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1.
Biol Chem ; 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33021958

RESUMO

N6-methyladenosine (m6A) is the most abundant modification in mRNA. The core of the human N6-methyltransferase complex (MTC) is formed by a heterodimer consisting of METTL3 and METTL14, which specifically catalyzes m6A formation within an RRACH sequence context. Using recombinant proteins in a site-specific methylation assay that allows determination of quantitative methylation yields, our results show that this complex methylates its target RNAs not only sequence but also secondary structure dependent. Furthermore, we demonstrate the role of specific protein domains on both RNA binding and substrate turnover, focusing on postulated RNA binding elements. Our results show that one zinc finger motif within the complex is sufficient to bind RNA, however, both zinc fingers are required for methylation activity. We show that the N-terminal domain of METTL3 alters the secondary structure dependence of methylation yields. Our results demonstrate that a cooperative effect of all RNA-binding elements in the METTL3-METTL14 complex is required for efficient catalysis, and that binding of further proteins affecting the NTD of METTL3 may regulate substrate specificity.

2.
Molecules ; 25(9)2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32354083

RESUMO

RNA-protein complexes (RNPs) are essential components in a variety of cellular processes, and oftentimes exhibit complex structures and show mechanisms that are highly dynamic in conformation and structure. However, biochemical and structural biology approaches are mostly not able to fully elucidate the structurally and especially conformationally dynamic and heterogeneous nature of these RNPs, to which end single molecule Förster resonance energy transfer (smFRET) spectroscopy can be harnessed to fill this gap. Here we summarize the advantages of strategic smFRET studies to investigate RNP dynamics, complemented by structural and biochemical data. Focusing on recent smFRET studies of three essential biological systems, we demonstrate that investigation of RNPs on a single molecule level can answer important functional questions that remained elusive with structural or biochemical approaches alone: The complex structural rearrangements throughout the splicing cycle, unwinding dynamics of the G-quadruplex (G4) helicase RHAU, and aspects in telomere maintenance regulation and synthesis.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Quadruplex G , RNA/química , Imagem Individual de Molécula , Animais , Bovinos , Análise por Conglomerados , Cristalografia por Raios X , Humanos , Cadeias de Markov , Conformação de Ácido Nucleico , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Splicing de RNA , Ribonucleoproteínas , Spliceossomos/química , Telomerase/química , Telômero/química , Telômero/ultraestrutura
3.
Biol Chem ; 402(1): 89-98, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33544495

RESUMO

N 6-methyladenosine (m6A) is the most abundant modification in mRNA. The core of the human N 6-methyltransferase complex (MTC) is formed by a heterodimer consisting of METTL3 and METTL14, which specifically catalyzes m6A formation within an RRACH sequence context. Using recombinant proteins in a site-specific methylation assay that allows determination of quantitative methylation yields, our results show that this complex methylates its target RNAs not only sequence but also secondary structure dependent. Furthermore, we demonstrate the role of specific protein domains on both RNA binding and substrate turnover, focusing on postulated RNA binding elements. Our results show that one zinc finger motif within the complex is sufficient to bind RNA, however, both zinc fingers are required for methylation activity. We show that the N-terminal domain of METTL3 alters the secondary structure dependence of methylation yields. Our results demonstrate that a cooperative effect of all RNA-binding elements in the METTL3-METTL14 complex is required for efficient catalysis, and that binding of further proteins affecting the NTD of METTL3 may regulate substrate specificity.


Assuntos
Metiltransferases/metabolismo , RNA Mensageiro/metabolismo , RNA/metabolismo , Humanos , Metilação , Metiltransferases/química , Metiltransferases/genética , Conformação de Ácido Nucleico , RNA/química , RNA Mensageiro/química , RNA Mensageiro/genética
5.
Nucleic Acids Res ; 46(20): 11099-11114, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30285154

RESUMO

A(syn)-U/T and G(syn)-C+ Hoogsteen (HG) base pairs (bps) are energetically more disfavored relative to Watson-Crick (WC) bps in A-RNA as compared to B-DNA by >1 kcal/mol for reasons that are not fully understood. Here, we used NMR spectroscopy, optical melting experiments, molecular dynamics simulations and modified nucleotides to identify factors that contribute to this destabilization of HG bps in A-RNA. Removing the 2'-hydroxyl at single purine nucleotides in A-RNA duplexes did not stabilize HG bps relative to WC. In contrast, loosening the A-form geometry using a bulge in A-RNA reduced the energy cost of forming HG bps at the flanking sites to B-DNA levels. A structural and thermodynamic analysis of purine-purine HG mismatches reveals that compared to B-DNA, the A-form geometry disfavors syn purines by 1.5-4 kcal/mol due to sugar-backbone rearrangements needed to sterically accommodate the syn base. Based on MD simulations, an additional penalty of 3-4 kcal/mol applies for purine-pyrimidine HG bps due to the higher energetic cost associated with moving the bases to form hydrogen bonds in A-RNA versus B-DNA. These results provide insights into a fundamental difference between A-RNA and B-DNA duplexes with important implications for how they respond to damage and post-transcriptional modifications.


Assuntos
Pareamento de Bases/fisiologia , DNA de Forma B/química , Conformação de Ácido Nucleico , Purinas/química , RNA/química , DNA/química , Metabolismo Energético , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Pirimidinas/química , Termodinâmica
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