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

Abstract

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'-O position of the first transcribed nucleotide (m7GpppNmpRNA) and sometimes on the adjoining nucleotide (m7GpppNmpNmpRNA). 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'-O-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'-O-methylated cap structure. Fluorescence spectroscopy assays also revealed that the presence of the 2'-O-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.

Fig 1. DXO hydrolyzes only capped RNAs without a 2’-O-methylation.

(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’-O-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) in vitro. 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.

Fig 2. Catalytically inactive DXO.

(A) Two residues involved in the coordination of Mg²⁺ ions in the active site of mouse DXO (pdb 4j7l) are shown. (B) Sequence conservation of residues coordinating Mg²⁺ ions between mouse and human DXO. (C) To ensure that the observed activity is indeed due to DXO, the catalytically inactive D236A/E253A mutant was used in a decapping assay using GpppG-RNA as substrate. Wild-type DXO readily hydrolyzes this cap structure in the presence of magnesium. However, no hydrolysis is observed with the catalytically inactive DXO mutant.

Fig 3. The presence of a 2’-O-methylation decreases affinity of DXO for capped RNA.

The affinity of DXO for cap0-RNA and cap1-RNA were measured using fluorescence spectroscopy. (A) Increasing amounts of cap0-RNA (0.46μM– 28μM) were added to a of 1.2μM solution of DXO, and the emission spectrum was scanned from 310 to 440nm. (B) A saturation isotherm can be generated from these data by plotting the change in fluorescence intensity at 332nm as a function of added RNA. Results are shown as changes in intrinsic fluorescence in the presence of capped RNA compared to the absence of RNA (ΔF/F₀).

Fig 4. The presence of a 2’-O-methylation blocks the exoribonuclease activity of DXO.

(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’-O-methylation, a 2’-O-methylation on the first nucleotide or a 2’-O-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.

Fig 5. DXO contains a functional NLS as shown by site-directed mutagenesis and immunofluorescence.

(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.

Fig 6. Model of DXO activity on RNA.

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.