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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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Global m6A reduction decreases hnRNP C binding at m6A-switchesa, Density plot showing negative enrichment at the U-tracts. b, Identification of HCS m6A-switches. c, Region distribution of HCS m6A-switches. d, Density plot showing m6A-switches distribution relative to exon/intron boundaries. e, m6A-switches in coding RNA were enriched in the 3’UTR and near the stop codon. f, Cumulative distribution of HCS m6A-switches (black) and control (orange) regarding the S1/V1 cleavage preference (data from4) at U-tracts and RRACH motif. U-tract can be 3’ (upper) or 5’ (lower) of the RRACH motif. *: p < 0.05, **: p < 10−4, Kolmogorov-Smirnov test. g, Phylogenetic conservation of HCS m6A-switches among primates and vertebrates. ***: p < 10−16, Mann-Whitney-Wilcoxon test.
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Figure 3: Global m6A reduction decreases hnRNP C binding at m6A-switchesa, Density plot showing negative enrichment at the U-tracts. b, Identification of HCS m6A-switches. c, Region distribution of HCS m6A-switches. d, Density plot showing m6A-switches distribution relative to exon/intron boundaries. e, m6A-switches in coding RNA were enriched in the 3’UTR and near the stop codon. f, Cumulative distribution of HCS m6A-switches (black) and control (orange) regarding the S1/V1 cleavage preference (data from4) at U-tracts and RRACH motif. U-tract can be 3’ (upper) or 5’ (lower) of the RRACH motif. *: p < 0.05, **: p < 10−4, Kolmogorov-Smirnov test. g, Phylogenetic conservation of HCS m6A-switches among primates and vertebrates. ***: p < 10−16, Mann-Whitney-Wilcoxon test.

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)

Global m6A reduction decreases hnRNP C binding at m6A-switchesa, Density plot showing negative enrichment at the U-tracts. b, Identification of HCS m6A-switches. c, Region distribution of HCS m6A-switches. d, Density plot showing m6A-switches distribution relative to exon/intron boundaries. e, m6A-switches in coding RNA were enriched in the 3’UTR and near the stop codon. f, Cumulative distribution of HCS m6A-switches (black) and control (orange) regarding the S1/V1 cleavage preference (data from4) at U-tracts and RRACH motif. U-tract can be 3’ (upper) or 5’ (lower) of the RRACH motif. *: p < 0.05, **: p < 10−4, Kolmogorov-Smirnov test. g, Phylogenetic conservation of HCS m6A-switches among primates and vertebrates. ***: p < 10−16, Mann-Whitney-Wilcoxon test.
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Figure 3: Global m6A reduction decreases hnRNP C binding at m6A-switchesa, Density plot showing negative enrichment at the U-tracts. b, Identification of HCS m6A-switches. c, Region distribution of HCS m6A-switches. d, Density plot showing m6A-switches distribution relative to exon/intron boundaries. e, m6A-switches in coding RNA were enriched in the 3’UTR and near the stop codon. f, Cumulative distribution of HCS m6A-switches (black) and control (orange) regarding the S1/V1 cleavage preference (data from4) at U-tracts and RRACH motif. U-tract can be 3’ (upper) or 5’ (lower) of the RRACH motif. *: p < 0.05, **: p < 10−4, Kolmogorov-Smirnov test. g, Phylogenetic conservation of HCS m6A-switches among primates and vertebrates. ***: p < 10−16, Mann-Whitney-Wilcoxon test.
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