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[Preprint]. 2024 Apr 24:2024.04.24.590974. [Version 1] doi: 10.1101/2024.04.24.590974

Figure 2. tRNA is a potential substrate of C12orf29.

Figure 2.

(A) Urea-PAGE and SYBR Gold staining depicting the nucleic acids that copurified with recombinant C12orf29 or mutants from E. coli. Samples were treated with DNase or RNase A and digested with proteinase K prior to analysis. (B) Urea-PAGE and SYBR Gold staining depicting the nucleic acids that copurified with α-Flag-immunoprecipitates from HEK293A cells expressing Flag-tagged C12orf29 or C12orf29E250A. Immunoprecipitates were treated with DNase or RNase A and digested with proteinase K prior to analysis. (C) Volcano plot depicting the enrichment of tRNA in the α-Flag-immunoprecipitates from HEK293A cells expressing Flag-tagged C12orf29E250A vs Flag-tagged C12orf29. RNAs were analyzed by AQ-seq. tRNA is labeled in red. The fold change cutoff is 2 and the p-value cutoff is 0.05. (D) Schematic depicting a segment of the tRNA anticodon stem-loop. Cleavage sites are shown, and the nucleotides are numbered according to the canonical tRNA nucleotide numbering system. (E) Autoradiograph depicting the reaction products of an in vitro tRNA-Lys-CTT fragment ligation assay using T4 Rnl1 (Rnl1) and human C12orf29 (WT) or C12orf29K57M (KM). A synthetic tRNA-Lys-CTT 3’ fragment was labeled with 32P at the 5’end, annealed to an unlabeled 5’ fragment and incubated with the ligases. Substrates are indicated in (D). (F) Autoradiograph depicting an in vitro exon ligation assay using Rnl1 and human C12orf29 or C12orf29K57M. Saccharomyces cerevisiae pre-tRNA-Phe-GAA was transcribed in vitro and cleaved with the TSEN endonuclease complex. Exons were purified following Urea-PAGE electrophoresis and were processed and labeled with T4 Pnk using [γ−32P]ATP, after which they were incubated with the ligases. (G) Northern blot depicting intron-containing tRNAs from WT or C12orf29 KO Hela cells. 5s rRNA is shown as a loading control. (H) Protein immunoblots depicting Lamin A/C, Vinculin, C12orf29 and RtcB. A549 cell lysates were fractionated to separate the nucleus from the cytoplasm. Lamin A/C is used as nuclear marker and Vinculin as cytosolic marker.