Structural analysis of human 2′-O-ribose methyltransferases involved in mRNA cap structure formation

Śmietański, Mirosław; Werner, Maria Fernanda de Paula; Purta, Elżbieta; Kamińska, Katarzyna; Stępiński, Janusz; Darżynkiewicz, Edward; Nowotny, Marcin; Bujnicki, Janusz M.

doi:10.1038/ncomms4004

Cited by 92 publications

(118 citation statements)

References 43 publications

(61 reference statements)

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“…This is especially reflected in the low number of structures of proteins in complex with capped RNA (>1 nt) that have been determined to date. Currently, these structures include the human 2 ′ -O-ribose methyltransferase CMTr1 with a capped 4-mer that was produced by chemical coupling (Smietanski et al 2014), the 2 ′ -O-ribose methyltransferase of vaccinia virus with a capped 6-mer that was produced by in vitro transcription in the presence of a cap analog (Hodel et al 1998), the 2 ′ -O-ribose methyltransferase in the NS5 protein from dengue virus with an 8-mer cap-0 viral RNA that was produced using a cap analog (Zhao et al 2015), the dengue virus methyltransferase in complex with a 5 ′ -capped RNA 8-mer that was chemically synthesized (Yap et al 2010), and the innate immune receptor RIG-I that contains a chemically synthesized 24-nt-long capped hairpin RNA (Devarkar et al 2016). The high-resolution structural data available is thus confined to a small subset of the numerous enzymes and proteins that directly interact with the mRNA cap structure.…”

Section: Thementioning

confidence: 99%

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

Fuchs

Neu

Sprangers

2016

RNA

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The eukaryotic mRNA 5 ′ cap structure is indispensible for pre-mRNA processing, mRNA export, translation initiation, and mRNA stability. Despite this importance, structural and biophysical studies that involve capped RNA are challenging and rare due to the lack of a general method to prepare mRNA in sufficient quantities. Here, we show that the vaccinia capping enzyme can be used to produce capped RNA in the amounts that are required for large-scale structural studies. We have therefore designed an efficient expression and purification protocol for the vaccinia capping enzyme. Using this approach, the reaction scale can be increased in a cost-efficient manner, where the yields of the capped RNA solely depend on the amount of available uncapped RNA target. Using a large number of RNA substrates, we show that the efficiency of the capping reaction is largely independent of the sequence, length, and secondary structure of the RNA, which makes our approach generally applicable. We demonstrate that the capped RNA can be directly used for quantitative biophysical studies, including fluorescence anisotropy and highresolution NMR spectroscopy. In combination with 13 C-methyl-labeled S-adenosyl methionine, the methyl groups in the RNA can be labeled for methyl TROSY NMR spectroscopy. Finally, we show that our approach can produce both cap-0 and cap-1 RNA in high amounts. In summary, we here introduce a general and straightforward method that opens new means for structural and functional studies of proteins and enzymes in complex with capped RNA.

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Section: Thementioning

confidence: 99%

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

Fuchs

Neu

Sprangers

2016

RNA

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“…S3) reveals that 3 residues, K1816, D1927, and K1962, are aligned to the conserved catalytic residues in the templates. 92 In addition, the "GEGAGA" motif at positions 1836-1841 of Ebolavirus protein L is aligned to the conserved S-adenosyl-L-methionine-binding motif in the templates. This motif is also conserved in sequences from Filoviridae, suggesting a similar function in co-factor binding.…”

Section: The Zinc-finger Domain Of Vp30mentioning

confidence: 99%

Predictive and comparative analysis of Ebolavirus proteins

2015

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Ebolavirus is the pathogen for Ebola Hemorrhagic Fever (EHF). This disease exhibits a high fatality rate and has recently reached a historically epidemic proportion in West Africa. Out of the 5 known Ebolavirus species, only Reston ebolavirus has lost human pathogenicity, while retaining the ability to cause EHF in long-tailed macaque. Significant efforts have been spent to determine the three-dimensional (3D) structures of Ebolavirus proteins, to study their interaction with host proteins, and to identify the functional motifs in these viral proteins. Here, in light of these experimental results, we apply computational analysis to predict the 3D structures and functional sites for Ebolavirus protein domains with unknown structure, including a zinc-finger domain of VP30, the RNA-dependent RNA polymerase catalytic domain and a methyltransferase domain of protein L. In addition, we compare sequences of proteins that interact with Ebolavirus proteins from RESTV-resistant primates with those from RESTV-susceptible monkeys. The host proteins that interact with GP and VP35 show an elevated level of sequence divergence between the RESTV-resistant and RESTV-susceptible species, suggesting that they may be responsible for host specificity. Meanwhile, we detect variable positions in protein sequences that are likely associated with the loss of human pathogenicity in RESTV, map them onto the 3D structures and compare their positions to known functional sites. VP35 and VP30 are significantly enriched in these potential pathogenicity determinants and the clustering of such positions on the surfaces of VP35 and GP suggests possible uncharacterized interaction sites with host proteins that contribute to the virulence of Ebolavirus.

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“…4 In those studies, KIAA0082 clustered with genes known to be induced by interferon and to exert antiviral activity. 5 More recently, the crystal structure of the catalytic methyltransferase domain of hMTr1 (residues 126-550) in complex with a capped oligoribonucleotide and S-adenosyl-methionine, which is the methyl group donor for this reaction, was described, 6 and it provided further information on the catalytic mechanism of cap1 synthesis. hMTr1 is a modular protein containing domains for RNA binding (G-patch, residues 85-131), methyltransferase (RrmJ/FtsJ, residues 252-451), DNA ligase/mRNA capping (residues 530-729), and protein interaction (WW, residues 735-786).…”

mentioning

confidence: 99%

Interactome analysis of the human Cap‐specific mRNA (nucleoside‐2′‐O‐)‐methyltransferase 1 (hMTr1) protein

Simabuco

Pavan

Pestana

et al. 2018

J of Cellular Biochemistry

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In a previous study, we have shown that the gene promoter of a protein termed KIAA0082 is regulated by interferon and that this protein interacts with the RNA polymerase II. It has been subsequently shown that KIAA0082 is the human cap‐specific messenger RNA (mRNA) (nucleoside‐2′‐O‐)‐methyltransferase 1 (hMTr1), which catalyzes methylation of the 2′‐O ‐ribose of the first nucleotide of capped mRNAs. Pre‐mRNAs are cotranscriptionally processed, requiring coordinate interactions or dissociations of hundreds of proteins. hMTr1 potentially binds to the 5′‐end of the whole cellular pre‐mRNA pool. Besides, it contains a WW protein interaction domain and thus is expected to be associated with several proteins. In this current study, we determined the composition of complexes isolated by hMTr1 immunoprecipitation from HEK293 cellular extracts. Consistently, a large set of proteins that function in pre‐mRNA maturation was identified, including splicing factors, spliceosome‐associated proteins, RNA helicases, heterogeneous nuclear ribonucleoproteins (HNRNPs), RNA‐binding proteins and proteins involved in mRNA 5′‐ and 3′‐end processing, forming an extensive interaction network. In total, 137 proteins were identified in two independent experiments, and some of them were validated by immunoblotting and immunofluorescence. Besides, we further characterized the nature of several hMTr1 interactions, showing that some are RNA dependent, including PARP1, ILF2, XRCC6, eIF2α, and NCL, and others are RNA independent, including FXR1, NPM1, PPM1B, and PRMT5. The data presented here are consistent with the important role played by hMTr1 in pre‐mRNA synthesis.

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Structural analysis of human 2′-O-ribose methyltransferases involved in mRNA cap structure formation

Cited by 92 publications

References 43 publications

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

A general method for rapid and cost-efficient large-scale production of 5′ capped RNA

Predictive and comparative analysis of Ebolavirus proteins

Interactome analysis of the human Cap‐specific mRNA (nucleoside‐2′‐O‐)‐methyltransferase 1 (hMTr1) protein

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