The human tRNA m (5) C methyltransferase Misu is multisite-specific

Abstract

The human tRNA m ( 5) C methyltransferase Misu is a novel downstream target of the proto-oncogene Myc that participates in controlling cell division and proliferation. Misu catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to carbon 5 of cytosines in tRNAs. It was previously shown to catalyze in vitro the intron-dependent formation of m ( 5) C at the first position of the anticodon (position 34) within the human pre-tRNA (Leu) (CAA). In addition, it was recently reported that C48 and C49 are methylated in vivo by Misu. We report here the expression of hMisu in Escherichia coli and its purification to homogeneity. We show that this enzyme methylates position 48 in tRNA (Leu) (CAA) with or without intron and positions 48, 49 and 50 in tRNA (Gly2) (GCC) in vitro. Therefore, hMisu is the enzyme responsible for the methylation of at least four cytosines in human tRNAs. By comparison, the orthologous yeast enzyme Trm4 catalyzes the methylation of carbon 5 of cytosine at positions 34, 40, 48 or 49 depending on the tRNAs.

Keywords: 5-methylcytosine; Misu; NSun2; RNA methyltransferase; Trm4; m5C; tRNA modification enzyme.

Figure 1. Formation of m⁵C in human tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$ catalyzed by purified hMisu. (A) 10% SDS PAGE analysis of hMisu purification. Lane 1: Ni-NTA purified His-GST fused hMisu (117 kDa). Lane 2: Proteins mixture after TEV protease digestion of Ni-NTA purified His-GST-hMisu (after digestion, the molecular weight of hMisu is 86 kDa). Lane 3: hMisu after TEV protease digestion and elution of Ni-NTA column with 20 mM imidazole. (B and C) The transcripts used as substrates were the intron-containing human pre- tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$ (B) and intron-less human tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$. (C) The arrows indicate the position of the intron. (D−F) Thin layer chromatography analyses of pre- tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$ incubated without protein (D) or with hMisu (E), and of tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$ incubated with hMisu (F) in the absence of magnesium. The main spot is C and the minor spot m⁵C. (G and H) Time course of m⁵C formation catalyzed by hMisu in pre- tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$ (G) or tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$ (H) in the presence of 50 mM NaCl, and in the absence (open circles) or presence (filled circles) of 10 mM MgCl₂.

Figure 2. MALDI mass spectrometry analysis of intron-containing human pre- tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$ for methylation at position 34. (A) MALDI mass spectrum of pre- tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$ methylated by hMisu and digested by RNase T1 that cleaves after guanosines. The spectral region around the ACUCAAGp fragment containing C34 is enlarged to show the peak of the nonmethylated ion (m/z 2265.32) in the control without enzyme and the ion methylated by hMisu (m/z 2279.35). (B) List of theoretical masses of RNase T1 fragments of singly protonated pre- tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$.

Figure 3. MALDI mass spectrometry analysis of intron-less human tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$ for methylation by hMisu at position 48. (A) MALDI mass spectrum of tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$ methylated by hMisu and digested by RNase A that cleaves after pyrimidines. The spectral region around the fragment GGAGGCp containing C48 is enlarged to show the peak of the nonmethylated ion (m/z 2031.18) from the control without enzyme and the ion methylated by hMisu (m/z 2045.62). (B) List of theoretical masses of RNase A fragments of singly protonated tRNA $\begin{array}{c}Leu\\ \left(CAA\right)\end{array}$.

Figure 4. MALDI mass spectrometry analysis of human tRNA $\begin{array}{c}\text{Gly2}\\ \text{GCC}\end{array}$ for methylation by hMisu. (A) Cloverleaf structure of the mature human tRNA$\begin{array}{c}\text{Gly2}\\ \text{GCC}\end{array}$ indicating its natural posttranscriptional modifications. (B) MALDI mass spectrum of tRNA $\begin{array}{c}\text{Gly2}\\ \text{GCC}\end{array}$ methylated by hMisu and digested by RNase T1 that cleaves after guanosines. The spectral region around the fragment CCCGp containing C48, C49 and C50 is enlarged to show the peak of the nonmethylated ion (m/z 1277.15) from the control without enzyme and the ion methylated by hMisu (m/z 1319.24), see also Figure S3. (C) List of theoretical masses of RNase T1 fragments of singly protonated tRNA$\begin{array}{c}\text{Gly2}\\ \text{GCC}\end{array}$. (D) Identification of the three methylation targets of hMisu at positions 48, 49 and 50 in human tRNA$\begin{array}{c}\text{Gly2}\\ \text{GCC}\end{array}$ by MS/MS. The fragment at m/z 1319.24 of the methylated sample shown in (B) was selected and fragmented by tandem mass spectrometry. The fragments assignment follows the scheme of McLuckey et al. The peaks corresponding to the c-ions and w-ions are generated by loss of the 3′ and 5′ nucleotides, respectively.