( F) Western blot of the coimmunoprecipitation of different F/H-METT元 and myc-METTL14 constructs. The lower panels show immunofluorescence stainings of myc-METTL14 and the predicted F/H-METT元-NLS mutant. ( E) For the upper panel, HEK293T cells were transfected with myc-METTL14 and F/H-METT元. The cartoon shows the position and sequence of the potential NLS in the protein.
( D) HeLa cells were transfected with myc-METTL14 WT ( upper panels) and the predicted F/H-NLS mutant ( lower panels). GFP containing either a myc- or a F/H-tag serves as control. ( C) Western blots of coimmunoprecipitations between F/H-METT元-NLS mutant and myc-WTAP. The computed NLS is predicted in the potential RBD between the leader helix and the MT-A70 domain as shown in the schematic view beneath the stainings. ( B) Immunofluorescence of HeLa cells which were transfected with WT ( upper panels) and NLS-mutated F/H-METT元 ( lower panels). The schematic cartoon under the immunofluorescences shows the very N-terminal location and the sequence of the NLS in the protein. ( A) Immunofluorescence staining of HeLa cells transfected with wild-type (WT) myc-WTAP ( upper panels) and myc-WTAP-NLS mutant (mut) ( lower panels). Localization studies of METT元, METTL14, and WTAP. ( J) Schematic cartoon of the regions of interaction between WTAP and METT元 based on the results of coimmunoprecipitation experiments. The upper panel shows the HA-antibody treated blot, the lower blot shows the α-myc-western blot. ( I) Western blots of coimmunoprecipitations of the N-terminal part of F/H-METT元 (leader helix, short LH) with C-terminally truncated constructs of myc-WTAP. ( H) Western blot of coimmunoprecipitated full-length myc-WTAP and LH-deletion mutants of F/H-METT元 (ΔLH-METT元). ( G) Western blots of coimmunoprecipitations as in F but with C-terminally truncated constructs of myc-WTAP. The arrows show the bands for the different constructs.
The upper panel was incubated with α-HA antibody, the lower one with α-myc antibody. ( F) Western blots of coimmunoprecipitations of full-length F/H-METT元 with N-terminal truncations of myc-WTAP. ( E) Scheme of the different WTAP N- and C-terminal truncation constructs. ( D) Schematic view of the interaction surface between METT元 and METTL14 revealed by coexpression studies. Complexes were purified via GST-METT元 (76–580) and GST was subsequently cleaved off during elution. ( C) Coomassie gel of recombinantly expressed and purified complexes composed of the indicated truncated METTL14 and METT元 (76–580) constructs. ( B) Schematic representation of the different truncation constructs of F/H-METT元 and myc-METTL14 used to narrow down the binding sites of these proteins. The complex was purified via the GST-tag on METT元. The coexpression was conducted in insect cells (SF21).
( A) Coomassie gel of purified recombinant full-length GST-METT元 and copurified METTL14. Published by Cold Spring Harbor Laboratory Press for the RNA Society.īinding studies of METT元, METTL14, and WTAP. METTL14 METT元 RNA modification m6A methyltransferase. Our biochemical work identifies characteristic features of METT元/14-WTAP and reveals novel insight into the overall architecture of this important enzyme complex. In addition, we show that the C-terminal RGG repeats of METTL14 are required for METT元/14 activity by contributing to RNA substrate binding. Using an in vitro methylation assay, we confirm that monomeric METT元 is soluble and inactive while the catalytic center of METTL14 is degenerated and thus also inactive.
Furthermore, we identify nuclear localization signals and identify phosphorylation sites on the endogenous proteins. Here, we used recombinant proteins and mapped binding surfaces within the METT元/14-WTAP complex. Although recent crystal structures revealed how the catalytic MT-A70 domains of METT元 and METTL14 interact with each other, a more global architecture including WTAP and RNA interactions has not been reported so far. METT元/14 is found in the nucleus where it is localized to nuclear speckles and the splicing regulator WTAP is required for this distinct nuclear localization pattern. A heterodimeric enzyme complex composed of METT元 and METTL14 generates m 6A on mRNAs. m 6A modification has been implicated in mRNA stability and turnover, localization, or translation efficiency. N 6-methyladenine (m 6A) is found on many eukaryotic RNAs including mRNAs.
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