Supplementary Figure 4: Dnmt2-depended m5C modification regulate biological properties of tsRNA.
(a) Dnmt2-dependent C38 methylation in testis. Bisulfite sequencing maps for the three known tRNA targets of Dnmt2 (tRNA-Asp, tRNA-Gly and tRNA-Val in mouse testis (Dnmt2+/+ and Dnmt2–/–). Each row represents one sequence read, each column a cytosine residue. Yellow boxes represent unmethylated cytosine residues; blue boxes indicate methylated cytosine residues (m5C), sequencing gaps are shown in white. Numbers above the maps indicate the number of reads. Cytosine C38 is labeled in red, other cytosine sites are in black. (b) The sequence of chemically synthesized 3’tsRNA-Gly that harbors five m5C according to the Dnmt2+/+ condition (5 × m5C), 3’tsRNA-Gly with four m5C, lacking a Dnmt2-mediated m5C at C38 position (4 × m5C), and 3’tsRNA-Gly without any RNA modifications (no m5C). (c) By transfecting the above three types of tsRNAs into 3T3 cell lines followed by transcriptome sequencing (n = 2 biologically independent RNA samples). Each dot in the box plot represent the geneset score of one transcriptome data, the upper boundary of the box represents the maxima, the lower boundary represents the minima, and the centre represent the average. We observed that the same tsRNAs sequence with different number of m5C modifications (no m5C, five m5C and four m5C respectively) can induced different cellular transcriptome changes compared to the empty transfection: while all three types of tsRNAs similarly induced an overall elevated gene expression of Citrate Acid Circle pathway (which might be induced by and dependent on their specific sequence); the three types of tsRNAs with different m5C modification induced distinct gene responses of Ribosome pathway (which might be sensitive to the RNA structural changes caused by m5C), which may reflect recent studies reporting the function of tsRNAs in regulating Ribosome functions6–9.
