleolar compartment and Cajal bodies are all involved in the production and assembly of human RNase P.

The Phylogenetic Position of “Acomyinae” (Rodentia, Mammalia) as Sister Group of a Murinae + Gerbillinae Clade: Evidence from the Nuclear Ribonuclease Gene

The phylogenetic relationships of Acomys and Uranomys within Muridae were investigated using nuclear pancreatic ribonuclease A gene sequences. The various kinds of substitutions in the data matrix (15 taxa×375 nucleotides) were examined for saturation, in order to apply a weighted parsimony approach. Phylogenies were derived by maximum parsimony (weighted and unweighted) and maximum likelihood procedures, using a dormouse (Gliridae) as outgroup. Maximum likelihood gave the most robust results. All analyses cluster some traditional taxa with a strong robustness, such as three species of the genus Mus, two South-East Asian rats, and two genera in each of the gerbil and vole families. When analyzed with those of other murid rodents representing Murinae, Gerbillinae, Arvicolinae, Cricetinae, and Sigmodontinae, sequences of the ribonuclease gene suggest that Acomys and Uranomys constitute a monophyletic clade at the subfamily level, denoted “Acomyinae.” The relationships between the six subfamilies of Muridae appear poorly resolved, except for a clade uniting Murinae, Acomyinae, and Gerbillinae. Within this clade, the sister group of Acomyinae could not be identified, as the branch length defining a Gerbillinae + Murinae cluster is extremely short. The poor resolution of our phylogenetic inferences is probably the result of two confounding factors, namely the limited size of the pancreatic ribonuclease sequence and the probable short time intervals during the radiation of the six murid subfamilies involved in this study.

Purification and characterization of the nuclear ribonuclease P of Aspergillus nidulans.

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.

Partial purification and characterization of nuclear ribonuclease P from wheat.

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(ethylenglycol) 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 Mg2+, which can be partly replaced by Mn2+. 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 Km and Vmax for an intronless plant pre-tRNA(Tyr) are 10.3 nM and 1.12 fmol/min, respectively.

Eukaryotic Nuclear RNase P: Structures and Functions

Over the past decade, ribonucleic acids (RNAs) have gained recognition as the catalysts for an increasing number of biochemical reactions. This chapter discusses the structures and functions of eukaryotic nuclear ribonuclease P (RNase P). The recognition and alignment of appropriate cleavage site proceeds through Watson–Crick base-pairing among the regions of complementary sequence within the same RNA chain. The function of RNase P in vivo is to bind and cleave all of the various transfer RNA (tRNA) precursors. To accomplish this in the absence of base-pairing, common determinants in the three-dimensional structures of the substrates become important. Because of the complex mode of substrate recognition by RNase P, the knowledge of the tertiary structures of both the catalytic and substrate RNAs as well as an understanding of the way these structured RNAs interact is necessary for the determination of its catalytic mechanism. Ribonuclease P is a ribonucleoprotein enzyme that endonucleolytically cleaves precursor-tRNA molecules, generating mature 5' termini. The enzyme RNase P is an essential, well-conserved functionally and has been found in every organism tested. Yeast is a single-cell eukaryotic organism that contains two versions of RNase P: a nuclear form that processes nuclear-encoded pre-tRNAs destined for the cytoplasm and a mitochondrial form that processes pre-tRNAs encoded by the mitochondrial genome.

Structure-sensitive RNA footprinting of yeast nuclear ribonuclease P.

Several enzymatic and chemical reagents were used to probe the secondary structure of Saccharomyces cerevisiae nuclear RNase P RNA in the presence and absence of its protein components. Double-stranded regions were detected with RNase V1 and single-stranded regions with RNase ONE (Escherichia coli RNase I). Nucleotides not paired at Watson-Crick positions were monitored with dimethyl sulfate, kethoxal, and 1-cyclohexyl-3-[2-(N-methylmorpholinio)ethyl]carbodiimide p-toluenesulfonate. The results supported most aspects of the previously proposed, phylogenetically-derived RNA secondary structure, although minor refinements allowed incorporation of both the biochemical and phylogenetic data. Digestion of the RNase P protein(s) with proteinase K gave enhanced reactivities to structure probes at selected positions, indicating regions of the RNA made inaccessible by the presence of the protein subunit(s). The regions of RNA protected in the yeast nuclear holoenzyme were considerably more extensive than that seen in the Escherichia coli holoenzyme, consistent with the observation that the protein moiety generally comprises a larger percentage of the RNase P holoenzyme in eukaryotes than in eubacteria.

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

Ribonuclease P from the fission yeast Schizosaccharomyces pombe has been purified to apparent homogeneity. A purification of 23,000-fold was achieved by four fractionation steps with DEAE-cellulose chromatography, phosphocellulose chromatography, glycerol-gradient fractionation and finally tRNA-affinity chromatography. A 100-kDa protein was present in the most pure preparations in amounts approximately stoichiometric with the previously identified RNA components of the enzyme, K1-RNA and K2-RNA [Krupp, G., Cherayil, B., Frendeway, D., Nishikawa, S. & Söll, D. (1986) EMBO J. 5, 1697-1703]. A cross-linking experiment utilizing a 4-thiouridine-substituted precursor tRNA demonstrated that the 100-kDa protein interacts with the ribonuclease P substrate in a specific fashion. We therefore conclude that the protein component of S. pombe ribonuclease P is a 100-kDa protein.

In vitro inhibition of HeLa cell nuclear ribonucleases by ADP-ribosylation

Ribonuclease activity in HeLa cell nuclei is markedly inhibited by ADP-ribosylation following incubation of intact isolated nuclei with [14C]NAD. Time course experiments demonstrate that [14C] incorporation into proteins is accompanied by a 50% inhibition of ribonuclease activity on single-strand and double-strand polynucleotides. Inhibition does not occur when 3-aminobenzamide, a potent (ADP-ribose) polymerase inhibitor, is present. Two enzymatic activities that degrade double-strand polynucleotides have been purified and partially characterized. A relevant level of radioactivity resulting from [14C]NAD incubation of nuclei was associated to the purified enzyme. The RNase F1 component, which shows maximal activity on polyU-polyA is demonstrated to be the major ADP-ribose acceptor protein.

The activity of neutral ribonucleases in nuclei of rat sympathetic ganglia and effects of nerve injury.

Nuclei were isolated from homogenates of rat superior cervical ganglion by a conventional differential centrifugation technique with approximately 60% recovery. Ribonuclease activity at pH 7.1 (neutral ribonuclease) was associated with the "nuclei fraction" and represented 19% of the overall activity in normal ganglia. Ribonuclease in the "nuclei fraction" was stimulated variably by the sulfhydryl blocker N-ethylmaleimide indicating that a proportion was bound to the endogenous ribonuclease inhibitor present in these ganglia. The total activity of nuclear ribonuclease was increased 2-6 days after postganglionic nerve injury, such that the inhibitor-bound form of the enzyme increased maximally by 600% at day 4. The percentage of the total ganglionic activity in the "nuclei fraction" decreased in injured ganglia as a result of a rise in the activity of non-nuclear components. The changes in nuclear ribonuclease activity were distinct from those in the 850 g supernatant indicating that specific nuclear enzymes are being affected during regeneration.

Inhibition of ribonuclease activity during RNA synthesis in isolated yeast nuclei by cadmium

We have developed an efficient transcription system in isolated yeast nuclei. If MnCl 2 is substituted by CdCl 2, degradation of newly synthesized RNA is markedly reduced. This effect is due to the inhibition of nuclear ribonuclease activity, since microsomal ribonuclease activity is less affected by the cation. The extent to which the addition of CdCl 2 to the in vitro transcription assay inhibits ribonuclease activity is demonstrated by the measurements of the size of newly synthesized RNA. Efficient RNA synthesis in this system is not affected up to a concentration of 0.1 M CdCl 2.

Partial purification and some properties of a ribonuclease associated with wheat leaf chromatin

A nuclear ribonuclease has been solubilized and partially purified from wheat leaf chromatin using column chromatography on DEAE Sephadex A-25 and hydroxyapatite. The RNase preparation was free from phosphodiesterase activity. The enzyme hydrolyzed poly(U) and poly(A) but has no activity with poly(C) and poly(G). The pH optimum was 7.0–7.2. These results show that the chromatographic behaviour and properties of chromatin associated RNase are different from previously described soluble RNases.