2'-O-methylation of the mRNA cap protects RNAs from decapping and degradation by DXO
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 16th 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.