Fig. 6: Splicing efficiency of late viral RNA is mediated by m6A.
a Nascent transcription was analyzed by labeling RNA with 1 mM 4-thiouridine (4sU) for exactly 10 min at 24 hpi for infections of A549 cells transfected with control (siCTRL) or siRNA-mediated knockdown of METTL3 (siMETTL3). Nascent 4sU-labeled RNA was extracted for use in qRT-PCR for analysis of relative transcription rates of two viral early genes (E1A and E4) and the tripartite leader (MLP) found in all Ad5 late transcripts. Samples include four biological replicates. b Transcriptional shut-off was performed by adding 60 µM of the RNA Pol II elongation inhibitor DRB to the media of cells infected with Ad5 for 24 h, and stability of labeled spliced transcripts was measured by qRT-PCR for 2, 4, or 8 h post shut-off. Samples include three biological replicates. c Schematic of the early transcript E1A and the late transcript Fiber that both contain m6A sites. Three primers allow for the distinction between spliced and unspliced PCR products that can be analyzed by qRT-PCR. d Splice efficiency as defined by the relative ratio of spliced to unspliced transcripts of E1A and Fiber were analyzed by qRT-PCR in A549 cells infected with Ad5 for 24 h after depletion of METTL3 or WTAP. Data represents three biological experiments. e Fiber splice efficiency was analyzed in Parental A549 cells or two independent METTL3 KO cell lines. f Splice efficiency of Fiber was analyzed after siRNA-mediated depletion of the nuclear m6A reader YTHDC1. g Immunoblot showing viral late proteins Hexon, Penton, and Fiber, as well as viral early proteins DBP and E1A. A549 cells were depleted of METTL3 (M3), WTAP, YTHDC1 (DC1) or control siRNA (siC) for 48 h prior to infection with Ad5 for 24 h. Immunoblot representative of three independent experiments. h Schematic design for a luciferase construct that expresses the third adenovirus tripartite leader to Fiber splice site with intervening L1 and L5 adenoviral intron. The 5’ fragment of Fiber that contains the m6A signal peak was fused in-frame to a Renilla luciferase transgene where all m6A DRACH motifs have been ablated by silent mutation. A matching construct was generated with all 15 potential m6A DRACH motifs ablated by silent or synonymous mutation (m6A Mut Fiber). i HeLa cells were control transfected (siC) or depleted of METTL3 (siM3) by siRNA for 48 h before transfection with either WT Fiber or m6A Mut Fiber plasmid. At 24 h after the second transfection, splicing efficiency of the transgene was assayed using Fiber-specific primers. Data represent four biological replicates. For all assays significance was determined by unpaired two-tailed Student’s T-test, *p ≤ 0.05, **p ≤ 0.01, ns=not significant. Exact p-Values are included in the source data file. Graphs represent mean +/− standard deviation.
