Genome-wide analysis of N1-methyl-adenosine modification in human tRNAs

Abstract

The N(1)-methyl-Adenosine (m(1)A58) modification at the conserved nucleotide 58 in the TPsiC loop is present in most eukaryotic tRNAs. In yeast, m(1)A58 modification is essential for viability because it is required for the stability of the initiator-tRNA(Met). However, m(1)A58 modification is not required for the stability of several other tRNAs in yeast. This differential m(1)A58 response for different tRNA species raises the question of whether some tRNAs are hypomodified at A58 in normal cells, and how hypomodification at A58 may affect the stability and function of tRNA. Here, we apply a genomic approach to determine the presence of m(1)A58 hypomodified tRNAs in human cell lines and show how A58 hypomodification affects stability and involvement of tRNAs in translation. Our microarray-based method detects the presence of m(1)A58 hypomodified tRNA species on the basis of their permissiveness in primer extension. Among five human cell lines examined, approximately one-quarter of all tRNA species are hypomodified in varying amounts, and the pattern of the hypomodified tRNAs is quite similar. In all cases, no hypomodified initiator-tRNA(Met) is detected, consistent with the requirement of this modification in stabilizing this tRNA in human cells. siRNA knockdown of either subunit of the m(1)A58-methyltransferase results in a slow-growth phenotype, and a marked increase in the amount of m(1)A58 hypomodified tRNAs. Most m(1)A58 hypomodified tRNAs can associate with polysomes in varying extents. Our results show a distinct pattern for m(1)A58 hypomodification in human tRNAs, and are consistent with the notion that this modification fine tunes tRNA functions in different contexts.

FIGURE 1.

A microarray method to study m¹A modifications in human tRNA. (A) Location of N¹-methyl-A modification in tRNA. The RT primers are complementary to the region in red. (B) The amount of RT product is proportional to the amount of unmodified template tRNA. The primer is complementary to nucleotides 76–59, and all four dNTPs are included. The small amount of hypomodified yeast tRNA^Phe at x = 0 is confirmed by another primer extension using the primer complementary to nucleotides 76–60 and a mixture of dATP, dTTP, dCTP, and ddGTP. A short exposure of the same lane on the left shows the separation of primer and primer stop at m¹A58 (+1). (C) Scheme of a microarray experiment for m¹A modification studies. (D) Detection of RT product using primers for individual human tRNA families. (E) Detection of RT products using a microarray. The array with a total human RNA sample (MCF10A cell line, left) shows much more products than the array with primers and standard tRNAs alone (middle). The array key is on the right. The array results correlates well with the gel analysis. For example, both methods show a very low amount of product for Met-i and a very large amount of product for Ala-hGC. (F) Specificity of the array method is indicated by the specific loss of the Ala-hGC signal when a large amount of the Ala-hGC oligo is added in array hybridization. The array key is on the right.

FIGURE 2.

Profiling m¹A58 hypomodification in human cell lines. (A) Hypomodified tRNA species in HeLa cells; the blue arrow indicates initiator-tRNA^Met, which is completely modified at A58. The relative signal (y-axis) = 1 are those of the two tRNA transcripts that are unmodified at A58. For this work, we defined that y < 0.5 has low, y = 0.5-2 has medium, and y > 2 has high levels of hypomodified tRNA. (B) Array image from cell line HeLA showing the signals for Asp-GTC and Glu-yTC, and the absence of signal for Met-i. (C) Heat map showing ranges (high, medium, low) of m¹A58 hypomodification in five human cell lines (HeLa, HEK 293T, MDA-MB-231, MCF-10A, and Neuroblastoma). The blue arrow indicates initiator-tRNA^Met (negative control) and the orange arrows indicate tRNA^Asp and tRNA^Glu (positive controls). (D) Determination of hypomodification at m¹G9 for tRNA^Gln, and tRNA^Pro. The detection limit for hypomodification is ∼2% in this experiment. The gel shows the primer extension reaction for tRNA^Gln in the total RNA from MDA-MB-231.

FIGURE 3.

Effect of siRNA knockdown of m¹A-methylase subunits, TRM6 and TRM61. (A) Western blot showing efficiency of siRNA treatment of MDA-MB231 cells. The siRNA treatment causes the decrease in protein level after 3 d of treatment. GAPDH level is used as a control. (B) Growth of control versus TRM6 and TRM61 siRNA-treated cells. (C) Representative array image from control and siTRM6-treated cells. (D) Heat map of hypomodification in control versus TRM6 and TRM61 knockdown cells. Purple arrows show tRNA species with significant increase in hypomodification in the siRNA-treated cells. Blue arrow indicates initiator-tRNA^Met, which, as expected, is not hypomodified at all times. (E) Heat map of relative tRNA abundance of the control and siRNA-treated cells shows no change in tRNA abundance in all cases.

FIGURE 4.

Effect of m¹A hypomodification on polysome association. The analysis was performed with HeLa and MDA-MB231 cells. (A) Scheme of the procedure to measure polysome association. (B) Array images showing differences between free and polysome samples from HeLa cells. The array key is on the right. (C) Heat map of polysome association. Color coding shows the comparison of polysome association of m¹A58 hypomodified tRNA with fully modified tRNA.