RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release

Abstract

Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m⁵C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m⁵C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.

Keywords: 5-methylcytosine RNA modification; CRISPR screening; RNA stability; RNA-binding protein; innate immunity; mitochondrial double-stranded RNA.

Figure 1.. mt-RBP CRISPR screening reveals NSUN4 as a pivotal regulator of mt-dsRNAs.

(A) Experimental scheme of the CRISPR screening. Eighty-nine mt-RBPs were individually downregulated using CRISPR, and extracted RNAs were analyzed by strand-specific RT-qPCR to assess the expression of both mt-mRNAs and their complementary lncRNAs. (B) Strand-specific and total mtRNA expression when the indicated mt-RBP was suppressed (n = 3). Each sample was normalized to cells transduced with sgNC. Due to decreased cell viability, siRNAs were used for GADD45GIP1, TACO1, TFAM, TRAP1, DDX20, and C1QBP (marked in blue). Group 1, increased both strand RNAs; Group 2, decreased both strand RNAs; Group 3, increased H-strand RNAs and decreased L-strand RNAs; Group 4, increased L-strand RNAs; Group 5, others. (C) Validation of the screening results using immunocytochemistry with anti-dsRNA (J2) antibody. The target genes were marked in green in (B). Scale bar indicates 50 μm. Quantification is shown on the right (n = 4). (D and E) mtRNA expression in NSUN4-deficient cells analyzed by strand-specific RT-qPCR (D, n = 3) and RNA-FISH (E). Scale bar, 50 μm. All error bars denote s.e.m. Statistical significance was calculated using one-tailed Student’s t-tests, n.s. not significant, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. See also Figure S1.

Figure 2.. Regulation of mtRNA stability by NSUN4.

(A) Expression levels of mtRNAs upon IMT1 treatment over time (n = 3). (B) Stability of H- and L-strands of ND5 and ND6 in cells transduced with sgNC and sgNSUN4 (n = 3). (C) NSUN4 protein expression following transfection of plasmids with WT or catalytic mutant NSUN4 tagged with EGFP into NSUN4-deficient cells. (D) Comparison of dsRNA expression after transfection of WT or catalytic mutant NSUN4 into NSUN4-deficient cells using immunocytochemistry with J2 antibody. Quantification is shown on the bottom (n = 3). Scale bar indicates 20 μm. (E) Strand-specific RT-qPCR analysis of H- and L-strands of mtRNAs after transfection of WT or catalytic mutant NSUN4 into NSUN4-deficient cells (n = 3). All error bars denote s.e.m. Statistical significance was calculated using one-tailed Student’s t-tests, n.s. not significant, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. See also Figure S2.

Figure 3.. m⁵C modification of mtRNAs by NSUN4.

(A) Scheme of bisulfite conversion. (B) Validation of bisulfite conversion using 28S rRNA through Sanger sequencing. (C and D) The overall methylation level of H- and L-strands of mtRNAs (C; Median, 6.45%) and of mRNAs encoded by the nuclear genome (D; Median, 4.81%). (E) The m⁵C enrichment of mtRNAs in sgNC or sgNSUN4-transduced cells analyzed by immunoprecipitation using anti-m⁵C antibody (n = 3). (F) The relative methylation level of mtRNAs in sgNC and sgNSUN4-transduced cells from Bis-seq (n = 2). The statistical significance was calculated using one-tailed paired Student’s t-tests. (G) A gene structure of ND5 L-strand and accumulation of Bis-seq reads at indicated methylation sites. (H) The m⁵C distribution along the RNA analyzed based on NSUN4-dependency. (I) Comparison of MFEs between NSUN4-dependent or independent m⁵C sites on mtRNAs with two different flanking lengths. The modified cytosine is positioned at the center of the analyzed sequences. All error bars denote s.e.m. Box plot features: center line, median; box limits, upper and lower quartiles; whiskers, 1.5x interquartile range; points, individual data. All statistical significances were calculated using one-tailed Student’s t-tests, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. See also Figure S3.

Figure 4.. Degradation of m⁵C-modified mtRNAs by C1QBP and PNPT1.

(A) Replot of an m⁵C interactome from a published paper. (B) Analysis of the interaction of C1QBP with synthetic mtRNAs containing m⁵C modification using biotin-streptavidin immunoprecipitation. (C) Analysis of mtRNAs pulled down with C1QBP in sgNSUN4-transduced cells normalized to that in sgNC-transduced cells (n = 3). (D) Bis-seq analysis on NSUN4-dependent methylation sites in siLuc or siC1QBP transfected cells (n = 2). (E) The strand-specific mtRNA expression in C1QBP downregulated cells (n = 3). (F) Stability analysis of selected mtRNAs in C1QBP- or PNPT1-deficient cells (n = 3). (G and H) Co-immunoprecipitation analysis for C1QBP-PNPT1 interaction in the presence (G) or absence (H) of nucleic acids. MNase was treated to digest nucleic acids. (I) Bis-seq analysis on NSUN4-dependent methylation sites in siLuc or siPNPT1 transfected cells (n = 3). All error bars denote s.e.m. All statistical significances were calculated using one-tailed Student’s t-tests, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. See also Figures S4 and S5.

Figure 5.. Cytosolic release and immune activation by m⁵C-modified mtRNAs.

(A) The expression of cytosolic mtRNAs in C1QBP-deficient cells (n = 3). (B and C) Analysis of innate immune response pathways through western blotting of key dsRNA sensors (B) and RT-qPCR of selected ISGs (C, n = 3) in PNPT1, C1QBP, or NSUN4-depleted cells. (D and E) Immune response pathways through western blotting of key dsRNA sensors (D) and RT-qPCR of selected ISGs (E, n = 3) in PNPT1, C1QBP, or NSUN4-depleted cells after depleting mtRNAs with 2-CM treatment. (F) The expression of cytosolic mtRNAs in NSUN4-deficient cells (n = 3). (G) Schematics of the m⁵C-modified RNA degradation and cytosolic release model via NSUN4-C1QBP-PNPT1 axis. All error bars denote s.e.m. Statistical significances were calculated using one-tailed Student’s t-tests, n.s. not significant, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001. See also Figure S6.