The human tRNA(m(2)(2)G(26))dimethyltransferase: functional expression and characterization of a cloned hTRM1 gene

Abstract

This paper presents the first example of a complete gene sequence coding for and expressing a biologically functional human tRNA methyltransferase: the hTRM1 gene product tRNA(m(2)(2)G)dimethyltransferase. We isolated a human cDNA (1980 bp) made from placental mRNA coding for the full-length (659 amino acids) human TRM1 polypeptide. The sequence was fairly similar to Saccharomyces cerevisiae Trm1p, to Caenorhabditis elegans TRM1p and to open reading frames (ORFs) found in mouse and a plant (Arabidopsis thaliana) DNA. The human TRM1 gene was expressed at low temperature in Escherichia coli as a functional recombinant protein, able to catalyze the formation of dimethylguanosine in E.coli tRNA in vivo. It targeted solely position G(26) in T7 transcribed spliced and unspliced human tRNA(Tyr) in vitro and in yeast trm1 mutant tRNA. Thus, the human TRM1 protein is a tRNA(m(2)(2)G(26))dimethyltransferase. Compared with yeast Trm1p, hTRM1p has a C-terminal protrusion of approximately 90 amino acids which shows similarities to a mouse protein related to RNA splicing. A deletion of these 90 C-terminal amino acids left the modification activity in vitro intact. Among point mutations in the hTRM1 gene, only those located in conserved regions of hTRM1p completely eliminated modification activity.

Figure 1

In vitro incorporation of methyl groups into G₂₆ methyl-deficient D4 tRNA catalyzed by the human TRM1 enzyme. The assay was performed at 30°C for different times in the presence of 5 µl crude enzyme extract from IPTG-induced E.coli BL21(DE3)plysS cells harboring the pET-hTRM1 plasmid, [¹⁴C]methyl-AdoMet, the methyl donor (53 mCi/mmol), and different amounts of tRNA substrates from S.cerevisiae strain D4 (trm1, filled symbols) or wild-type strain YF⁺ (TRM1, + symbol).

Figure 2

HPLC analyses of modified nucleosides in E.coli tRNA from wild-type cells and from E.coli carrying a plasmid with a human TRM1 gene. Isolated tRNA was digested with both nuclease P1 and bacterial alkaline phosphatase before HPLC chromatography of the nucleosides. (A) Escherichia coli BL21(DE3)plysS carrying the pET15b plasmid. (B) IPTG-induced E.coli BL21(DE3)plysS harboring the pET-hTRM1 plasmid. Nucleosides were identified by their UV spectra and their relative retention times (only times between 15 and 50 min are shown). The positions for m²G and m²₂G are indicated by arrows.

Figure 3

Autoradiograms of 2-dimensional TLC showing the formation of modified nucleotides m²₂G and m²G in human tRNA^Tyr and pre-tRNA^Tyr molecules with recombinant hTRM1 enzyme. Unmodified [α-³²P]GTP-labeled or [α-³²P]ATP-labeled in vitro transcripts of human tRNA^Tyr and pre-tRNA^Tyr were incubated with methyl donor and cell extracts containing or lacking the hTRM1 gene product. The full-length tRNA was isolated, degraded with RNase T₂, analyzed by 2-dimensional TLC and spots were identified (see Materials and Methods). The [α-³²P] label in the tRNA transcripts and the nuclease used are indicated at the top of each panel. Spots corresponding to m²G and m²₂G are indicated. (A) [α-³²P]GTP-labeled tRNA^Tyr incubated with extract from E.coli pET15b without (A1) and with (A2) the hTRM1 gene. (B) [α-³²P]GTP-labeled pre-tRNA^Tyr incubated with extract from E.coli pET15b without (B1) and with (B2) the hTRM1 gene. (C) [α-³²P]ATP-labeled tRNA^Tyr incubated with extract from E.coli pET15b without (C1) and with (C2) the hTRM1 gene. (D) [α-³²P]ATP-labeled pre-tRNA^Tyr incubated with extract from E.coli pET15b without (D1) and with (D2) the hTRM1 gene. Note that the m¹G spots in (C1) and (C2) originated from position G₃₇ (label came from the neighboring A₃₈) and were modified by an inherent E.coli enzyme. In unspliced tRNA G₃₇ is adjacent to the intron and not to a labeled A and therefore no m¹G was found in (D1) and (D2).

Figure 4

Autoradiograms of 2-dimensional TLC showing the formation of modified nucleotides of m²₂G and m²G by human HeLa cell extracts. Unmodified [α-³²P]GTP-labeled or [α-³²P]ATP-labeled in vitro transcripts of human tRNA^Tyr were incubated with HeLa cell extracts at 30°C for 60 min. The isolated tRNA was purified, degraded with nuclease T₂, analyzed by 2-dimensional TLC and the spots identified (see Materials and Methods). The spots corresponding to m²G and m²₂G are indicated. (A) [α-³²P]GTP-labeled tRNA^Tyr. (B) [α-³²P]ATP-labeled tRNA^Tyr.