Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 15 de 15
Filtrar
1.
Nat Commun ; 15(1): 4683, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38824131

RESUMO

The human mitochondrial genome is transcribed into two RNAs, containing mRNAs, rRNAs and tRNAs, all dedicated to produce essential proteins of the respiratory chain. The precise excision of tRNAs by the mitochondrial endoribonucleases (mt-RNase), P and Z, releases all RNA species from the two RNA transcripts. The tRNAs then undergo 3'-CCA addition. In metazoan mitochondria, RNase P is a multi-enzyme assembly that comprises the endoribonuclease PRORP and a tRNA methyltransferase subcomplex. The requirement for this tRNA methyltransferase subcomplex for mt-RNase P cleavage activity, as well as the mechanisms of pre-tRNA 3'-cleavage and 3'-CCA addition, are still poorly understood. Here, we report cryo-EM structures that visualise four steps of mitochondrial tRNA maturation: 5' and 3' tRNA-end processing, methylation and 3'-CCA addition, and explain the defined sequential order of the tRNA processing steps. The methyltransferase subcomplex recognises the pre-tRNA in a distinct mode that can support tRNA-end processing and 3'-CCA addition, likely resulting from an evolutionary adaptation of mitochondrial tRNA maturation complexes to the structurally-fragile mitochondrial tRNAs. This subcomplex can also ensure a tRNA-folding quality-control checkpoint before the sequential docking of the maturation enzymes. Altogether, our study provides detailed molecular insight into RNA-transcript processing and tRNA maturation in human mitochondria.


Assuntos
Mitocôndrias , RNA de Transferência , Ribonuclease P , tRNA Metiltransferases , Humanos , RNA de Transferência/metabolismo , RNA de Transferência/genética , RNA de Transferência/química , Mitocôndrias/metabolismo , Ribonuclease P/metabolismo , Ribonuclease P/genética , Ribonuclease P/química , tRNA Metiltransferases/metabolismo , tRNA Metiltransferases/genética , tRNA Metiltransferases/química , Processamento Pós-Transcricional do RNA , Microscopia Crioeletrônica , RNA Mitocondrial/metabolismo , RNA Mitocondrial/genética , RNA Mitocondrial/química , Metilação , Conformação de Ácido Nucleico , Modelos Moleculares , Precursores de RNA/metabolismo , Precursores de RNA/genética
2.
Nucleic Acids Res ; 50(10): 5793-5806, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35580049

RESUMO

Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in RNA biology. To gain insights into poorly understood RNA nucleotide methylation processes, we developed a new method to synthesize RNA-conjugates for the study of RNA recognition and methyl-transfer mechanisms of SAM-dependent m6A RNA methyltransferases. These RNA conjugates contain a SAM cofactor analogue connected at the N6-atom of an adenosine within dinucleotides, a trinucleotide or a 13mer RNA. Our chemical route is chemo- and regio-selective and allows flexible modification of the RNA length and sequence. These compounds were used in crystallization assays with RlmJ, a bacterial m6A rRNA methyltransferase. Two crystal structures of RlmJ in complex with RNA-SAM conjugates were solved and revealed the RNA-specific recognition elements used by RlmJ to clamp the RNA substrate in its active site. From these structures, a model of a trinucleotide bound in the RlmJ active site could be built and validated by methyltransferase assays on RlmJ mutants. The methyl transfer by RlmJ could also be deduced. This study therefore shows that RNA-cofactor conjugates are potent molecular tools to explore the active site of RNA modification enzymes.


Assuntos
Metiltransferases , RNA , Adenosina , Domínio Catalítico , Metilação , Metiltransferases/metabolismo , RNA/metabolismo
3.
Am J Hum Genet ; 108(11): 2195-2204, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34715011

RESUMO

Human mitochondrial RNase P (mt-RNase P) is responsible for 5' end processing of mitochondrial precursor tRNAs, a vital step in mitochondrial RNA maturation, and is comprised of three protein subunits: TRMT10C, SDR5C1 (HSD10), and PRORP. Pathogenic variants in TRMT10C and SDR5C1 are associated with distinct recessive or x-linked infantile onset disorders, resulting from defects in mitochondrial RNA processing. We report four unrelated families with multisystem disease associated with bi-allelic variants in PRORP, the metallonuclease subunit of mt-RNase P. Affected individuals presented with variable phenotypes comprising sensorineural hearing loss, primary ovarian insufficiency, developmental delay, and brain white matter changes. Fibroblasts from affected individuals in two families demonstrated decreased steady state levels of PRORP, an accumulation of unprocessed mitochondrial transcripts, and decreased steady state levels of mitochondrial-encoded proteins, which were rescued by introduction of the wild-type PRORP cDNA. In mt-tRNA processing assays performed with recombinant mt-RNase P proteins, the disease-associated variants resulted in diminished mitochondrial tRNA processing. Identification of disease-causing variants in PRORP indicates that pathogenic variants in all three subunits of mt-RNase P can cause mitochondrial dysfunction, each with distinct pleiotropic clinical presentations.


Assuntos
Alelos , Pleiotropia Genética , Mitocôndrias/enzimologia , RNA Mitocondrial/genética , RNA de Transferência/genética , Ribonuclease P/genética , Adulto , Feminino , Humanos , Masculino , Linhagem
4.
J Virol ; 95(14): e0066321, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-33963053

RESUMO

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.


Assuntos
COVID-19 , Genoma Viral , Motivos de Nucleotídeos , Dobramento de RNA , RNA Viral , SARS-CoV-2/fisiologia , Replicação Viral , Animais , COVID-19/genética , COVID-19/metabolismo , Chlorocebus aethiops , Células HEK293 , Humanos , RNA Viral/genética , RNA Viral/metabolismo , Células Vero
5.
Nucleic Acids Res ; 49(13): 7239-7255, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34023900

RESUMO

Gene expression is regulated at many levels including co- or post-transcriptionally, where chemical modifications are added to RNA on riboses and bases. Expression control via RNA modifications has been termed 'epitranscriptomics' to keep with the related 'epigenomics' for DNA modification. One such RNA modification is the N6-methylation found on adenosine (m6A) and 2'-O-methyladenosine (m6Am) in most types of RNA. The N6-methylation can affect the fold, stability, degradation and cellular interaction(s) of the modified RNA, implicating it in processes such as splicing, translation, export and decay. The multiple roles played by this modification explains why m6A misregulation is connected to multiple human cancers. The m6A/m6Am writer enzymes are RNA methyltransferases (MTases). Structures are available for functionally characterized m6A RNA MTases from human (m6A mRNA, m6A snRNA, m6A rRNA and m6Am mRNA MTases), zebrafish (m6Am mRNA MTase) and bacteria (m6A rRNA MTase). For each of these MTases, we describe their overall domain organization, the active site architecture and the substrate binding. We identify areas that remain to be investigated, propose yet unexplored routes for structural characterization of MTase:substrate complexes, and highlight common structural elements that should be described for future m6A/m6Am RNA MTase structures.


Assuntos
Adenosina/análogos & derivados , Metiltransferases/química , Adenosina/metabolismo , Animais , Bactérias/enzimologia , Humanos , Metiltransferases/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/metabolismo
6.
APMIS ; 129(7): 393-400, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33730407

RESUMO

The SARS-CoV-2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS-CoV-2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two-step RT-qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one-step RT-qPCR against SARS-CoV-2 using NP and OP samples. We furthermore tested a SARS-CoV-2 dilution series to determine the detection threshold. The method enables downstream detection of SARS-CoV-2 by RT-qPCR with high sensitivity (~4 viral RNA copies per RT-qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR-ready viral RNA and circumvents the need for commercial RNA purification kits.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/isolamento & purificação , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Humanos , Kit de Reagentes para Diagnóstico
7.
RNA Biol ; 18(11): 1996-2006, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33541205

RESUMO

All species transcribe ribosomal RNA in an immature form that requires several enzymes for processing into mature rRNA. The number and types of enzymes utilized for these processes vary greatly between different species. In low G + C Gram-positive bacteria including Bacillus subtilis and Geobacillus stearothermophilus, the endoribonuclease (RNase) M5 performs the final step in 5S rRNA maturation, by removing the 3'- and 5'-extensions from precursor (pre) 5S rRNA. This cleavage activity requires initial complex formation between the pre-rRNA and a ribosomal protein, uL18, making the full M5 substrate a ribonucleoprotein particle (RNP). M5 contains a catalytic N-terminal Toprim domain and an RNA-binding C-terminal domain, respectively, shown to assist in processing and binding of the RNP. Here, we present structural data that show how two Mg2+ ions are accommodated in the active site pocket of the catalytic Toprim domain and investigate the importance of these ions for catalysis. We further perform solution studies that support the previously proposed 3'-before-5' order of removal of the pre-5S rRNA extensions and map the corresponding M5 structural rearrangements during catalysis.


Assuntos
Bacillus subtilis/enzimologia , Endorribonucleases/química , Endorribonucleases/metabolismo , Geobacillus stearothermophilus/enzimologia , Magnésio/metabolismo , Precursores de RNA/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Ribossômico 5S/metabolismo , Sequência de Aminoácidos , Endorribonucleases/genética , Conformação de Ácido Nucleico , Precursores de RNA/genética , RNA de Cadeia Dupla/genética , RNA Ribossômico 5S/genética , Ribossomos/genética , Ribossomos/metabolismo , Especificidade por Substrato
8.
Mol Cell ; 80(2): 227-236.e5, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32991829

RESUMO

The pathways for ribosomal RNA (rRNA) maturation diverge greatly among the domains of life. In the Gram-positive model bacterium, Bacillus subtilis, the final maturation steps of the two large ribosomal subunit (50S) rRNAs, 23S and 5S pre-rRNAs, are catalyzed by the double-strand specific ribonucleases (RNases) Mini-RNase III and RNase M5, respectively. Here we present a protocol that allowed us to solve the 3.0 and 3.1 Å resolution cryoelectron microscopy structures of these RNases poised to cleave their pre-rRNA substrates within the B. subtilis 50S particle. These data provide the first structural insights into rRNA maturation in bacteria by revealing how these RNases recognize and process double-stranded pre-rRNA. Our structures further uncover how specific ribosomal proteins act as chaperones to correctly fold the pre-rRNA substrates and, for Mini-III, anchor the RNase to the ribosome. These r-proteins thereby serve a quality-control function in the process from accurate ribosome assembly to rRNA processing.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Precursores de RNA/metabolismo , Ribonucleases/química , Subunidades Ribossômicas Maiores de Bactérias/metabolismo , Bacillus subtilis/ultraestrutura , Proteínas de Bactérias/ultraestrutura , Sequência de Bases , Microscopia Crioeletrônica , Modelos Moleculares , Precursores de RNA/ultraestrutura , Ribonucleases/ultraestrutura , Subunidades Ribossômicas Maiores de Bactérias/ultraestrutura , Especificidade por Substrato
9.
RNA Biol ; 16(6): 798-808, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30879411

RESUMO

RNA methyltransferases (MTases) catalyse the transfer of a methyl group to their RNA substrates using most-often S-adenosyl-L-methionine (SAM) as cofactor. Only few RNA-bound MTases structures are currently available due to the difficulties in crystallising RNA:protein complexes. The lack of complex structures results in poorly understood RNA recognition patterns and methylation reaction mechanisms. On the contrary, many cofactor-bound MTase structures are available, resulting in well-understood protein:cofactor recognition, that can guide the design of bisubstrate analogues that mimic the state at which both the substrate and the cofactor is bound. Such bisubstrate analogues were recently synthesized for proteins monomethylating the N6-atom of adenine (m6A). These proteins include, amongst others, RlmJ in E. coli and METLL3:METT14 and METTL16 in human. As a proof-of-concept, we here test the ability of the bisubstrate analogues to mimic the substrate:cofactor bound state during catalysis by studying their binding to RlmJ using differential scanning fluorimetry, isothermal titration calorimetry and X-ray crystallography. We find that the methylated adenine base binds in the correct pocket, and thus these analogues could potentially be used broadly to study the RNA recognition and catalytic mechanism of m6A MTases. Two bisubstrate analogues bind RlmJ with micro-molar affinity, and could serve as starting scaffolds for inhibitor design against m6A RNA MTases. The same analogues cause changes in the melting temperature of the m1A RNA MTase, TrmK, indicating non-selective protein:compound complex formation. Thus, optimization of these molecular scaffolds for m6A RNA MTase inhibition should aim to increase selectivity, as well as affinity.


Assuntos
Adenina/análogos & derivados , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/química , Metiltransferases/química , Adenina/metabolismo , Domínio Catalítico , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Temperatura
10.
J Biol Chem ; 293(33): 12862-12876, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29880640

RESUMO

Mitochondrial tRNAs are transcribed as long polycistronic transcripts of precursor tRNAs and undergo posttranscriptional modifications such as endonucleolytic processing and methylation required for their correct structure and function. Among them, 5'-end processing and purine 9 N1-methylation of mitochondrial tRNA are catalyzed by two proteinaceous complexes with overlapping subunit composition. The Mg2+-dependent RNase P complex for 5'-end cleavage comprises the methyltransferase domain-containing protein tRNA methyltransferase 10C, mitochondrial RNase P subunit (TRMT10C/MRPP1), short-chain oxidoreductase hydroxysteroid 17ß-dehydrogenase 10 (HSD17B10/MRPP2), and metallonuclease KIAA0391/MRPP3. An MRPP1-MRPP2 subcomplex also catalyzes the formation of 1-methyladenosine/1-methylguanosine at position 9 using S-adenosyl-l-methionine as methyl donor. However, a lack of structural information has precluded insights into how these complexes methylate and process mitochondrial tRNA. Here, we used a combination of X-ray crystallography, interaction and activity assays, and small angle X-ray scattering (SAXS) to gain structural insight into the two tRNA modification complexes and their components. The MRPP1 N terminus is involved in tRNA binding and monomer-monomer self-interaction, whereas the C-terminal SPOUT fold contains key residues for S-adenosyl-l-methionine binding and N1-methylation. The entirety of MRPP1 interacts with MRPP2 to form the N1-methylation complex, whereas the MRPP1-MRPP2-MRPP3 RNase P complex only assembles in the presence of precursor tRNA. This study proposes low-resolution models of the MRPP1-MRPP2 and MRPP1-MRPP2-MRPP3 complexes that suggest the overall architecture, stoichiometry, and orientation of subunits and tRNA substrates.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/química , Metiltransferases/química , Modelos Moleculares , Complexos Multienzimáticos/química , RNA Mitocondrial/química , RNA de Transferência/química , Ribonuclease P/química , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Cristalografia por Raios X , Humanos , Metiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , RNA Mitocondrial/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Espalhamento a Baixo Ângulo
11.
Biochim Biophys Acta Mol Basis Dis ; 1863(12): 3294-3302, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28888424

RESUMO

MRPP2 (also known as HSD10/SDR5C1) is a multifunctional protein that harbours both catalytic and non-catalytic functions. The protein belongs to the short-chain dehydrogenase/reductases (SDR) family and is involved in the catabolism of isoleucine in vivo and steroid metabolism in vitro. MRPP2 also moonlights in a complex with the MRPP1 (also known as TRMT10C) protein for N1-methylation of purines at position 9 of mitochondrial tRNA, and in a complex with MRPP1 and MRPP3 (also known as PRORP) proteins for 5'-end processing of mitochondrial precursor tRNA. Inherited mutations in the HSD17B10 gene encoding MRPP2 protein lead to a childhood disorder characterised by progressive neurodegeneration, cardiomyopathy or both. Here we report two patients with novel missense mutations in the HSD17B10 gene (c.34G>C and c.526G>A), resulting in the p.V12L and p.V176M substitutions. Val12 and Val176 are highly conserved residues located at different regions of the MRPP2 structure. Recombinant mutant proteins were expressed and characterised biochemically to investigate their effects towards the functions of MRPP2 and associated complexes in vitro. Both mutant proteins showed significant reduction in the dehydrogenase, methyltransferase and tRNA processing activities compared to wildtype, associated with reduced stability for protein with p.V12L, whereas the protein carrying p.V176M showed impaired kinetics and complex formation. This study therefore identified two distinctive molecular mechanisms to explain the biochemical defects for the novel missense patient mutations.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Mitocôndrias/metabolismo , RNA de Transferência/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/química , Feminino , Expressão Gênica , Humanos , Lactente , Masculino , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease P/genética , Ribonuclease P/metabolismo
12.
Biomolecules ; 7(1)2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28230814

RESUMO

To date, about 90 post-transcriptional modifications have been reported in tRNA expanding their chemical and functional diversity. Methylation is the most frequent post-transcriptional tRNA modification that can occur on almost all nitrogen sites of the nucleobases, on the C5 atom of pyrimidines, on the C2 and C8 atoms of adenosine and, additionally, on the oxygen of the ribose 2'-OH. The methylation on the N1 atom of adenosine to form 1-methyladenosine (m1A) has been identified at nucleotide position 9, 14, 22, 57, and 58 in different tRNAs. In some cases, these modifications have been shown to increase tRNA structural stability and induce correct tRNA folding. This review provides an overview of the currently known m1A modifications, the different m1A modification sites, the biological role of each modification, and the enzyme responsible for each methylation in different species. The review further describes, in detail, two enzyme families responsible for formation of m1A at nucleotide position 9 and 58 in tRNA with a focus on the tRNA binding, m1A mechanism, protein domain organisation and overall structures.


Assuntos
Adenosina/análogos & derivados , Processamento Pós-Transcricional do RNA , RNA de Transferência/metabolismo , Adenosina/metabolismo , Animais , Enzimas/metabolismo , Humanos , Metilação , Modelos Moleculares , RNA de Transferência/química , RNA de Transferência/genética
13.
Chem Biol Interact ; 234: 114-25, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25526675

RESUMO

Short-chain dehydrogenases/reductases (SDRs) constitute a large, functionally diverse branch of enzymes within the class of NAD(P)(H) dependent oxidoreductases. In humans, over 80 genes have been identified with distinct metabolic roles in carbohydrate, amino acid, lipid, retinoid and steroid hormone metabolism, frequently associated with inherited genetic defects. Besides metabolic functions, a subset of atypical SDR proteins appears to play critical roles in adapting to redox status or RNA processing, and thereby controlling metabolic pathways. Here we present an update on the human SDR superfamily and a ligand identification strategy using differential scanning fluorimetry (DSF) with a focused library of oxidoreductase and metabolic ligands to identify substrate classes and inhibitor chemotypes. This method is applicable to investigate structure-activity relationships of oxidoreductases and ultimately to better understand their physiological roles.


Assuntos
Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Humanos , Ligantes , Oxirredutases/genética , Oxirredutases/metabolismo , Filogenia , Relação Estrutura-Atividade , Especificidade por Substrato/genética
14.
Hum Mol Genet ; 23(13): 3618-28, 2014 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-24549042

RESUMO

17ß-Hydroxysteroid dehydrogenase type 10 (HSD10) is multifunctional protein coded by the X-chromosomal HSD17B10 gene. Mutations in this gene cause HSD10 disease characterized by progressive neurological abnormalities and cardiomyopathy. Disease progression and severity of symptoms is unrelated to the protein's dehydrogenase activity. Recently, it was shown that HSD10 is an essential component of mitochondrial Ribonuclease P (RNase P), an enzyme required for mitochondrial tRNA processing, but little is known about the role of HSD10 in RNase P function. RNase P consists of three different proteins MRPP1, MRPP2 (HSD10) and MRPP3, each of which is essential for RNase P function. Here, we show that HSD10 protein levels are significantly reduced in fibroblasts from patients carrying the HSD17B10 mutation p.R130C. A reduction in HSD10 levels was accompanied by a reduction in MRPP1 protein but not MRPP3 protein. In HSD10 knock-down cells, MRPP1 protein content was also reduced, indicating that HSD10 is important for the maintenance of normal MRPP1 protein levels. Ectopic expression of HSD10 partially restored RNA processing in HSD10 knock-down cells and fibroblasts, and also expression of MRPP1 protein was restored to values comparable to controls. In both, patient fibroblasts and HSD10 knock-down cells, there was evidence of impaired processing of precursor tRNA transcripts of the mitochondrial heavy strand but not the light strand compared with controls. Our findings indicate that HSD10 is important for the maintenance of the MRPP1-HSD10 subcomplex of RNase P and that loss of HSD10 causes impaired mitochondrial precursor transcript processing which may explain mitochondrial dysfunction observed in HSD10 disease.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/metabolismo , Metiltransferases/metabolismo , 3-Hidroxiacil-CoA Desidrogenases/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Metiltransferases/genética , Mutação , RNA de Transferência/genética , Ribonuclease P/genética , Ribonuclease P/metabolismo
15.
Dalton Trans ; 42(1): 292-8, 2013 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-23168834

RESUMO

Highly stereoselective and rapid (<1 min) addition reactions to the imine double bond of 2-(methylimino)acetate complexes [L(4)Co(O(2)CCH=NCH(3))](2+) [L(4) = (en)(2) (7), (tren) (11)] were achieved in aqueous solution with nitromethane, ethyl 3-oxobutanoate or diethyl malonate. The molecular structures of two product complexes, rac-(Δ*-R(C)*-S(N)*)-[Co(en)(2)(O(2)CCH[CH(2)NO(2)]NHCH(3))]ZnCl(4) and rac-(Δ*-R(C)*-S(N)*)-[Co(en)(2)(O(2)CCH[CH(2)COCH(3)]NHCH(3))]ZnCl(4), were established by X-ray diffraction.


Assuntos
Cobalto/química , Complexos de Coordenação/química , Bases de Mannich/química , Água/química , Acetoacetatos/química , Complexos de Coordenação/síntese química , Cristalografia por Raios X , Malonatos/química , Metano/análogos & derivados , Metano/química , Conformação Molecular , Nitroparafinas/química , Estereoisomerismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA