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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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Increased accessibility of U-tracts enhances hnRNP C bindinga, Structure probing of the 2,577A-to-U mutated MALAT1 hairpin (2,577-U), same annotation as in Fig. 1d. b, Quantification of the RNase V1 cleavage signal for the U-tract region from RNA structural mapping assays as in a. To correct for sample loading difference, each band signal was normalized to the band signal of the 3’ most U of the U-tract. n = 2, technical replicates. c, Filter-binding curves displaying the binding affinities between recombinant hnRNP C1 and 2,577-U/A oligos. n = 3, ± s.d., technical replicates. d, Filter-binding results showing the binding affinities between recombinant hnRNP C1 and four mutated MALAT1 oligos. (i) Mutate G-C to C-C, A2,577: predicted to weaken the hairpin stem and increase hnRNP C binding. Results: binding improved from 722 nM Kd to 142 nM (5-fold); (ii) Mutate G-C to C-C, m6A2,577: in this context of weaker stem, m6A is predicted to confer a smaller effect compared to wild-type hairpin. Result: improved binding only 2-fold instead of 8-fold; (iii) Restore C-C to C-G, A2,577: predicted to restore the hairpin stem and decrease hnRNP C binding compared to C-C mutant. Result: binding decreased by 6.4-fold; (iv) Restore C-C to C-G, m6A2,577: in this context of restored stem, m6A is again predicted to confer increased binding compared to A2,577 hairpin. Result: improved binding by 2.5-fold. n = 3 each, ± s.d., technical replicates. e, RNA alkaline hydrolysis terminal truncation assay showing recombinant hnRNP C1 binding to terminal truncated MALAT1 hairpin oligos (2,577 site m6A methylated or unmethylated). In this assay, 3′ radiolabeled MALAT1 2,577 hairpin oligos were terminal truncated by alkaline hydrolysis into RNA fragments which were then incubated with hnRNP C1 protein followed by filter binding wash steps. The remaining RNA on the filter paper was isolated and analyzed by denaturing gel electrophoresis, as indicated in the lane “C1-bound or C1-B”. “Input” refers to alkaline hydrolysis truncated RNA oligos used for incubation with hnRNP C1; “G-L or G-ladder” was generated from RNase T1 digestion; “Ctrl” refers to the intact MALAT1 hairpin without alkaline hydrolysis truncation. One pair of methylated/unmethylated truncated oligos (CUT1, marked by green arrows) was selected for subsequent biochemical analysis, due to their strong interaction with hnRNP C1. f, RNA terminal truncation assay as in e except 5′ 32P-labeled oligos were used. One pair of methylated/unmethylated truncated oligos (CUT2, marked by green arrows) was selected for subsequent biochemical analysis. g, Structure probing of the CUT1 oligos using RNase V1 and nuclease S1 digestion, same annotation as in Fig. 1e. The red dot marks the m6A site and the red line marks the U-tract region. h, Structure probing of the CUT2 oligos using RNase V1 and nuclease S1 digestion, same annotation as in g. i, Truncated oligos with exposed U-tracts increased hnRNP C binding regardless of m6A. n = 3, ± s.d., technical replicates.
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Figure 6: Increased accessibility of U-tracts enhances hnRNP C bindinga, Structure probing of the 2,577A-to-U mutated MALAT1 hairpin (2,577-U), same annotation as in Fig. 1d. b, Quantification of the RNase V1 cleavage signal for the U-tract region from RNA structural mapping assays as in a. To correct for sample loading difference, each band signal was normalized to the band signal of the 3’ most U of the U-tract. n = 2, technical replicates. c, Filter-binding curves displaying the binding affinities between recombinant hnRNP C1 and 2,577-U/A oligos. n = 3, ± s.d., technical replicates. d, Filter-binding results showing the binding affinities between recombinant hnRNP C1 and four mutated MALAT1 oligos. (i) Mutate G-C to C-C, A2,577: predicted to weaken the hairpin stem and increase hnRNP C binding. Results: binding improved from 722 nM Kd to 142 nM (5-fold); (ii) Mutate G-C to C-C, m6A2,577: in this context of weaker stem, m6A is predicted to confer a smaller effect compared to wild-type hairpin. Result: improved binding only 2-fold instead of 8-fold; (iii) Restore C-C to C-G, A2,577: predicted to restore the hairpin stem and decrease hnRNP C binding compared to C-C mutant. Result: binding decreased by 6.4-fold; (iv) Restore C-C to C-G, m6A2,577: in this context of restored stem, m6A is again predicted to confer increased binding compared to A2,577 hairpin. Result: improved binding by 2.5-fold. n = 3 each, ± s.d., technical replicates. e, RNA alkaline hydrolysis terminal truncation assay showing recombinant hnRNP C1 binding to terminal truncated MALAT1 hairpin oligos (2,577 site m6A methylated or unmethylated). In this assay, 3′ radiolabeled MALAT1 2,577 hairpin oligos were terminal truncated by alkaline hydrolysis into RNA fragments which were then incubated with hnRNP C1 protein followed by filter binding wash steps. The remaining RNA on the filter paper was isolated and analyzed by denaturing gel electrophoresis, as indicated in the lane “C1-bound or C1-B”. “Input” refers to alkaline hydrolysis truncated RNA oligos used for incubation with hnRNP C1; “G-L or G-ladder” was generated from RNase T1 digestion; “Ctrl” refers to the intact MALAT1 hairpin without alkaline hydrolysis truncation. One pair of methylated/unmethylated truncated oligos (CUT1, marked by green arrows) was selected for subsequent biochemical analysis, due to their strong interaction with hnRNP C1. f, RNA terminal truncation assay as in e except 5′ 32P-labeled oligos were used. One pair of methylated/unmethylated truncated oligos (CUT2, marked by green arrows) was selected for subsequent biochemical analysis. g, Structure probing of the CUT1 oligos using RNase V1 and nuclease S1 digestion, same annotation as in Fig. 1e. The red dot marks the m6A site and the red line marks the U-tract region. h, Structure probing of the CUT2 oligos using RNase V1 and nuclease S1 digestion, same annotation as in g. i, Truncated oligos with exposed U-tracts increased hnRNP C binding regardless of m6A. n = 3, ± s.d., technical replicates.

Mentions: Since m6A residues within RNA stems can destabilize the thermo-stability of model RNA duplexes28, we hypothesized that the m6A2,577 residue destabilizes this MALAT1 hairpin-stem to make its opposing U-tract more single-stranded or accessible, thus enhancing its interaction with hnRNP C. We performed several experiments to validate this hypothesis. First, according to the RNA structural probing assays, the m6A-modified hairpin showed significantly increased nuclease S1 digestion (single-stranded specific) at the GAC (A= m6A) motif as well as markedly decreased RNase V1 digestion (double-stranded/stacking specific) at the U-tract opposing the GAC motif (Fig. 1d). The m6A residue markedly destabilized the stacking properties of the region centered around the U-residue that pairs with A/m6A2,577 (Extended data Fig. 1f-g), which was also supported by the increased reactivity between CMCT and the U-tract bases in the presence of m6A (Extended Data Fig. 1h). Second, the A2,577-to-U mutation increased hnRNP C pull down amount from nuclear extract, whereas U-to-C mutations in the U-tract significantly reduced hnRNP C pull down amount regardless of m6A modification (Fig. 1e). Third, the A2,577-to-U mutation increased the accessibility of U-tract and enhanced hnRNP C binding by ~4-fold (Extended data Fig. 2a-c). Binding results with 4 other mutated A/m6A oligos also supported the U-tract with increased accessibility alone being sufficient to enhance hnRNP C binding (Extended data Fig. 2d). Fourth, RNA terminal truncation followed by hnRNP C binding identified two pairs of truncated hairpins with highly accessible U-tracts, which improved hnRNP C binding significantly but independent of the m6A modification (Extended data Fig. 2ei). All these results confirmed that m6A modification can alter its local RNA structure and enhance the accessibility of its base-paired residues or nearby regions to modulate protein binding (Fig. 1f). We term this mechanism that regulates RNA-protein interactions through m6A-dependent RNA structural remodeling as “m6A-switch”.


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)

Increased accessibility of U-tracts enhances hnRNP C bindinga, Structure probing of the 2,577A-to-U mutated MALAT1 hairpin (2,577-U), same annotation as in Fig. 1d. b, Quantification of the RNase V1 cleavage signal for the U-tract region from RNA structural mapping assays as in a. To correct for sample loading difference, each band signal was normalized to the band signal of the 3’ most U of the U-tract. n = 2, technical replicates. c, Filter-binding curves displaying the binding affinities between recombinant hnRNP C1 and 2,577-U/A oligos. n = 3, ± s.d., technical replicates. d, Filter-binding results showing the binding affinities between recombinant hnRNP C1 and four mutated MALAT1 oligos. (i) Mutate G-C to C-C, A2,577: predicted to weaken the hairpin stem and increase hnRNP C binding. Results: binding improved from 722 nM Kd to 142 nM (5-fold); (ii) Mutate G-C to C-C, m6A2,577: in this context of weaker stem, m6A is predicted to confer a smaller effect compared to wild-type hairpin. Result: improved binding only 2-fold instead of 8-fold; (iii) Restore C-C to C-G, A2,577: predicted to restore the hairpin stem and decrease hnRNP C binding compared to C-C mutant. Result: binding decreased by 6.4-fold; (iv) Restore C-C to C-G, m6A2,577: in this context of restored stem, m6A is again predicted to confer increased binding compared to A2,577 hairpin. Result: improved binding by 2.5-fold. n = 3 each, ± s.d., technical replicates. e, RNA alkaline hydrolysis terminal truncation assay showing recombinant hnRNP C1 binding to terminal truncated MALAT1 hairpin oligos (2,577 site m6A methylated or unmethylated). In this assay, 3′ radiolabeled MALAT1 2,577 hairpin oligos were terminal truncated by alkaline hydrolysis into RNA fragments which were then incubated with hnRNP C1 protein followed by filter binding wash steps. The remaining RNA on the filter paper was isolated and analyzed by denaturing gel electrophoresis, as indicated in the lane “C1-bound or C1-B”. “Input” refers to alkaline hydrolysis truncated RNA oligos used for incubation with hnRNP C1; “G-L or G-ladder” was generated from RNase T1 digestion; “Ctrl” refers to the intact MALAT1 hairpin without alkaline hydrolysis truncation. One pair of methylated/unmethylated truncated oligos (CUT1, marked by green arrows) was selected for subsequent biochemical analysis, due to their strong interaction with hnRNP C1. f, RNA terminal truncation assay as in e except 5′ 32P-labeled oligos were used. One pair of methylated/unmethylated truncated oligos (CUT2, marked by green arrows) was selected for subsequent biochemical analysis. g, Structure probing of the CUT1 oligos using RNase V1 and nuclease S1 digestion, same annotation as in Fig. 1e. The red dot marks the m6A site and the red line marks the U-tract region. h, Structure probing of the CUT2 oligos using RNase V1 and nuclease S1 digestion, same annotation as in g. i, Truncated oligos with exposed U-tracts increased hnRNP C binding regardless of m6A. n = 3, ± s.d., technical replicates.
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Figure 6: Increased accessibility of U-tracts enhances hnRNP C bindinga, Structure probing of the 2,577A-to-U mutated MALAT1 hairpin (2,577-U), same annotation as in Fig. 1d. b, Quantification of the RNase V1 cleavage signal for the U-tract region from RNA structural mapping assays as in a. To correct for sample loading difference, each band signal was normalized to the band signal of the 3’ most U of the U-tract. n = 2, technical replicates. c, Filter-binding curves displaying the binding affinities between recombinant hnRNP C1 and 2,577-U/A oligos. n = 3, ± s.d., technical replicates. d, Filter-binding results showing the binding affinities between recombinant hnRNP C1 and four mutated MALAT1 oligos. (i) Mutate G-C to C-C, A2,577: predicted to weaken the hairpin stem and increase hnRNP C binding. Results: binding improved from 722 nM Kd to 142 nM (5-fold); (ii) Mutate G-C to C-C, m6A2,577: in this context of weaker stem, m6A is predicted to confer a smaller effect compared to wild-type hairpin. Result: improved binding only 2-fold instead of 8-fold; (iii) Restore C-C to C-G, A2,577: predicted to restore the hairpin stem and decrease hnRNP C binding compared to C-C mutant. Result: binding decreased by 6.4-fold; (iv) Restore C-C to C-G, m6A2,577: in this context of restored stem, m6A is again predicted to confer increased binding compared to A2,577 hairpin. Result: improved binding by 2.5-fold. n = 3 each, ± s.d., technical replicates. e, RNA alkaline hydrolysis terminal truncation assay showing recombinant hnRNP C1 binding to terminal truncated MALAT1 hairpin oligos (2,577 site m6A methylated or unmethylated). In this assay, 3′ radiolabeled MALAT1 2,577 hairpin oligos were terminal truncated by alkaline hydrolysis into RNA fragments which were then incubated with hnRNP C1 protein followed by filter binding wash steps. The remaining RNA on the filter paper was isolated and analyzed by denaturing gel electrophoresis, as indicated in the lane “C1-bound or C1-B”. “Input” refers to alkaline hydrolysis truncated RNA oligos used for incubation with hnRNP C1; “G-L or G-ladder” was generated from RNase T1 digestion; “Ctrl” refers to the intact MALAT1 hairpin without alkaline hydrolysis truncation. One pair of methylated/unmethylated truncated oligos (CUT1, marked by green arrows) was selected for subsequent biochemical analysis, due to their strong interaction with hnRNP C1. f, RNA terminal truncation assay as in e except 5′ 32P-labeled oligos were used. One pair of methylated/unmethylated truncated oligos (CUT2, marked by green arrows) was selected for subsequent biochemical analysis. g, Structure probing of the CUT1 oligos using RNase V1 and nuclease S1 digestion, same annotation as in Fig. 1e. The red dot marks the m6A site and the red line marks the U-tract region. h, Structure probing of the CUT2 oligos using RNase V1 and nuclease S1 digestion, same annotation as in g. i, Truncated oligos with exposed U-tracts increased hnRNP C binding regardless of m6A. n = 3, ± s.d., technical replicates.
Mentions: Since m6A residues within RNA stems can destabilize the thermo-stability of model RNA duplexes28, we hypothesized that the m6A2,577 residue destabilizes this MALAT1 hairpin-stem to make its opposing U-tract more single-stranded or accessible, thus enhancing its interaction with hnRNP C. We performed several experiments to validate this hypothesis. First, according to the RNA structural probing assays, the m6A-modified hairpin showed significantly increased nuclease S1 digestion (single-stranded specific) at the GAC (A= m6A) motif as well as markedly decreased RNase V1 digestion (double-stranded/stacking specific) at the U-tract opposing the GAC motif (Fig. 1d). The m6A residue markedly destabilized the stacking properties of the region centered around the U-residue that pairs with A/m6A2,577 (Extended data Fig. 1f-g), which was also supported by the increased reactivity between CMCT and the U-tract bases in the presence of m6A (Extended Data Fig. 1h). Second, the A2,577-to-U mutation increased hnRNP C pull down amount from nuclear extract, whereas U-to-C mutations in the U-tract significantly reduced hnRNP C pull down amount regardless of m6A modification (Fig. 1e). Third, the A2,577-to-U mutation increased the accessibility of U-tract and enhanced hnRNP C binding by ~4-fold (Extended data Fig. 2a-c). Binding results with 4 other mutated A/m6A oligos also supported the U-tract with increased accessibility alone being sufficient to enhance hnRNP C binding (Extended data Fig. 2d). Fourth, RNA terminal truncation followed by hnRNP C binding identified two pairs of truncated hairpins with highly accessible U-tracts, which improved hnRNP C binding significantly but independent of the m6A modification (Extended data Fig. 2ei). All these results confirmed that m6A modification can alter its local RNA structure and enhance the accessibility of its base-paired residues or nearby regions to modulate protein binding (Fig. 1f). We term this mechanism that regulates RNA-protein interactions through m6A-dependent RNA structural remodeling as “m6A-switch”.

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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