RESUMO
PARN encodes poly(A)-specific ribonuclease. Biallelic and monoallelic PARN variants are associated with Hoyeraal-Hreidarsson syndrome/dyskeratosis congenita and idiopathic pulmonary fibrosis (IPF), respectively. The molecular features associated with incomplete penetrance of PARN-associated IPF have not been described. We report a family with a rare missense, p.Y91C, and a novel insertion, p.(I274*), PARN variant. We found PARN p.Y91C had reduced deadenylase activity and the p.(I274*) transcript was depleted. Detailed analysis of the consequences of these variants revealed that, while PARN protein was lowest in the severely affected biallelic child who had the shortest telomeres, it was also reduced in his mother with the p.(I274*) variant but telomeres at the 50th percentile. Increased adenylation of telomerase RNA, human telomerase RNA, and certain small nucleolar RNAs, and impaired ribosomal RNA maturation were observed in cells derived from the severely affected biallelic carrier, but not in the other, less affected biallelic carrier, who had less severely shortened telomeres, nor in the monoallelic carriers who were unaffected and had telomeres ranging from the 1st to the 50th percentiles. We identified hsa-miR-202-5p as a potential negative regulator of PARN. We propose one or more genetic modifiers influence the impact of PARN variants on its targets and this underlies incomplete penetrance of PARN-associated disease.
Assuntos
Disceratose Congênita/genética , Exorribonucleases/genética , Retardo do Crescimento Fetal/genética , Deficiência Intelectual/genética , MicroRNAs/genética , Microcefalia/genética , Mutagênese Insercional , Mutação de Sentido Incorreto , Adolescente , Linhagem Celular , Pré-Escolar , Regulação para Baixo , Exorribonucleases/metabolismo , Feminino , Humanos , Masculino , Linhagem , Penetrância , Encurtamento do TelômeroRESUMO
BACKGROUND: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Förster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them. RESULTS: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, meffBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coli BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors. CONCLUSION: Availability of 14 engineered CP genes compared in E. coli, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.
RESUMO
The interaction between the Staphylococcal Protein A (SpA) domain B (the basis of the Affibody) molecule and the Fc of IgG is key to the use of Affibodies in affinity chromatography and in potential therapies against certain inflammatory diseases. Despite its importance and four-decade history, to our knowledge this interaction has never been affinity matured. We elucidate reasons why single-substitutions in the SpA which improve affinity to Fc may be very rare, and also discover substitutions which potentially serve several engineering purposes. We used a variation of FoldX to predict changes in protein-protein-binding affinity, and produce a list of 41 single-amino acid substitutions on the SpA molecule, of which four are near wild type (wt) and five are at most a factor of four from wt affinity. The nine substitutions include one which removes lysine, and several others which change charge. Subtle modulations in affinity may be useful for modifying column elution conditions. The method is applicable to other protein-protein systems, providing molecular insights with lower workload than existing experimental techniques.
Assuntos
Substituição de Aminoácidos , Fragmentos Fc das Imunoglobulinas/química , Lisina/química , Proteína Estafilocócica A/química , Afinidade de Anticorpos , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Cinética , Lisina/metabolismo , Modelos Moleculares , Plasmídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Staphylococcus aureus/química , Eletricidade Estática , TermodinâmicaRESUMO
The mRNA 5' cap structure plays a pivotal role in coordination of eukaryotic translation and mRNA degradation. Poly(A)-specific ribonuclease (PARN) is a dimeric exoribonuclease that efficiently degrades mRNA 3' poly(A) tails while also simultaneously interacting with the mRNA 5' cap. The cap binding amplifies the processivity of PARN action. We used surface plasmon resonance kinetic analysis, quantitative equilibrium fluorescence titrations and circular dichroism to study the cap binding properties of PARN. The molecular mechanism of 5' cap recognition by PARN has been demonstrated to differ from interactions seen for other known cap-binding proteins in that: i) the auxiliary biological function of 5' cap binding by the 3' degrading enzyme is accomplished by negative cooperativity of PARN dimer subunits; ii) non-coulombic interactions are major factors in the complex formation; and iii) PARN has versatile activity toward alternative forms of the cap. These characteristics contribute to stabilization of the PARN-cap complex needed for the deadenylation processivity. Our studies provide a consistent biophysical basis for elucidation of the processive mechanism of PARN-mediated 3' mRNA deadenylation and provide a new framework to interpret the role of the 5' cap in mRNA degradation.
Assuntos
Exorribonucleases/química , Proteínas de Ligação ao Cap de RNA/química , Capuzes de RNA/química , Cinética , Concentração Osmolar , Conformação Proteica , RNA Mensageiro/metabolismo , TermodinâmicaRESUMO
BACKGROUND: Deadenylation regulates RNA function and fate. Poly(A)-specific ribonuclease (PARN) is a deadenylase that processes mRNAs and non-coding RNA. Little is known about the biological significance of germline mutations in PARN. METHODS: We identified mutations in PARN in patients with haematological and neurological manifestations. Genomic, biochemical and knockdown experiments in human marrow cells and in zebrafish have been performed to clarify the role of PARN in the human disease. RESULTS: We identified large monoallelic deletions in PARN in four patients with developmental delay or mental illness. One patient in particular had a severe neurological phenotype, central hypomyelination and bone marrow failure. This patient had an additional missense mutation on the non-deleted allele and severely reduced PARN protein and deadenylation activity. Cells from this patient had impaired oligoadenylation of specific H/ACA box small nucleolar RNAs. Importantly, PARN-deficient patient cells manifested short telomeres and an aberrant ribosome profile similar to those described in some variants of dyskeratosis congenita. Knocking down PARN in human marrow cells and zebrafish impaired haematopoiesis, providing further evidence for a causal link with the human disease. CONCLUSIONS: Large monoallelic mutations of PARN can cause developmental/mental illness. Biallelic PARN mutations cause severe bone marrow failure and central hypomyelination.
Assuntos
Doenças da Medula Óssea/genética , Deficiências do Desenvolvimento/genética , Exorribonucleases/genética , Mutação de Sentido Incorreto , Deleção de Sequência , Alelos , Animais , Doenças da Medula Óssea/metabolismo , Criança , Análise Mutacional de DNA , Deficiências do Desenvolvimento/metabolismo , Feminino , Testes Genéticos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Bainha de Mielina/genética , Bainha de Mielina/patologia , Homeostase do Telômero/genética , Adulto Jovem , Peixe-ZebraRESUMO
Deadenylation of eukaryotic mRNA is a mechanism critical for mRNA function by influencing mRNA turnover and efficiency of protein synthesis. Here, we review poly(A)-specific ribonuclease (PARN), which is one of the biochemically best characterized deadenylases. PARN is unique among the currently known eukaryotic poly(A) degrading nucleases, being the only deadenylase that has the capacity to directly interact during poly(A) hydrolysis with both the m(7)G-cap structure and the poly(A) tail of the mRNA. In short, PARN is a divalent metal-ion dependent poly(A)-specific, processive and cap-interacting 3'-5' exoribonuclease that efficiently degrades poly(A) tails of eukaryotic mRNAs. We discuss in detail the mechanisms of its substrate recognition, catalysis, allostery and processive mode of action. On the basis of biochemical and structural evidence, we present and discuss a working model for PARN action. Models of regulation of PARN activity by trans-acting factors are discussed as well as the physiological relevance of PARN.
Assuntos
Exorribonucleases/química , Exorribonucleases/metabolismo , Adenina/química , Adenina/metabolismo , Animais , Evolução Molecular , Exorribonucleases/genética , Humanos , Modelos Moleculares , Poli A/metabolismo , Conformação Proteica , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Ribose/química , Ribose/metabolismo , Especificidade por SubstratoRESUMO
Deadenylation is the initial and often rate-limiting step in the main pathways of eukaryotic mRNA decay. Poly(A)-specific ribonuclease (PARN) is a eukaryotic enzyme that efficiently degrades mRNA poly(A) tails. Structural and functional studies have shown that human PARN is composed of at least three functional domains, i.e. the catalytic nuclease domain and two RNA binding domains, the R3H and the RNA recognition motif (RRM), respectively. However, the complete structure of the full length protein is still unknown. We have investigated the global architecture of human PARN by atomic force microscopy (AFM) imaging in buffered milieu and report for the first time the dimensions of the full length protein at subnanometer resolution. The AFM images of single PARN molecules reveal compact ellipsoidal dimers (10.9 × 7.6 × 4.6nm). The dimeric form of PARN was confirmed by dynamic light scattering (DLS) measurements that rendered a molecular weight of 161 kDa, in accordance with previous crystal structures of PARN fragments showing a dimeric composition. We discuss a putative internal arrangement of three functional domains within the full length PARN dimer.
Assuntos
Exorribonucleases/química , Microscopia de Força Atômica/métodos , Sequência de Aminoácidos , Humanos , Luz , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Terciária de Proteína , Espalhamento de RadiaçãoRESUMO
mRNA polyadenylation and deadenylation are important processes that allow rapid regulation of gene expression in response to different cellular conditions. Almost all eukaryotic mRNA precursors undergo a co-transcriptional cleavage followed by polyadenylation at the 3' end. After the signals are selected, polyadenylation occurs to full extent, suggesting that this first round of polyadenylation is a default modification for most mRNAs. However, the length of these poly(A) tails changes by the activation of deadenylation, which might regulate gene expression by affecting mRNA stability, mRNA transport, or translation initiation. The mechanisms behind deadenylation activation are highly regulated and associated with cellular conditions such as development, mRNA surveillance, DNA damage response, cell differentiation and cancer. After deadenylation, depending on the cellular response, some mRNAs might undergo an extension of the poly(A) tail or degradation. The polyadenylation/deadenylation machinery itself, miRNAs, or RNA binding factors are involved in the regulation of polyadenylation/deadenylation. Here, we review the mechanistic connections between polyadenylation and deadenylation and how the two processes are regulated in different cellular conditions. It is our conviction that further studies of the interplay between polyadenylation and deadenylation will provide critical information required for a mechanistic understanding of several diseases, including cancer development.
Assuntos
Poliadenilação , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Citoplasma/metabolismo , Exorribonucleases/metabolismo , MicroRNAs/metabolismo , MicroRNAs/fisiologia , RNA Polimerase II/metabolismo , Estabilidade de RNA , Saccharomyces cerevisiae/metabolismoRESUMO
Heterozygous mutations in the ribosomal protein S19 (RPS19) gene are associated with Diamond-Blackfan anemia (DBA). The mechanism by which RPS19 mediates anemia are still unclear, as well as the regulation of RPS19 expression. We show herein that RPS19 binds specifically to the 5' untranslated region of its own mRNA with an equilibrium binding constant (K(D)) of 4.1+/-1.9 nM. We investigated the mRNA binding properties of two mutant RPS19 proteins (W52R and R62W) identified in DBA patients. We observed a significant increase in K(D) for both proteins (16.1+/-2.1 and 14.5+/-4.9 nM, respectively), indicating a reduced RNA binding capability (p<0.05). We suggest that the binding of RPS19 to its mRNA has a regulatory function and hypothesize that the weaker RNA binding of mutant rRPS19 may have implications for the pathophysiological mechanisms in DBA.
Assuntos
Anemia de Diamond-Blackfan/metabolismo , Mutação de Sentido Incorreto , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Regiões 5' não Traduzidas , Anemia de Diamond-Blackfan/genética , Humanos , Ligação ProteicaRESUMO
We previously showed that mRNA 3' end cleavage reaction in cell extracts is strongly but transiently inhibited under DNA-damaging conditions. The cleavage stimulation factor-50 (CstF-50) has a role in this response, providing a link between transcription-coupled RNA processing and DNA repair. In this study, we show that CstF-50 interacts with nuclear poly(A)-specific ribonuclease (PARN) using in vitro and in extracts of UV-exposed cells. The CstF-50/PARN complex formation has a role in the inhibition of 3' cleavage and activation of deadenylation upon DNA damage. Extending these results, we found that the tumour suppressor BARD1, which is involved in the UV-induced inhibition of 3' cleavage, strongly activates deadenylation by PARN in the presence of CstF-50, and that CstF-50/BARD1 can revert the cap-binding protein-80 (CBP80)-mediated inhibition of PARN activity. We also provide evidence that PARN along with the CstF/BARD1 complex participates in the regulation of endogenous transcripts under DNA-damaging conditions. We speculate that the interplay between polyadenylation, deadenylation and tumour-suppressor factors might prevent the expression of prematurely terminated messengers, contributing to control of gene expression under different cellular conditions.
Assuntos
Núcleo Celular/metabolismo , Dano ao DNA , Poliadenilação , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Reparo do DNA , Exorribonucleases/metabolismo , Regulação da Expressão Gênica , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Modelos Biológicos , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Raios UltravioletaRESUMO
Poly(A)-specific ribonuclease (PARN) is a mammalian 3'-exoribonuclease that degrades poly(A) with high specificity. To reveal mechanisms by which poly(A) is recognized by the active site of PARN, we have performed a kinetic analysis using a large repertoire of trinucleotide substrates. Our analysis demonstrated that PARN harbors specificity for adenosine recognition in its active site and that the nucleotides surrounding the scissile bond are critical for adenosine recognition. We propose that two binding pockets, which interact with the nucleotides surrounding the scissile bond, play a pivotal role in providing specificity for the recognition of adenosine residues by the active site of PARN. In addition, we show that PARN, besides poly(A), also quite efficiently degrades poly(U), approximately 10-fold less efficiently than poly(A). The poly(U)-degrading property of PARN could be of biological significance as oligo(U) tails recently have been proposed to play a role in RNA stabilization and destabilization.
Assuntos
Adenosina/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Aminoácidos/metabolismo , Biopolímeros/metabolismo , Domínio Catalítico , Humanos , Cinética , Nucleotídeos/metabolismo , Poli A/metabolismo , Estabilidade de RNA , Especificidade por SubstratoRESUMO
Poly(A)-specific ribonuclease (PARN) is a homodimeric, processive, and cap-interacting 3' exoribonuclease that efficiently degrades eukaryotic mRNA poly(A) tails. The crystal structure of a C-terminally truncated PARN in complex with m(7)GpppG reveals that, in one subunit, m(7)GpppG binds to a cavity formed by the RRM domain and the nuclease domain, whereas in the other subunit, it binds almost exclusively to the RRM domain. Importantly, our structural and competition data show that the cap-binding site overlaps with the active site in the nuclease domain. Mutational analysis demonstrates that residues involved in m(7)G recognition are crucial for cap-stimulated deadenylation activity, and those involved in both cap and poly(A) binding are important for catalysis. A modeled PARN, which shows that the RRM domain from one subunit and the R3H domain from the other subunit enclose the active site, provides a structural foundation for further studies to elucidate the mechanism of PARN-mediated deadenylation.
Assuntos
Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/metabolismo , Exorribonucleases/química , Exorribonucleases/metabolismo , Animais , Sítios de Ligação , Camundongos , Modelos Moleculares , Conformação de Ácido Nucleico , Poli A/química , Poli A/metabolismo , Ligação Proteica , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Capuzes de RNA/química , Capuzes de RNA/metabolismoRESUMO
Genome data are increasingly important in the computational identification of novel regulatory non-coding RNAs (ncRNAs). However, most ncRNA gene-finders are either specialized to well-characterized ncRNA gene families or require comparisons of closely related genomes. We developed a method for de novo screening for ncRNA genes with a nucleotide composition that stands out against the background genome based on a partial sum process. We compared the performance when assuming independent and first-order Markov-dependent nucleotides, respectively, and used Karlin-Altschul and Karlin-Dembo statistics to evaluate the significance of hits. We hypothesized that a first-order Markov-dependent process might have better power to detect ncRNA genes since nearest-neighbor models have been shown to be successful in predicting RNA structures. A model based on a first-order partial sum process (analyzing overlapping dinucleotides) had better sensitivity and specificity than a zeroth-order model when applied to the AT-rich genome of the amoeba Dictyostelium discoideum. In this genome, we detected 94% of previously known ncRNA genes (at this sensitivity, the false positive rate was estimated to be 25% in a simulated background). The predictions were further refined by clustering candidate genes according to sequence similarity and/or searching for an ncRNA-associated upstream element. We experimentally verified six out of 10 tested ncRNA gene predictions. We conclude that higher-order models, in combination with other information, are useful for identification of novel ncRNA gene families in single-genome analysis of D. discoideum. Our generalizable approach extends the range of genomic data that can be searched for novel ncRNA genes using well-grounded statistical methods.
Assuntos
Dictyostelium/genética , Genômica/métodos , RNA não Traduzido/genética , Adenina/análise , Animais , Composição de Bases , Sequência de Bases , Sequência Conservada , Genes de Protozoários , Genoma de Protozoário , Cadeias de Markov , Dados de Sequência Molecular , Família Multigênica , Nucleotídeos/análise , RNA não Traduzido/química , RNA não Traduzido/metabolismo , Timina/análiseRESUMO
Poly(A)-specific ribonuclease (PARN) is an oligomeric, processive and cap-interacting 3' exoribonuclease that efficiently degrades mRNA poly(A) tails. Here we show that the RNA recognition motif (RRM) of PARN harbors both poly(A) and cap binding properties, suggesting that the RRM plays an important role for the two critical and unique properties that are tightly associated with PARN activity, i.e. recognition and dependence on both the cap structure and poly(A) tail during poly(A) hydrolysis. We show that PARN and its RRM have micromolar affinity to the cap structure by using fluorescence spectroscopy and nanomolar affinity for poly(A) by using filter binding assay. We have identified one tryptophan residue within the RRM that is essential for cap binding but not required for poly(A) binding, suggesting that the cap- and poly(A)-binding sites associated with the RRM are both structurally and functionally separate from each other. RRM is one of the most commonly occurring RNA-binding domains identified so far, suggesting that other RRMs may have both cap and RNA binding properties just as the RRM of PARN.
Assuntos
Exorribonucleases/química , Exorribonucleases/metabolismo , Poli A/química , Poli A/metabolismo , RNA/química , RNA/metabolismo , Adenina/metabolismo , Motivos de Aminoácidos , Animais , Sítios de Ligação , Sequência Conservada , Exorribonucleases/genética , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Alinhamento de SequênciaRESUMO
Aminoglycosides are potent inhibitors of bacterial growth and are used clinically as antibiotics. However, their usage has declined in recent years due to the emergence of resistance and severe toxic side effects. Here we show that human poly(A) polymerase gamma (PAPgamma) is inhibited by aminoglycosides. The inhibition was pH dependent and could be released by Mg(II) ions in a competitive manner suggesting that electrostatic interactions are important for inhibition and that the binding sites for aminoglycosides overlap with Mg(II) ion binding sites. Kinetic analysis revealed that aminoglycosides of the neomycin and kanamycin families behaved as mixed non-competitive inhibitors for the PAPgamma substrates oligoA15 and ATP. Interestingly, sisomicin and 5-epi-sisomycin showed a competitive mechanism of inhibition for the oligoA15 whereas they inhibited the ATP substrate mixed non-competitive. This implies that different aminoglycosides bind in different ways to a common binding pocket and suggests that the binding sites for related aminoglycosides are not overlapping even if they may share molecular determinants. Our study emphasizes the possibility that aminoglycoside toxicity could be due to interference with housekeeping enzymes involved in breaking and forming phosphodiester bonds.
Assuntos
Aminoglicosídeos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Trifosfato de Adenosina/metabolismo , Aminoglicosídeos/química , Antibacterianos/química , Antibacterianos/farmacologia , Catálise/efeitos dos fármacos , Relação Dose-Resposta a Droga , Framicetina/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Estrutura Molecular , Paromomicina/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
We have detected a surprising heterogeneity among human spliceosomal U1 small nuclear RNA (snRNA). Most interestingly, we have identified three U1 snRNA variants that lack complementarity to the canonical 5' splice site (5'SS) GU dinucleotide. Furthermore, we have observed heterogeneity among the identified variant U1 snRNA genes caused by single nucleotide polymorphism (SNP). The identified snRNAs were ubiquitously expressed in a variety of human tissues representing different stages of development and displayed features of functional spliceosomal snRNAs, i.e., trimethylated cap structures, association with Sm proteins and presence in nuclear RNA-protein complexes. The unanticipated heterogeneity among spliceosomal snRNAs could contribute to the complexity of vertebrates by expanding the coding capacity of their genomes.
Assuntos
Sítios de Splice de RNA/genética , Splicing de RNA , RNA Nuclear Pequeno/genética , Spliceossomos/metabolismo , Pareamento de Bases , Sequência de Bases , Sequência Conservada , DNA Complementar/genética , Evolução Molecular , Variação Genética , Genoma Humano , Células HeLa , Humanos , Íntrons , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Filogenia , RNA Nuclear Pequeno/química , RNA Nuclear Pequeno/metabolismo , Análise de Sequência de RNA , Homologia de Sequência do Ácido Nucleico , Spliceossomos/genéticaRESUMO
Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5' cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5' end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.
Assuntos
Dictyostelium/genética , Poliadenilação , RNA de Protozoário/genética , RNA de Protozoário/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Spliceossomos/química , Animais , Sequência de Bases , Biologia Computacional , Sequência Conservada , Citoplasma/química , Dictyostelium/química , Dictyostelium/crescimento & desenvolvimento , Dictyostelium/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Imunoprecipitação , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA de Protozoário/química , RNA Nuclear Pequeno/química , Homologia de Sequência do Ácido Nucleico , Spliceossomos/metabolismoRESUMO
PARN is a poly(A)-specific ribonuclease that degrades the poly(A) tail of mRNA. We have established conditions for expressing soluble recombinant human PARN. We investigated different Escherichia coli strains, expression vectors, media and growth conditions. We found that PARN expressed from pET33 in BL21(DE3) grown in TB and induced at OD595 approximately 1 with 1 mM IPTG yielded mg amounts of soluble PARN per litre culture. Further, a purification protocol was established to purify PARN. We use His-tag affinity chromatography, HiTrap Q HP ion exchange chromatography and 7-Me-GTP-Sepharose affinity chromatography. This purification procedure render a 90-95% pure PARN. Purified recombinant PARN has enzymatic activity and will be used for further mechanistic and structural studies.
Assuntos
Exorribonucleases/isolamento & purificação , Cromatografia de Afinidade/métodos , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Exorribonucleases/genética , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
Poly(A)-specific ribonuclease (PARN) is a cap-interacting and poly(A)-specific 3'-exoribonuclease. Here we have investigated how the cap binding complex (CBC) affects human PARN activity. We showed that CBC, via its 80-kDa subunit (CBP80), inhibited PARN, suggesting that CBC can regulate mRNA deadenylation. The CBC-mediated inhibition of PARN was cap-independent, and in keeping with this, the CBP80 subunit alone inhibited PARN. Our data suggested a new function for CBC, identified CBC as a potential regulator of PARN, and emphasized the importance of communication between the two extreme ends of the mRNA as a key strategy to regulate mRNA degradation. Based on our data, we have proposed a model for CBC-mediated regulation of PARN, which relies on an interaction between CBP80 and PARN. Association of CBC with PARN might have importance in the regulated recruitment of PARN to the nonsense-mediated decay pathway during the pioneer round of translation.
Assuntos
Exorribonucleases/antagonistas & inibidores , Complexo Proteico Nuclear de Ligação ao Cap/fisiologia , RNA Mensageiro/metabolismo , Exorribonucleases/fisiologia , Humanos , Poliadenilação , Subunidades Proteicas , Capuzes de RNA/fisiologiaRESUMO
Poly(A)-specific ribonuclease (PARN) is a highly poly(A)-specific 3'-exoribonuclease that efficiently degrades mRNA poly(A) tails. PARN belongs to the DEDD family of nucleases, and four conserved residues are essential for PARN activity, i.e. Asp-28, Glu-30, Asp-292, and Asp-382. Here we have investigated how catalytically important divalent metal ions are coordinated in the active site of PARN. Each of the conserved amino acid residues was substituted with cysteines, and it was found that all four mutants were inactive in the presence of Mg2+. However, in the presence of Mn2+, Zn2+, Co2+, or Cd2+, PARN activity was rescued from the PARN(D28C), PARN(D292C), and PARN(D382C) variants, suggesting that these three amino acids interact with catalytically essential metal ions. It was found that the shortest sufficient substrate for PARN activity was adenosine trinucleotide (A3) in the presence of Mg2+ or Cd2+. Interestingly, adenosine dinucleotide (A) was efficiently hydrolyzed in the presence of Mn2+, Zn2+, or Co2+, suggesting that the substrate length requirement for PARN can be modulated by the identity of the divalent metal ion. Finally, introduction of phosphorothioate modifications into the A substrate demonstrated that the scissile bond non-bridging phosphate oxygen in the pro-R position plays an important role during cleavage, most likely by coordinating a catalytically important divalent metal ion. Based on our data we discuss binding and coordination of divalent metal ions in the active site of PARN.