Fig. 6: Effects of DUS1L overexpression on tRNA^Tyr(GUA) maturation.

a Northern blot analysis of tRNA^Tyr(GUA) in DUS1L OE and control LNZ308 cells. Each lane was derived from different cell dishes. The lower band corresponds to mature tRNA^Tyr(GUA) and the upper band is elongated tRNA^Tyr(GUA). The positions of single-stranded DNA size markers are shown on the left. b Comparison of D levels in mature tRNA^Tyr(GUA) and the elongated band, which were enriched using an oligo DNA probe against tRNA^Tyr(GUA) and gel excision, followed by nuclease digestion and mass spectrometry analysis, normalized by cytidine levels. Means ± s.e.m. from n = 4 samples each. *P = 0.0286 by the Mann–Whitney test. c Outline of the method to identify the sequence of the elongated tRNA^Tyr(GUA). d Schematic of the gene structure, transcription, and post-transcriptional processing of human tRNA^Tyr(GUA). Pol III transcription terminates after producing the oligo(U) stretch⁶³. The tRNA^Tyr(GUA) precursor contains the 5′ leader, intron, and 3′ trailer sequences, which are removed by RNase P, the tRNA splicing complex, and RNase Z, respectively, followed by the 3′ terminal CCA addition⁶⁴. e Sanger sequencing results of the elongated tRNA^Tyr(GUA). The upper four clones were analyzed by PCR using a forward primer designed to target the 5′ leader (underlined sequences) and a reverse primer designed against the 3′ adapter. The lower four clones were analyzed by PCR using a forward primer designed to target the 5′ end inside the tRNA^Tyr(GUA) sequence (underlined sequences) and a reverse primer designed against the 3′ adapter. f Northern blot analysis using a probe designed against the tRNA^Tyr(GUA) trailer sequence. Each lane was derived from different cell dishes.