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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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m6A alters RNA structure to enhance hnRNP C bindinga, m6A increases binding with nuclear proteins. b, RNA pull down showing hnRNP C preferably binds methylated RNA. n = 4, ± s.d., biological replicates. c, Filter binding showing m6A increases hnRNP C1 binding with respective apparent dissociation constant (Kd) indicated at lower right; n = 3, ± s.d., technical replicates. d, RNA structural probing showing m6A disrupts local RNA structure highlighted in yellow. Ctrl, no nuclease added; V1, RNase V1 digestion; S1, nuclease S1 digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. The orange bars mark the structurally altered RNA regions in the presence of m6A (red dot). e, RNA pull down with mutated oligos. n = 3, ± s.d., technical replicates. f, Illustration of the m6A-switch model.
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Figure 1: m6A alters RNA structure to enhance hnRNP C bindinga, m6A increases binding with nuclear proteins. b, RNA pull down showing hnRNP C preferably binds methylated RNA. n = 4, ± s.d., biological replicates. c, Filter binding showing m6A increases hnRNP C1 binding with respective apparent dissociation constant (Kd) indicated at lower right; n = 3, ± s.d., technical replicates. d, RNA structural probing showing m6A disrupts local RNA structure highlighted in yellow. Ctrl, no nuclease added; V1, RNase V1 digestion; S1, nuclease S1 digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. The orange bars mark the structurally altered RNA regions in the presence of m6A (red dot). e, RNA pull down with mutated oligos. n = 3, ± s.d., technical replicates. f, Illustration of the m6A-switch model.

Mentions: Post-transcriptional m6A RNA modification is indispensable for cell viability and development, yet its functional mechanisms are still poorly understood8-19. We recently identified one m6A site in a hairpin-stem on the human lncRNA MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript)25 (Extended Data Fig. 1a). Native gel shift assay indicated that this m6A residue increases the interaction of this RNA hairpin with proteins in the HeLa nuclear extract (Fig. 1a). RNA pull down assays identified heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) as the protein component of the nuclear extract that binds more strongly with the m6A-modified hairpin (Fig. 1b and Extended Data Fig. 1b, c). Stronger binding of the methylated hairpin was validated qualitatively by UV crosslinking and quantitatively (~8-fold increase) by filter-binding using recombinant hnRNP C1 protein (Fig. 1c and Extended Data Fig. 1d).


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)

m6A alters RNA structure to enhance hnRNP C bindinga, m6A increases binding with nuclear proteins. b, RNA pull down showing hnRNP C preferably binds methylated RNA. n = 4, ± s.d., biological replicates. c, Filter binding showing m6A increases hnRNP C1 binding with respective apparent dissociation constant (Kd) indicated at lower right; n = 3, ± s.d., technical replicates. d, RNA structural probing showing m6A disrupts local RNA structure highlighted in yellow. Ctrl, no nuclease added; V1, RNase V1 digestion; S1, nuclease S1 digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. The orange bars mark the structurally altered RNA regions in the presence of m6A (red dot). e, RNA pull down with mutated oligos. n = 3, ± s.d., technical replicates. f, Illustration of the m6A-switch model.
© Copyright Policy - premission-link
Related In: Results  -  Collection

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Figure 1: m6A alters RNA structure to enhance hnRNP C bindinga, m6A increases binding with nuclear proteins. b, RNA pull down showing hnRNP C preferably binds methylated RNA. n = 4, ± s.d., biological replicates. c, Filter binding showing m6A increases hnRNP C1 binding with respective apparent dissociation constant (Kd) indicated at lower right; n = 3, ± s.d., technical replicates. d, RNA structural probing showing m6A disrupts local RNA structure highlighted in yellow. Ctrl, no nuclease added; V1, RNase V1 digestion; S1, nuclease S1 digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. The orange bars mark the structurally altered RNA regions in the presence of m6A (red dot). e, RNA pull down with mutated oligos. n = 3, ± s.d., technical replicates. f, Illustration of the m6A-switch model.
Mentions: Post-transcriptional m6A RNA modification is indispensable for cell viability and development, yet its functional mechanisms are still poorly understood8-19. We recently identified one m6A site in a hairpin-stem on the human lncRNA MALAT1 (Metastasis Associated Lung Adenocarcinoma Transcript)25 (Extended Data Fig. 1a). Native gel shift assay indicated that this m6A residue increases the interaction of this RNA hairpin with proteins in the HeLa nuclear extract (Fig. 1a). RNA pull down assays identified heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNP C) as the protein component of the nuclear extract that binds more strongly with the m6A-modified hairpin (Fig. 1b and Extended Data Fig. 1b, c). Stronger binding of the methylated hairpin was validated qualitatively by UV crosslinking and quantitatively (~8-fold increase) by filter-binding using recombinant hnRNP C1 protein (Fig. 1c and Extended Data Fig. 1d).

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
Related in: MedlinePlus