The protective role of m1A during stress-induced granulation

Abstract

Post-transcriptional methylation of N6-adenine and N1-adenine can affect transcriptome turnover and translation. Furthermore, the regulatory function of N6-methyladenine (m6A) during heat shock has been uncovered, including the enhancement of the phase separation potential of RNAs. In response to acute stress, e.g. heat shock, the orderly sequestration of mRNAs in stress granules (SGs) is considered important to protect transcripts from the irreversible aggregation. Until recently, the role of N1-methyladenine (m1A) on mRNAs during acute stress response remains largely unknown. Here we show that the methyltransferase complex TRMT6/61A, which generates the m1A tag, is involved in transcriptome protection during heat shock. Our bioinformatics analysis indicates that occurrence of the m1A motif is increased in mRNAs known to be enriched in SGs. Accordingly, the m1A-generating methyltransferase TRMT6/61A accumulated in SGs and mass spectrometry confirmed enrichment of m1A in the SG RNAs. The insertion of a single methylation motif in the untranslated region of a reporter RNA leads to more efficient recovery of protein synthesis from that transcript after the return to normal temperature. Our results demonstrate far-reaching functional consequences of a minimal RNA modification on N1-adenine during acute proteostasis stress.

Keywords: N1-methyladenine; stress granules; stress response.

Figure 1

The m¹A motif is enriched in SG mRNAs. (A) TRMT6/61A methyltrasferase binds free RNAs in heat-shocked cytosol of HeLa cells (Alriquet et al., 2019). (B) tRNA, TΨC arm of tRNA where adenine 58 (A*) is N1-methylated by TRMT6/61A. mRNA, mRNA motif (red) targeted by TRMT6/61A for adenine N1-methylation (Li et al., 2017). This study, motif used here to predict mRNA targets of TRMT6/61A-mediated adenine N1-methylation. (C) Fraction of mRNAs enriched or depleted in SGs (Khong et al., 2017) and containing the m¹A motif: 150 out of 1434 and 60 out of 1353, respectively. Control, mRNA set neither enriched nor depleted in SG; ***P < 0.001, chi-square analysis. (D) The number of m¹A motifs in the mRNAs analyzed in Figure 1C. (E) Cumulative fraction of mRNAs at different transcript size cutoff.

Figure 2

Partial knockout of TRMT6/61A sensitizes cells to heat shock and arsenite stress. (A) TRMT61A amount determined by western blotting. GAPDH was used as loading control. WT, wild-type HeLa cells; pKOi, partial knockout with intermediate level of TRMT61A; pKO, partial knockout with strong reduction of TRMT61A level. **P < 0.01, ***P < 0.001, two-tailed t-test; N = 3 independent experiments (mean + SD). (B) High-pressure liquid chromatography (HPLC) analysis of N1-methylated adenosine (m¹A) from cellular tRNAs in wild-type HeLa cells (WT) and TRMT61A partial knockout cells (pKO). One representative out of three independent experiments is shown. (C) Reduced amounts of the methyltransferase correlate with the increased sensitivity to heat shock. WT, wild-type cells with normal amount of TRMT61A; pKO and pKOi, partial knockout cells with strongly reduced and intermediate amounts of TRMT61A, respectively. *P < 0.05, two-tailed t-test; N = 3 independent experiments (mean + SD). (D) Viability of TRMT61A knockout cells after arsenite treatment for 1 h in comparison (%) to wild-type cells at the same concentrations of arsenite. *P < 0.05, **P < 0.01, two-tailed t-test; #, not significant difference. N = 3 independent experiments (mean + SD).

Figure 3

TRMT6/61A is involved in stressed-induced granulation. (A) Immunofluorescence staining of TIAR (red) to detect SG formation upon arsenite treatment for 30 min in serum-free medium. DAPI staining (blue), nuclei. Scale bar, 20 μm. WT, wild-type HeLa cells; pKO, partial knockout of TRMT61A. *P < 0.05, two-tailed t-test; N = 3 independent experiments (mean + SD). (B) Localization of TRMT6/61A in arsenite-induced SGs that are detected with anti-TIAR antibody. A representative image from three independent experiments. Scale bar, 20 μm. The white bar in the lower picture indicates the line where the fluorescence rel. intensity extracted. Red, TIAR; green, TRMT6/61A.

Figure 4

m¹A accumulates in SGs. (A) Extracted ion chromatograms of indicated m/z with a range of ±0.002 Da. Top, a mixture of four standard ribonucleosides plus N1-methyladenosine and N6-methyladenosine; middle, N6-methyadenosine only; bottom, N1-methyladenosine only. A, adenosine. *, Dimroth rearrangement of m¹A to m⁶A. (B) The fraction of methylated adenine in SG RNAs (SG) is increased compared to that in the cytosolic mRNA pool (cytosol) as measured by selective ion monitoring MS. **P < 0.01, two-tailed t-test. N = 3 independent experiments (mean + SD). (C) m¹A enriched in SG RNA is stronger than m⁶A. m¹A, fraction of N1-methyladenosine over unmodified adenosine; m⁶A, fraction of N6-methyladenosine over unmodified adenosine. The fractions of m¹A and m⁶A in cytosolic mRNAs were set as 1. ***P < 0.001, two-tailed t-test; N = 3 independent experiments (mean + SD).

Figure 5

m¹A safeguards mRNAs during heat shock. (A) Schematic depiction of the m¹A motif-containing reporter. UTR, untranslated region. The position of the motif on the transcript is indicated. The nucleotide sequence of the motif is shown in Supplementary Figure S5B. (B) Accumulation of UbE from WT-UbE and MUT-UbE transcripts during 3 h-long inhibition of proteasomal degradation with 20 μM MG132 (+MG132). One representative anti-EGFP western blot from three independent experiments is shown. GAPDH was used as loading control. #, not significant difference; two-tailed t-test; N = 4 independent experiments (mean + SD). (C) Accumulation of UbE protein from WT-UbE and MUT-UbE transcripts during recovery after heat shock in the presence of actinomycin D to block synthesis of new mRNA molecules. UbE amount at time point 0 was subtracted from all values during recovery. One representative anti-EGFP western blot is shown. GAPDH was used as loading control. ***P < 0.001, **P < 0.01, two-tailed t-test; N = 4 independent experiments (mean + SD).

Figure 6

Different fates of free RNA during proteostasis stress. Polysome disassembly during stress releases free RNA into cytosol. The aberrant RNA–RNA and RNA–protein associations might lead to irreversible co-aggregates. We hypothesize that an orderly and reversible sequestration of free RNAs into RNA–protein granules has evolved to counteract the irreversible aggregation.