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N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions.

Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T - Nature (2015)

Bottom Line: We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing.We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation.Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs). However, RBMs can be buried within their local RNA structures, thus inhibiting RNA-protein interactions. N(6)-methyladenosine (m(6)A), the most abundant and dynamic internal modification in eukaryotic messenger RNA, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs, but how m(6)A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m(6)A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m(6)A-switch'. We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m(6)A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m(6)A-switches among HNRNPC-binding sites; and global m(6)A reduction decreased HNRNPC binding at 2,798 high-confidence m(6)A-switches. We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.

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Molecular features of HCS m6A-switchesa, Western blot showing stable hnRNP C protein abundance upon METTL3/L14 KD. b, Volcano plot of the METTL3/L14 KD data depicting RRACH-U-tracts coupling events (unfilled red circles) as defined in Extended Data Fig. 4b, according to their p-values39 (P, y-axis) and fold change values at the U-tracts (E, x-axis). c, Overlap of RRACH-U-tract coupling events with decreased hnRNP C binding by METTL3 and METTL14 KD. d, The intron fraction of HCS m6A-switches in coding RNA and non-coding RNA. e, Density plot displaying the distribution of exonic m6A-switches/HNRNPC PAR-CLIP peaks according to exon length. f, Inter-motif (RRACH-U-tract) distance distributions suggest that m6A-switches have a preference for shorter distances between the RRACH and U-tract (> 5xU) motifs. The distribution curves are from PARCLIP-MeRIP data (green), METTL3/L14 KD (red) and HCS m6A-switches (black). g, Analysis of the inter-motif (U-tract-U-tract) distance patterns, previously identified by iCLIP20, in PARCLIP-MeRIP, METTL3/L14 KD and HCS m6A-switch data. The peaks at ~165 and ~300 nucleotides are clearly present. For the 2,798 high confident switches, we analyzed those in which the other U-tract motif is also in a PAR-CLIP-identified sequence; the long-range peaks seem to have shifted to longer distances (~220 and ~370 nucleotides). h,METTL3/L14 KD does not affect the inter-motif (U-tract-U-tract) distance distributions for U-tracts (≥5x U) in HEK293T cells. i, EVOfold analysis for the 2,798 HCS m6A-switches. The chances for HCS m6A-switches to have EVOfold records are significantly higher than random genomic sequences. We first calculated the number of HCS sites in the EVO database if occurring in random to be ~1.7 HCS sites. We found 18 HCS sites are present in EVO database, resulting in ~11x enrichment. This result is further divided into intronic and exonic regions.
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Figure 10: Molecular features of HCS m6A-switchesa, Western blot showing stable hnRNP C protein abundance upon METTL3/L14 KD. b, Volcano plot of the METTL3/L14 KD data depicting RRACH-U-tracts coupling events (unfilled red circles) as defined in Extended Data Fig. 4b, according to their p-values39 (P, y-axis) and fold change values at the U-tracts (E, x-axis). c, Overlap of RRACH-U-tract coupling events with decreased hnRNP C binding by METTL3 and METTL14 KD. d, The intron fraction of HCS m6A-switches in coding RNA and non-coding RNA. e, Density plot displaying the distribution of exonic m6A-switches/HNRNPC PAR-CLIP peaks according to exon length. f, Inter-motif (RRACH-U-tract) distance distributions suggest that m6A-switches have a preference for shorter distances between the RRACH and U-tract (> 5xU) motifs. The distribution curves are from PARCLIP-MeRIP data (green), METTL3/L14 KD (red) and HCS m6A-switches (black). g, Analysis of the inter-motif (U-tract-U-tract) distance patterns, previously identified by iCLIP20, in PARCLIP-MeRIP, METTL3/L14 KD and HCS m6A-switch data. The peaks at ~165 and ~300 nucleotides are clearly present. For the 2,798 high confident switches, we analyzed those in which the other U-tract motif is also in a PAR-CLIP-identified sequence; the long-range peaks seem to have shifted to longer distances (~220 and ~370 nucleotides). h,METTL3/L14 KD does not affect the inter-motif (U-tract-U-tract) distance distributions for U-tracts (≥5x U) in HEK293T cells. i, EVOfold analysis for the 2,798 HCS m6A-switches. The chances for HCS m6A-switches to have EVOfold records are significantly higher than random genomic sequences. We first calculated the number of HCS sites in the EVO database if occurring in random to be ~1.7 HCS sites. We found 18 HCS sites are present in EVO database, resulting in ~11x enrichment. This result is further divided into intronic and exonic regions.

Mentions: To assess the effect of global m6A reduction on RNA-hnRNP C interactions, we performed hnRNP C PAR-CLIP experiments in METTL3 and METTL14 knockdown (KD) cells (Extended Data Fig. 6a). We identified 16,582 coupling events with decreased U-tracts-hnRNP C interactions upon METTL3 KD and METTL14 KD (METTL3/L14 KD) with significant overlaps at FDR ≤5% (Fig. 3a and Extended Data Fig. 6b, c). In total, 2,798 m6A-switches identified by PARCLIP-MeRIP experiments showed decreased hnRNP C binding upon METTL3/L14 KD (Fig. 3b) and this number is likely under-estimated due to the fact that METTL3/L14 KD reduces the global m6A level by only ~30-40% 11,12. These sites composed the high confidence m6A-switches (HCS) that were used for subsequent analysis.


N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions.

Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T - Nature (2015)

Molecular features of HCS m6A-switchesa, Western blot showing stable hnRNP C protein abundance upon METTL3/L14 KD. b, Volcano plot of the METTL3/L14 KD data depicting RRACH-U-tracts coupling events (unfilled red circles) as defined in Extended Data Fig. 4b, according to their p-values39 (P, y-axis) and fold change values at the U-tracts (E, x-axis). c, Overlap of RRACH-U-tract coupling events with decreased hnRNP C binding by METTL3 and METTL14 KD. d, The intron fraction of HCS m6A-switches in coding RNA and non-coding RNA. e, Density plot displaying the distribution of exonic m6A-switches/HNRNPC PAR-CLIP peaks according to exon length. f, Inter-motif (RRACH-U-tract) distance distributions suggest that m6A-switches have a preference for shorter distances between the RRACH and U-tract (> 5xU) motifs. The distribution curves are from PARCLIP-MeRIP data (green), METTL3/L14 KD (red) and HCS m6A-switches (black). g, Analysis of the inter-motif (U-tract-U-tract) distance patterns, previously identified by iCLIP20, in PARCLIP-MeRIP, METTL3/L14 KD and HCS m6A-switch data. The peaks at ~165 and ~300 nucleotides are clearly present. For the 2,798 high confident switches, we analyzed those in which the other U-tract motif is also in a PAR-CLIP-identified sequence; the long-range peaks seem to have shifted to longer distances (~220 and ~370 nucleotides). h,METTL3/L14 KD does not affect the inter-motif (U-tract-U-tract) distance distributions for U-tracts (≥5x U) in HEK293T cells. i, EVOfold analysis for the 2,798 HCS m6A-switches. The chances for HCS m6A-switches to have EVOfold records are significantly higher than random genomic sequences. We first calculated the number of HCS sites in the EVO database if occurring in random to be ~1.7 HCS sites. We found 18 HCS sites are present in EVO database, resulting in ~11x enrichment. This result is further divided into intronic and exonic regions.
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Figure 10: Molecular features of HCS m6A-switchesa, Western blot showing stable hnRNP C protein abundance upon METTL3/L14 KD. b, Volcano plot of the METTL3/L14 KD data depicting RRACH-U-tracts coupling events (unfilled red circles) as defined in Extended Data Fig. 4b, according to their p-values39 (P, y-axis) and fold change values at the U-tracts (E, x-axis). c, Overlap of RRACH-U-tract coupling events with decreased hnRNP C binding by METTL3 and METTL14 KD. d, The intron fraction of HCS m6A-switches in coding RNA and non-coding RNA. e, Density plot displaying the distribution of exonic m6A-switches/HNRNPC PAR-CLIP peaks according to exon length. f, Inter-motif (RRACH-U-tract) distance distributions suggest that m6A-switches have a preference for shorter distances between the RRACH and U-tract (> 5xU) motifs. The distribution curves are from PARCLIP-MeRIP data (green), METTL3/L14 KD (red) and HCS m6A-switches (black). g, Analysis of the inter-motif (U-tract-U-tract) distance patterns, previously identified by iCLIP20, in PARCLIP-MeRIP, METTL3/L14 KD and HCS m6A-switch data. The peaks at ~165 and ~300 nucleotides are clearly present. For the 2,798 high confident switches, we analyzed those in which the other U-tract motif is also in a PAR-CLIP-identified sequence; the long-range peaks seem to have shifted to longer distances (~220 and ~370 nucleotides). h,METTL3/L14 KD does not affect the inter-motif (U-tract-U-tract) distance distributions for U-tracts (≥5x U) in HEK293T cells. i, EVOfold analysis for the 2,798 HCS m6A-switches. The chances for HCS m6A-switches to have EVOfold records are significantly higher than random genomic sequences. We first calculated the number of HCS sites in the EVO database if occurring in random to be ~1.7 HCS sites. We found 18 HCS sites are present in EVO database, resulting in ~11x enrichment. This result is further divided into intronic and exonic regions.
Mentions: To assess the effect of global m6A reduction on RNA-hnRNP C interactions, we performed hnRNP C PAR-CLIP experiments in METTL3 and METTL14 knockdown (KD) cells (Extended Data Fig. 6a). We identified 16,582 coupling events with decreased U-tracts-hnRNP C interactions upon METTL3 KD and METTL14 KD (METTL3/L14 KD) with significant overlaps at FDR ≤5% (Fig. 3a and Extended Data Fig. 6b, c). In total, 2,798 m6A-switches identified by PARCLIP-MeRIP experiments showed decreased hnRNP C binding upon METTL3/L14 KD (Fig. 3b) and this number is likely under-estimated due to the fact that METTL3/L14 KD reduces the global m6A level by only ~30-40% 11,12. These sites composed the high confidence m6A-switches (HCS) that were used for subsequent analysis.

Bottom Line: We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing.We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation.Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, The University of Chicago, Chicago, Illinois 60637, USA.

ABSTRACT
RNA-binding proteins control many aspects of cellular biology through binding single-stranded RNA binding motifs (RBMs). However, RBMs can be buried within their local RNA structures, thus inhibiting RNA-protein interactions. N(6)-methyladenosine (m(6)A), the most abundant and dynamic internal modification in eukaryotic messenger RNA, can be selectively recognized by the YTHDF2 protein to affect the stability of cytoplasmic mRNAs, but how m(6)A achieves its wide-ranging physiological role needs further exploration. Here we show in human cells that m(6)A controls the RNA-structure-dependent accessibility of RBMs to affect RNA-protein interactions for biological regulation; we term this mechanism 'the m(6)A-switch'. We found that m(6)A alters the local structure in mRNA and long non-coding RNA (lncRNA) to facilitate binding of heterogeneous nuclear ribonucleoprotein C (HNRNPC), an abundant nuclear RNA-binding protein responsible for pre-mRNA processing. Combining photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and anti-m(6)A immunoprecipitation (MeRIP) approaches enabled us to identify 39,060 m(6)A-switches among HNRNPC-binding sites; and global m(6)A reduction decreased HNRNPC binding at 2,798 high-confidence m(6)A-switches. We determined that these m(6)A-switch-regulated HNRNPC-binding activities affect the abundance as well as alternative splicing of target mRNAs, demonstrating the regulatory role of m(6)A-switches on gene expression and RNA maturation. Our results illustrate how RNA-binding proteins gain regulated access to their RBMs through m(6)A-dependent RNA structural remodelling, and provide a new direction for investigating RNA-modification-coded cellular biology.

Show MeSH