The synergic effects between Mto1 and Mtpap on the polyadenylation of mRNAs. (A, B) Immunoprecipitation analysis of MTO1 with MTPAP. HEK 293T cells transiently expressing with or without MTO1-FLAG were solubilized with a lysis buffer and lysate proteins were immuno-precipitated with immunocapture buffer (left) (input) and FLAG-antibody (right) (IP), respectively. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting using anti-FLAG, anti-MTPAP and TOM20 antibodies, respectively. (C) Western blot analysis. Twenty micrograms of total proteins from mto1−/− and WT zebrafish were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with antibodies for Mtpap and Gapdh as a loading control. (D) Polyadenylation profiles of mitochondrial mRNAs upon mto1−/− and WT zebrafish hearts. The 3′ termini of nd1, cytb, cox1 and cox3 mRNAs were assessed by RT-PCR amplifications from polyadenylated and oligoadenylated RNAs of mto1−/− and WT zebrafish hearts and 3% agarose gel electrophoresis. Arrows indicated the positions of PCR products from the polyadenylated and oligoadenylated RNAs, respectively. (E) Quantification of poly(A) proportions of nd1, cytb, cox1 and cox3 transcripts in the WT and mto1−/− zebrafish. (F) Poly(A) tail lengths from individually sequenced clones after 3’ end RACE analysis of nd1 transcripts in the mto1−/− and WT zebrafish hearts. Graph details and symbols are explained in the legend to Figure 2.
|
