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, 45 (10), 6051-6063

N6-methyladenosine Alters RNA Structure to Regulate Binding of a Low-Complexity Protein

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N6-methyladenosine Alters RNA Structure to Regulate Binding of a Low-Complexity Protein

Nian Liu et al. Nucleic Acids Res.

Abstract

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA), and affects almost every stage of the mRNA life cycle. The YTH-domain proteins can specifically recognize m6A modification to control mRNA maturation, translation and decay. m6A can also alter RNA structures to affect RNA-protein interactions in cells. Here, we show that m6A increases the accessibility of its surrounding RNA sequence to bind heterogeneous nuclear ribonucleoprotein G (HNRNPG). Furthermore, HNRNPG binds m6A-methylated RNAs through its C-terminal low-complexity region, which self-assembles into large particles in vitro. The Arg-Gly-Gly repeats within the low-complexity region are required for binding to the RNA motif exposed by m6A methylation. We identified 13,191 m6A sites in the transcriptome that regulate RNA-HNRNPG interaction and thereby alter the expression and alternative splicing pattern of target mRNAs. Low-complexity regions are pervasive among mRNA binding proteins. Our results show that m6A-dependent RNA structural alterations can promote direct binding of m6A-modified RNAs to low-complexity regions in RNA binding proteins.

Figures

Figure 1.
Figure 1.
HNRNPG preferentially binds an m6A-modified hairpin in MALAT1. (A) Secondary structure of the 34-nt hairpin derived from positions 2,505–2,538 of MALAT1, including the m6A site at position 2,515. The methylated form of the hairpin is termed 2,515-m6A and the unmethylated form is termed 2,515-A. (B) Gel shift showing binding of HeLa nuclear extract to the MALAT1 hairpin in both its unmethylated (2,515-A) and methylated (2,515-m6A) forms. (C) Left: denaturing gel of the proteins pulled down by the unmethylated and methylated MALAT1 hairpins. In the control, no RNA was used as bait. Right: quantification of relative HNRNPG pull-down with the unmethylated and methylated hairpins, normalized to pulled-down Histone H1.2 (HIST1H1C). Data shown as mean; error bar = standard deviation; n = 4 biological replicates.
Figure 2.
Figure 2.
HNRNPG uses a low-complexity region to bind the MALAT1 hairpin. (A) Domain structure of HNRNPG, including an N-terminal RNA recognition motif (RRM) and an SRGP-rich low-complexity region, which contains the nascent transcripts targeting domain (NTD) and a C-terminal RNA binding domain (RBD). (B) Electron microscopy images of the N-terminal RRM (N-RRM) and C-terminal RBD (C-RBD) of HNRNPG at 5 μM concentration. C-RBD aggregates are marked by arrows. (C) Gel shift showing the ribonucleoprotein (RNP) complexes that form upon binding of the C-RBD of HNRNPG (0–20 μM) to the unmethylated and methylated MALAT1 hairpins. The free RNA is not shown, as it has run much farther down the gel. Top: 32P-labeled RNA gel; bottom: same gel stained for protein. (D) Ultraviolet cross-linking of the HNRNPG C-RBD, C-RBD mutant and N-RRM (0–5 μM) to the unmethylated and methylated MALAT1 hairpins. In the C-RBD mutant, all three RGG repeats in the C-RBD were mutated to FGG repeats.
Figure 3.
Figure 3.
m6A alters RNA structure to recruit HNRNPG. (A) Sequence logo of the most enriched motif within HNRNPG PAR-CLIP peaks. (B) Left: secondary structure of the MALAT1 hairpin, showing the A-2,515-to-G/C/U mutations that were introduced at the m6A site. Right: quantification of relative HNRNPG pull-down with the original (2,515-A) and mutated (2,515-G/C/U) MALAT1 hairpins, normalized to pulled-down HIST1H1C. Data shown as mean; error bar = standard deviation; n = 3 biological replicates. (C) Left: structural probing of the unmethylated and methylated MALAT1 hairpins. The orange lines indicate regions with marked differences between the unmethylated and methylated hairpins. The location of the m6A residue is indicated by a red dot. Ctrl, no nuclease added; V1; RNase V1 digestion; S1, S1 nuclease digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. Right: secondary structure of the unmethylated and methylated MALAT1 hairpins, marked at their S1 nuclease (red lines) and V1 nuclease (green lines) cleavage sites. (D) Model showing that m6A disrupts RNA structure, exposes a motif that includes the m6A site, and recruits an RNA binding protein.
Figure 4.
Figure 4.
HNRNPG binds m6A-modified RNAs transcriptome-wide. (A) PAR-CLIP–MeRIP input and IP (m6A-IP) read counts in a region of the MALAT1 transcript. The red arrowhead indicates the m6A site at position 2,515. (B) Identification of high-confidence HNRNPG-bound m6A sites (purple) as the overlap between m6A-modified HNRNPG binding sites, identified by HNRNPG PAR-CLIP–MeRIP (pink) and m6A methyltransferase-dependent HNRNPG-bound AGRAC sites, identified by HNRNPG PAR-CLIP in m6A methyltransferase (METTL3 and METTL14) knockdown HEK293T cells (blue). (C) Regional distribution of high-confidence HNRNPG-bound m6A sites. (D) Comparison of the structure of AGRAC sequences at high-confidence HNRNPG-bound m6A sites (red) versus random AGRAC sequences (black) in human polyadenylated RNAs, based on parallel analysis of RNA structure (PARS) data (48). The x-axis denotes nucleotide position; the y-axis shows the PARS score. Positive PARS scores indicate double-stranded conformation; negative scores indicate single-stranded conformation. P–value, Mann–Whitney U test.
Figure 5.
Figure 5.
m6A-dependent HNRNPG binding regulates mRNA abundance. (A) Western blot showing depletion of HNRNPG with two different siRNAs (KD1 and KD2) for mRNA-seq experiments. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (B) Number of genes with correlated changes in expression upon HNRNPG knockdown and m6A methyltransferase (METTL3 or METTL14) knockdown. HCG-m6A, high-confidence HNRNPG-bound m6A site. mRNA-seq data from HNRNPU knockdown HEK293T cells (Gene Expression Omnibus, GSE34995 (49)) were analyzed as a control. (C and D) mRNA-seq reads for SPOPL (speckle-type POZ protein-like) transcripts in control, HNRNPG knockdown (C) and m6A methyltransferase knockdown (D) cells. The arrowhead indicates the m6A site.
Figure 6.
Figure 6.
m6A-dependent HNRNPG binding regulates alternative splicing. (A) Splicing changes of annotated differentially expressed exons upon HNRNPG knockdown with siRNA KD2 (x-axis) and m6A methyltransferase (METTL3 or METTL14) knockdown (y-axis), by log ratio of normalized counts relative to control knockdown, log2 (KD/Control). Pearson's correlation coefficient r and P-values are shown for each panel. (B) Number of exons for which changes in exon usage are correlated upon HNRNPG, HNRNPC, and/or m6A methyltransferase (METTL3 or METTL14) knockdown. For HNRNPG knockdown, only exons in genes with high-confidence HNRNPG-bound m6A sites were counted. For HNRNPC knockdown, only exons in genes with high-confidence m6A-switches were counted (20). (C and D) mRNA-seq reads for NASP transcripts in control, HNRNPG knockdown (C) and m6A methyltransferase knockdown (D) cells. The yellow arrowhead indicates the alternatively spliced exon; the red arrowhead indicates the m6A site. (E) Reverse transcription and PCR (RT-PCR) validating differential exon usage in NASP. Data shown as mean; error bar = standard deviation; n = 3 biological replicates.

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