Figure 2: YTHDF2 destabilizes mRNA by hastening deadenylation as an initial step. | Nature Communications

Figure 2: YTHDF2 destabilizes mRNA by hastening deadenylation as an initial step.

From: YTHDF2 destabilizes m6A-containing RNA through direct recruitment of the CCR4–NOT deadenylase complex

Figure 2

(a) Construct of BG-1boxB, which can bind to proteins fused with a λN peptide through the boxB sequence. (b) Decay of BG-1boxB mRNA in the presence of λN-FLAG-YTHDF2 or FLAG-YTHDF2. AG-GAPDH, a constitutively transcribed internal standard was co-transfected. (c) Graphs of the concentration of BG mRNA described in b as a function of time. The abundance of BG-1boxB mRNA remaining at each time point was normalized to that of AG-GAPDH. Error bars, mean±s.d., n=3, biological replicates. (d) Deadenylation assay of BG-1boxB mRNA in the presence of λN-FLAG-YTHDF2 or FLAG-YTHDF2. (e) Retention of the 5′-cap on BG-1boxB mRNA in the presence of λN-FLAG-YTHDF2 or FLAG-YTHDF2. (f) Deadenylation assay of BG-1boxB mRNA tethered with an N-terminal P/Q/N-rich region (YTHDF2-N) or a C-terminal YTH domain (YTHDF2-C). (g) Construct of BG-2boxB-ORF, which has two boxB sequences inserted in-frame to the ORF of the BG reporter. (h) Deadenylation assay of BG-2boxB-ORF mRNA in the presence of λN-FLAG-FL, λN-FLAG-YTHDF2 or FLAG-YTHDF2.

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