Investigation de la spécificité nucléotidique de l’hélicase DHX36 lors du déroulement de structures d’ARN G-quadruplex.

La déstabilisation des structures G-quadruplex au niveau des acides nucléiques a des répercussions physiologiques importantes. L’accentuation des connaissances concernant les processus cellulaires associés au métabolisme des structures des G4 est primordiale. Une panoplie d’hélicases à G4 est impliquée dans le métabolisme des structures G4, notamment l’hélicase humaine DHX36. Il a été déterminé au préalable par certains groupes de recherche que l’hélicase DHX36 se lie à son substrat l’ARN G4 et utilise des nucléosides triphosphates afin de catalyser le dépliement de la structure G-quadruplex. Toutefois, l’interaction avec l’ARN G4 a été sommairement caractérisée et la spécificité nucléotidique n’a toujours pas été évaluée. Ainsi, nous avons décidé d’approfondir les connaissances du mécanisme de dépliement de la structure du G4 d’ARN par l’hélicase DHX36. Notamment, en évaluant la thermodynamique de l’interaction entre l’hélicase et l’ARN G4 afin de révéler particulièrement l’efficacité de liaison mais également en évaluant la spécificité nucléotidique de l’hélicase DHX36 afin d’effectuer le dépliement de l’ARN G4. La combinaison des analogues de nucléotides et le modèle structural permettent de révéler les caractéristiques structurales et fonctionnelles de l’interaction entre l’hélicase humaine DHX36 et l’ATP. Nos analyses permettent de constater que l’enzyme DHX36 est en mesure d’utiliser autant l’ATP que GTP afin de dérouler les structures G4 d’ARN ayant, par contre, une spécificité accrue pour la molécule d’ATP

Investigation de la spécificité nucléotidique de l’hélicase DHX36 lors du déroulement de structures d’ARN G-quadruplex.

La déstabilisation des structures G-quadruplex au niveau des acides nucléiques a des répercussions physiologiques importantes. L’accentuation des connaissances concernant les processus cellulaires associés au métabolisme des structures des G4 est primordiale. Une panoplie d’hélicases à G4 est impliquée dans le métabolisme des structures G4, notamment l’hélicase humaine DHX36. Il a été déterminé au préalable par certains groupes de recherche que l’hélicase DHX36 se lie à son substrat l’ARN G4 et utilise des nucléosides triphosphates afin de catalyser le dépliement de la structure G-quadruplex. Toutefois, l’interaction avec l’ARN G4 a été sommairement caractérisée et la spécificité nucléotidique n’a toujours pas été évaluée. Ainsi, nous avons décidé d’approfondir les connaissances du mécanisme de dépliement de la structure du G4 d’ARN par l’hélicase DHX36. Notamment, en évaluant la thermodynamique de l’interaction entre l’hélicase et l’ARN G4 afin de révéler particulièrement l’efficacité de liaison mais également en évaluant la spécificité nucléotidique de l’hélicase DHX36 afin d’effectuer le dépliement de l’ARN G4. La combinaison des analogues de nucléotides et le modèle structural permettent de révéler les caractéristiques structurales et fonctionnelles de l’interaction entre l’hélicase humaine DHX36 et l’ATP. Nos analyses permettent de constater que l’enzyme DHX36 est en mesure d’utiliser autant l’ATP que GTP afin de dérouler les structures G4 d’ARN ayant, par contre, une spécificité accrue pour la molécule d’ATP

Correction: 2'-O-methylation of the mRNA cap protects RNAs from decapping and degradation by DXO.

[This corrects the article DOI: 10.1371/journal.pone.0193804.]

DXO hydrolyzes only capped RNAs without a 2’-<i>O</i>-methylation.

<p>(A) The RNA 5’ cap structure is composed of a guanosine (blue) linked to the RNA (black) through a 5’-5’ triphosphate bridge. The subsequent N7-methylation of the guanosine (magenta) confers a positive charge to the cap structure. Additional 2’-<i>O</i>-methylations (orange) can be found on the first few nucleotides. (B) Nomenclature of the different cap structures. (C) Aliquots (2μg) of the purified preparations of DXO and mutant DXO protein (D236A/E253A) were analyzed by electrophoresis through a 12.5% polyacrylamide gel containing 0.1% SDS and visualized with Coomassie Blue Dye. The positions and sizes (in kDa) of the size markers are indicated on the left. (D) RNAs harbouring different cap structures were transcribed and capped (incorporation of [α-³²P]GTP) <i>in vitro</i>. They were then subjected to degradation by different enzymes, and reaction products were separated by thin layer chromatography. Lanes 1–4 show reaction products after treatment of differently capped RNAs with Nuclease P1. Degradation products after incubation of differently capped RNAs with purified DXO are shown in lanes 5–12. The origin of spotting and dinucleotide identities are listed on the left. NOTE: During the preparation of differently capped RNAs, only approximately 30% of GpppN-RNA was methylated to form GpppN_m-RNA (lanes 2,7–8), resulting in a mixture of GpppN-RNA and GpppN_m-RNA. Degradation products observed in lane 8 are due to the degradation of GpppN-RNA.</p

2'-<i>O</i>-methylation of the mRNA cap protects RNAs from decapping and degradation by DXO

<div><p>The 5' RNA cap structure (^m7GpppRNA) is a key feature of eukaryotic mRNAs with important roles in stability, splicing, polyadenylation, mRNA export, and translation. Higher eukaryotes can further modify this minimal cap structure with the addition of a methyl group on the ribose 2'-<i>O</i> position of the first transcribed nucleotide (^m7GpppN_mpRNA) and sometimes on the adjoining nucleotide (^m7GpppN_mpN_mpRNA). In higher eukaryotes, the DXO protein was previously shown to be responsible for both decapping and degradation of RNA transcripts harboring aberrant 5’ ends such as pRNA, pppRNA, GpppRNA, and surprisingly, ^m7GpppRNA. It was proposed that the interaction of the cap binding complex with the methylated cap would prevent degradation of ^m7GpppRNAs by DXO. However, the critical role of the 2’-<i>O</i>-methylation found in higher eukaryotic cap structures was not previously addressed. In the present study, we demonstrate that DXO possesses both decapping and exoribonuclease activities toward incompletely capped RNAs, only sparing RNAs with a 2’-<i>O</i>-methylated cap structure. Fluorescence spectroscopy assays also revealed that the presence of the 2’-<i>O</i>-methylation on the cap structure drastically reduces the affinity of DXO for RNA. Moreover, immunofluorescence and structure-function assays also revealed that a nuclear localisation signal is located in the amino-terminus region of DXO. Overall, these results are consistent with a quality control mechanism in which DXO degrades incompletely capped RNAs.</p></div

The presence of a 2’-<i>O</i>-methylation blocks the exoribonuclease activity of DXO.

<p>(A) The exoribonuclease activity of DXO toward different substrates was studied. 2μM of DXO were incubated with 100nM of the 30‐nt 3′‐radiolabelled RNA substrate harbouring either no 2’-<i>O</i>-methylation, a 2’-<i>O</i>-methylation on the first nucleotide or a 2’-<i>O</i>-methylation on the 16^th nucleotide. The reactions were incubated at 37°C for 0 to 64 minutes before being stopped by adding 100mM EDTA. Products were separated on a 20% denaturing polyacrylamide gel. (B) To ensure that the observed exoribonuclease activity is specific to the DXO protein, a catalytically inactive mutant (D236A-E253A) was used in an exoribonuclease assay with 5’ monophosphorylated RNA. Wild-type DXO readily degrades this RNA substrate, whereas almost no cleavage products are observed with the inactive mutant.</p

Model of DXO activity on RNA.

<p>DXO removes incomplete cap structures such as capG (right) and cap0 (middle) and degrades the resulting uncapped mRNA, whereas RNAs harboring a cap1 structure (left) are unaffected. RNAs with capG and cap0 structures can be either capping intermediates or non-self RNAs.</p

DXO contains a functional NLS as shown by site-directed mutagenesis and immunofluorescence.

<p>(A) A schematic representation of a classical bipartite NLS consensus sequence and the corresponding sequence at the N-terminal end of DXO. (B) HeLa cells were transfected with pcDNA3.1+/DXO and pcDNA3.1+/DXO-K7A-R8A (mutations in the NLS) using Lipofectamine 2000. DXO localization was monitored 48 hours post-transfection by immunofluorescence using a rabbit polyclonal DXO antibody and a polyclonal anti-rabbit antibody coupled to Alexa Fluor 488. Fluorescent images were gathered using an epifluorescence microscope with a 60x objective. Exposure times were identical in all three conditions.</p