2017 Jun 2
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
Nucleic Acids Res
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.
© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
HNRNPG preferentially binds an m
6A-modified hairpin in MALAT1. ( A) Secondary structure of the 34-nt hairpin derived from positions 2,505–2,538 of MALAT1, including the m 6A site at position 2,515. The methylated form of the hairpin is termed 2,515-m 6A 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-m 6A) 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.
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.
6A 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 m 6A 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 m 6A 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 m 6A disrupts RNA structure, exposes a motif that includes the m 6A site, and recruits an RNA binding protein.
HNRNPG binds m
6A-modified RNAs transcriptome-wide. ( A) PAR-CLIP–MeRIP input and IP (m 6A-IP) read counts in a region of the MALAT1 transcript. The red arrowhead indicates the m 6A site at position 2,515. ( B) Identification of high-confidence HNRNPG-bound m 6A sites (purple) as the overlap between m 6A-modified HNRNPG binding sites, identified by HNRNPG PAR-CLIP–MeRIP (pink) and m 6A methyltransferase-dependent HNRNPG-bound AGRAC sites, identified by HNRNPG PAR-CLIP in m 6A methyltransferase ( METTL3 and METTL14) knockdown HEK293T cells (blue). ( C) Regional distribution of high-confidence HNRNPG-bound m 6A sites. ( D) Comparison of the structure of AGRAC sequences at high-confidence HNRNPG-bound m 6A 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.
6A-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 m 6A methyltransferase ( METTL3 or METTL14) knockdown. HCG-m 6A, high-confidence HNRNPG-bound m 6A 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 m 6A methyltransferase knockdown (D) cells. The arrowhead indicates the m 6A site.
6A-dependent HNRNPG binding regulates alternative splicing. ( A) Splicing changes of annotated differentially expressed exons upon HNRNPG knockdown with siRNA KD2 ( x-axis) and m 6A methyltransferase ( METTL3 or METTL14) knockdown ( y-axis), by log ratio of normalized counts relative to control knockdown, log 2 (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 m 6A methyltransferase ( METTL3 or METTL14) knockdown. For HNRNPG knockdown, only exons in genes with high-confidence HNRNPG-bound m 6A sites were counted. For HNRNPC knockdown, only exons in genes with high-confidence m 6A-switches were counted (20). ( C and D) mRNA-seq reads for NASP transcripts in control, HNRNPG knockdown (C) and m 6A methyltransferase knockdown (D) cells. The yellow arrowhead indicates the alternatively spliced exon; the red arrowhead indicates the m 6A 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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Adenosine / analogs & derivatives
Gene Knockdown Techniques
Heterogeneous-Nuclear Ribonucleoproteins / metabolism
Methyltransferases / antagonists & inhibitors
Methyltransferases / genetics
Nucleic Acid Conformation
Oligoribonucleotides / chemical synthesis
Oligoribonucleotides / chemistry
Oligoribonucleotides / metabolism
RNA, Long Noncoding / metabolism
RNA, Small Interfering / genetics
MALAT1 long non-coding RNA, human
heterogeneous nuclear ribonucleoprotein G, human
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