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1.
Sci Rep ; 7: 44854, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28327545

RESUMO

Quantitative Polymerase Chain Reaction (qPCR) is one of central techniques in molecular biology and important tool in medical diagnostics. While being a golden standard qPCR techniques depend on reference measurements and are susceptible to large errors caused by even small changes of reaction efficiency or conditions that are typically not marked by decreased precision. Digital PCR (dPCR) technologies should alleviate the need for calibration by providing absolute quantitation using binary (yes/no) signals from partitions provided that the basic assumption of amplification a single target molecule into a positive signal is met. Still, the access to digital techniques is limited because they require new instruments. We show an analog-digital method that can be executed on standard (real-time) qPCR devices. It benefits from real-time readout, providing calibration-free assessment. The method combines advantages of qPCR and dPCR and bypasses their drawbacks. The protocols provide for small simplified partitioning that can be fitted within standard well plate format. We demonstrate that with the use of synergistic assay design standard qPCR devices are capable of absolute quantitation when normal qPCR protocols fail to provide accurate estimates. We list practical recipes how to design assays for required parameters, and how to analyze signals to estimate concentration.


Assuntos
Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Calibragem , Humanos , Método de Monte Carlo , Reprodutibilidade dos Testes
2.
Anal Chem ; 87(16): 8203-9, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26189596

RESUMO

Optimum algorithm for digital assays treats chemical compartments as bits of probabilistic information and arranges these bits in a fractional positional system. Maximization of information gain reduces, by orders of magnitude, the number of partitions required to achieve the requested dynamic range and precision of the assay. The method simplifies the execution of digital analytical methods providing for more accessible use of absolute quantization in research and in diagnostics.

3.
Mol Cell Biol ; 35(7): 1169-81, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25605335

RESUMO

Little is known about the RNA polymerase III (Pol III) complex assembly and its transport to the nucleus. We demonstrate that a missense cold-sensitive mutation, rpc128-1007, in the sequence encoding the C-terminal part of the second largest Pol III subunit, C128, affects the assembly and stability of the enzyme. The cellular levels and nuclear concentration of selected Pol III subunits were decreased in rpc128-1007 cells, and the association between Pol III subunits as evaluated by coimmunoprecipitation was also reduced. To identify the proteins involved in Pol III assembly, we performed a genetic screen for suppressors of the rpc128-1007 mutation and selected the Rbs1 gene, whose overexpression enhanced de novo tRNA transcription in rpc128-1007 cells, which correlated with increased stability, nuclear concentration, and interaction of Pol III subunits. The rpc128-1007 rbs1Δ double mutant shows a synthetic growth defect, indicating that rpc128-1007 and rbs1Δ function in parallel ways to negatively regulate Pol III assembly. Rbs1 physically interacts with a subset of Pol III subunits, AC19, AC40, and ABC27/Rpb5. Additionally, Rbs1 interacts with the Crm1 exportin and shuttles between the cytoplasm and nucleus. We postulate that Rbs1 binds to the Pol III complex or subcomplex and facilitates its translocation to the nucleus.


Assuntos
RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Mapas de Interação de Proteínas , Subunidades Proteicas/análise , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , RNA Polimerase III/análise , RNA Polimerase III/genética , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/análise , Regulação para Cima
4.
Bioarchitecture ; 2(4): 134-7, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22964977

RESUMO

The THO complex is a nuclear structure whose architecture is conserved among all kingdoms and plays an important role in mRNP biogenesis connecting transcription elongation with mRNA maturation and export. Recent data indicates that the THO complex is necessary for the proper expression of some genes, assurance of genetic stability by preventing transcription-associated recombination. Yeast THO has been described as a heterotetramer (Tho2, Hpr1, Mft1 and Thp2) that performs several functions through the interaction with other proteins like Tex1 or the mRNA export factors Sub2 and Yra1, with which it forms the TRanscription and EXport complex (TREX). In this article we review the cellular role of THO, which we show to be composed of five subunits with Tex1 being also an integral part of the complex. We also show a low-resolution structure of THO and localize some of its components. We discuss the consequences of THO interaction with nucleic acids through the unfolded C-terminal region of Tho2, highlighting the importance of unfolded regions in eukaryotic proteins. Finally, we comment on THO recruitment to active chromatin, a role that is linked to mRNA biogenesis.


Assuntos
Ácidos Nucleicos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
5.
EMBO J ; 31(6): 1605-16, 2012 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-22314234

RESUMO

The THO complex is a key factor in co-transcriptional formation of export-competent messenger ribonucleoprotein particles, yet its structure and mechanism of chromatin recruitment remain unknown. In yeast, this complex has been described as a heterotetramer (Tho2, Hpr1, Mft1, and Thp2) that interacts with Tex1 and mRNA export factors Sub2 and Yra1 to form the TRanscription EXport (TREX) complex. In this study, we purified yeast THO and found Tex1 to be part of its core. We determined the three-dimensional structures of five-subunit THO complex by electron microscopy and located the positions of Tex1, Hpr1, and Tho2 C-terminus using various labelling techniques. In the case of Tex1, a ß-propeller protein, we have generated an atomic model which docks into the corresponding part of the THO complex envelope. Furthermore, we show that THO directly interacts with nucleic acids through the unfolded C-terminal region of Tho2, whose removal reduces THO recruitment to active chromatin leading to mRNA biogenesis defects. In summary, this study describes the THO architecture, the structural basis for its chromatin targeting, and highlights the importance of unfolded regions of eukaryotic proteins.


Assuntos
Ácidos Nucleicos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Gênica/genética
6.
Structure ; 18(9): 1075-82, 2010 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-20826334

RESUMO

For high-throughput structural studies of protein complexes of composition inferred from proteomics data, it is crucial that candidate complexes are selected accurately. Herein, we exemplify a procedure that combines a bioinformatics tool for complex selection with in vivo validation, to deliver structural results in a medium-throughout manner. We have selected a set of 20 yeast complexes, which were predicted to be feasible by either an automated bioinformatics algorithm, by manual inspection of primary data, or by literature searches. These complexes were validated with two straightforward and efficient biochemical assays, and heterologous expression technologies of complex components were then used to produce the complexes to assess their feasibility experimentally. Approximately one-half of the selected complexes were useful for structural studies, and we detail one particular success story. Our results underscore the importance of accurate target selection and validation in avoiding transient, unstable, or simply nonexistent complexes from the outset.


Assuntos
Biologia Computacional/métodos , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Bases de Dados de Proteínas , Proteômica , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Nucleic Acids Res ; 38(1): 279-98, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19864255

RESUMO

The mechanism of human mitochondrial RNA turnover and surveillance is still a matter of debate. We have obtained a cellular model for studying the role of hSuv3p helicase in human mitochondria. Expression of a dominant-negative mutant of the hSUV3 gene which encodes a protein with no ATPase or helicase activity results in perturbations of mtRNA metabolism and enables to study the processing and degradation intermediates which otherwise are difficult to detect because of their short half-lives. The hSuv3p activity was found to be necessary in the regulation of stability of mature, properly formed mRNAs and for removal of the noncoding processing intermediates transcribed from both H and L-strands, including mirror RNAs which represent antisense RNAs transcribed from the opposite DNA strand. Lack of hSuv3p function also resulted in accumulation of aberrant RNA species, molecules with extended poly(A) tails and degradation intermediates truncated predominantly at their 3'-ends. Moreover, we present data indicating that hSuv3p co-purifies with PNPase; this may suggest participation of both proteins in mtRNA metabolism.


Assuntos
RNA Helicases DEAD-box/fisiologia , Processamento Pós-Transcricional do RNA , RNA/metabolismo , Sequência de Bases , Processos de Crescimento Celular , Linhagem Celular , Forma Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/isolamento & purificação , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Poliadenilação , Polirribonucleotídeo Nucleotidiltransferase/isolamento & purificação , RNA/química , Estabilidade de RNA , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismo , RNA Mitocondrial , RNA de Transferência/metabolismo , RNA não Traduzido/metabolismo
8.
Acta Biochim Pol ; 53(1): 179-88, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16505900

RESUMO

Human mitochondrial polynucleotide phosphorylase (hPNPase) is an exoribonuclease localized in mitochondria. The exact physiological function of this enzyme is unknown. Recent studies have revealed the existence of a relationship between induction of hPNPase mRNA and both cellular senescence and growth arrest of melanoma cells following beta-interferon treatment. The aim of this study was to verify whether the augmented hPNPase mRNA level results in increase of the protein level. In several cell lines established from five metastatic melanoma patients we did not find any such correlation. However, an elevated level of hPNPase protein was observed in interferon-induced HeLa and Jurkat cells. This increase was correlated with a slight shortening of poly(A) tails of mitochondrial ND3 transcript.


Assuntos
Regulação Neoplásica da Expressão Gênica , Interferon beta/metabolismo , Melanoma/metabolismo , Mitocôndrias/enzimologia , Polirribonucleotídeo Nucleotidiltransferase/biossíntese , RNA Mensageiro/metabolismo , Regulação para Cima , Sequência de Bases , Linhagem Celular Tumoral , Exorribonucleases/metabolismo , Células HeLa , Humanos , Células Jurkat , Dados de Sequência Molecular
9.
Acta Biochim Pol ; 53(1): 157-68, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16389406

RESUMO

The physiological significance and metabolism of oligoadenylated and polyadenylated human mitochondrial mRNAs are not known to date. After study of eight mitochondrial transcripts (ND1, ND2, ND3, ND5, CO1, CO2, ATP6/8 and Cyt. b) we found a direct correlation between the half-lives of mitochondrial mRNAs and their steady-state levels. Investigation of the mt-mRNA decay after thiamphenicol treatment indicated that three transcripts (ND2, ND3 and Cyt. b) are significantly stabilized after inhibition of mitochondrial translation. Careful analysis one of them, ND3, showed that inaccurate processing of the H-strand RNA precursor may occasionally occur between the ND3 and tRNA(Arg) locus leading to synthesis of ND3 mRNAs lacking the STOP codon. However, analysis of the oligo(A) fraction observed in case of the ND3 indicates that partially polyadenylated mRNAs are linked rather to the transcription process than to the translation-dependent deadenylation. Analysis of ND3 mRNA turnover in cells with siRNA-mediated knock-down of the mitochondrial poly(A) polymerase shows that strongly decreased polyadenylation does not markedly affect the decay of this transcript. We present a model where oligoadenylated mitochondrial transcripts are precursors of molecules containing full length poly(A) tails.


Assuntos
RNA Mensageiro/química , RNA/química , Northern Blotting , Códon de Terminação , Células HeLa , Humanos , Mitocôndrias/metabolismo , Desnaturação de Ácido Nucleico , Polinucleotídeo Adenililtransferase/química , Biossíntese de Proteínas , Interferência de RNA , RNA Mitocondrial , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tianfenicol/farmacologia , Transcrição Gênica
10.
Nucleic Acids Res ; 32(20): 6001-14, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15547249

RESUMO

We report here on the identification of a novel human nuclear-encoded mitochondrial poly(A) polymerase. Immunocytochemical experiments confirm that the enzyme indeed localizes to mitochondrial compartment. Inhibition of expression of the enzyme by RNA interference results in significant shortening of the poly(A) tails of the mitochondrial ND3, COX III and ATP 6/8 transcripts, suggesting that the investigated protein represents a bona fide mitochondrial poly(A) polymerase. This is in agreement with our sequencing data which show that poly(A) tails of several mitochondrial messengers are composed almost exclusively of adenosine residues. Moreover, the data presented here indicate that all analyzed mitochondrial transcripts with profoundly shortened poly(A) tails are relatively stable, which in turn argues against the direct role of long poly(A) extensions in the stabilization of human mitochondrial messengers.


Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/fisiologia , Mitocôndrias/enzimologia , Processamento de Terminações 3' de RNA , RNA/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Núcleo Celular/genética , Chlorocebus aethiops , Clonagem Molecular , DNA Polimerase Dirigida por DNA/análise , Células HeLa , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , RNA/química , Interferência de RNA , Estabilidade de RNA , RNA Mitocondrial , Alinhamento de Sequência , Análise de Sequência de RNA
11.
Toxicol Mech Methods ; 14(1-2): 53-7, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-20021123

RESUMO

RNA turnover in yeast mitochondria is controlled by the complex called degradosome, which consists of two nuclear-encoded proteins: the SUV3 gene codes for an RNA helicase and the DSS1 gene codes for an RNase. In contrast to yeast, much less is known about RNA degradation in human mitochondria. We suggest that the key enzyme involved in this process is nuclear-encoded polynucleotide phosphorylase, hPNPase.

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