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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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Validation of two identified m6A-switchesa-b, PARCLIP-MeRIP data detected positive IP/Input enrichment at the RRACH sites (red arrowheads) on the DNAJC25-GNG10 gene (a) and HNRNPH1 gene (b) in HEK293T cells. c-d, Quantification of RNase V1 cleavage signals around the U-tract region of m6A-switches on the DNAJC25-GNG10 (c) and HNRNPH1 (d) transcript, related to Fig. 2g-h. n = 3, ± s.d., technical replicates each. e, Quantitative CMCT mapping of DNAJC25-GNG10 m6A-switch shows increased band signals around the uridine base that pairs with m6A. The red vertical line marks the U-tract region. Quantitation of bands signal for the U-tract region is shown on the right. n = 3, ± s.d., technical replicates. HNRNPH1 m6A-switch hairpin is not suitable for CMCT probing, because its reverse transcription binding primer region is too short. f-g,In vivo DMS mapping of the DNAJC25-GNG10 hairpin (f) and HNRNPH1 (g); data are from7. A and C residues are marked with orange dots and the m6A residue is marked with a red dot. The hairpin loops are indicated by red bars. h, Transcriptome-wide S1/V1 mapping around the HNRNPH1 m6A-switch site. Blue bars represent V1 signal; magenta bars represent S1 signal. The hairpin loop is indicated by a red bar; data are from4. Not enough reads could be collected to make a plot for the DNAJC25-GNG10 m6A-switch region.
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Figure 9: Validation of two identified m6A-switchesa-b, PARCLIP-MeRIP data detected positive IP/Input enrichment at the RRACH sites (red arrowheads) on the DNAJC25-GNG10 gene (a) and HNRNPH1 gene (b) in HEK293T cells. c-d, Quantification of RNase V1 cleavage signals around the U-tract region of m6A-switches on the DNAJC25-GNG10 (c) and HNRNPH1 (d) transcript, related to Fig. 2g-h. n = 3, ± s.d., technical replicates each. e, Quantitative CMCT mapping of DNAJC25-GNG10 m6A-switch shows increased band signals around the uridine base that pairs with m6A. The red vertical line marks the U-tract region. Quantitation of bands signal for the U-tract region is shown on the right. n = 3, ± s.d., technical replicates. HNRNPH1 m6A-switch hairpin is not suitable for CMCT probing, because its reverse transcription binding primer region is too short. f-g,In vivo DMS mapping of the DNAJC25-GNG10 hairpin (f) and HNRNPH1 (g); data are from7. A and C residues are marked with orange dots and the m6A residue is marked with a red dot. The hairpin loops are indicated by red bars. h, Transcriptome-wide S1/V1 mapping around the HNRNPH1 m6A-switch site. Blue bars represent V1 signal; magenta bars represent S1 signal. The hairpin loop is indicated by a red bar; data are from4. Not enough reads could be collected to make a plot for the DNAJC25-GNG10 m6A-switch region.

Mentions: To map the m6A sites around hnRNP C binding sites, we performed Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP)29 to isolate all hnRNP C bound RNA regions (Input control) followed by anti-m6A immunoprecipitation (MeRIP)13,14 to enrich m6A-containing hnRNP C bound RNA regions (IP). Both the Input control and IP samples from two biological replicates were sent for RNA-seq (Fig. 2c and Extended data Fig. 3b, c). This approach, termed PARCLIP-MeRIP, identified transcriptome-wide the m6A proximal hnRNP C binding site, such as the enriched peak around the MALAT1-2,577 site (Fig. 2d). Remarkably, hnRNP C PARCLIP-MeRIP peaks harbored two consensus motifs, the hnRNP C RBM (U-tracts) and the m6A consensus motif GRACH (a subset of RRACH13,14) (Fig. 2e). Both motifs were located mostly within 50 residues, suggesting transcriptome-wide RRACH-U-tract coupling events within the hnRNP C binding sites (Extended Data Fig. 4a, b). About 62% of all RRACH-U-tracts coupling events within hnRNP C binding sites are enriched at the RRACH motif (Fig. 2f). Our PARCLIP-MeRIP approach identified a total of 39,060 hnRNP C m6A-switches which corresponded to m6A-modified RRACH-U-tracts coupling events at FDR ≤ 5% (Extended Data Fig. 4c). These switches account for ~7% of 592,477 hnRNP C binding sites identified by PAR-CLIP. The majority (87%) of m6A-switches occur within introns (Extended Data Fig. 4d, e), consistent with the literature that hnRNP C is nuclear localized and primarily binds nascent transcripts20,23. We validated two intronic m6A-switches in hairpin structures where m6A residues increase the U-tract accessibility, and enhance hnRNP C binding by ~3-4 fold (Fig. 2g, h and Extended data Fig. 5).


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)

Validation of two identified m6A-switchesa-b, PARCLIP-MeRIP data detected positive IP/Input enrichment at the RRACH sites (red arrowheads) on the DNAJC25-GNG10 gene (a) and HNRNPH1 gene (b) in HEK293T cells. c-d, Quantification of RNase V1 cleavage signals around the U-tract region of m6A-switches on the DNAJC25-GNG10 (c) and HNRNPH1 (d) transcript, related to Fig. 2g-h. n = 3, ± s.d., technical replicates each. e, Quantitative CMCT mapping of DNAJC25-GNG10 m6A-switch shows increased band signals around the uridine base that pairs with m6A. The red vertical line marks the U-tract region. Quantitation of bands signal for the U-tract region is shown on the right. n = 3, ± s.d., technical replicates. HNRNPH1 m6A-switch hairpin is not suitable for CMCT probing, because its reverse transcription binding primer region is too short. f-g,In vivo DMS mapping of the DNAJC25-GNG10 hairpin (f) and HNRNPH1 (g); data are from7. A and C residues are marked with orange dots and the m6A residue is marked with a red dot. The hairpin loops are indicated by red bars. h, Transcriptome-wide S1/V1 mapping around the HNRNPH1 m6A-switch site. Blue bars represent V1 signal; magenta bars represent S1 signal. The hairpin loop is indicated by a red bar; data are from4. Not enough reads could be collected to make a plot for the DNAJC25-GNG10 m6A-switch region.
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Figure 9: Validation of two identified m6A-switchesa-b, PARCLIP-MeRIP data detected positive IP/Input enrichment at the RRACH sites (red arrowheads) on the DNAJC25-GNG10 gene (a) and HNRNPH1 gene (b) in HEK293T cells. c-d, Quantification of RNase V1 cleavage signals around the U-tract region of m6A-switches on the DNAJC25-GNG10 (c) and HNRNPH1 (d) transcript, related to Fig. 2g-h. n = 3, ± s.d., technical replicates each. e, Quantitative CMCT mapping of DNAJC25-GNG10 m6A-switch shows increased band signals around the uridine base that pairs with m6A. The red vertical line marks the U-tract region. Quantitation of bands signal for the U-tract region is shown on the right. n = 3, ± s.d., technical replicates. HNRNPH1 m6A-switch hairpin is not suitable for CMCT probing, because its reverse transcription binding primer region is too short. f-g,In vivo DMS mapping of the DNAJC25-GNG10 hairpin (f) and HNRNPH1 (g); data are from7. A and C residues are marked with orange dots and the m6A residue is marked with a red dot. The hairpin loops are indicated by red bars. h, Transcriptome-wide S1/V1 mapping around the HNRNPH1 m6A-switch site. Blue bars represent V1 signal; magenta bars represent S1 signal. The hairpin loop is indicated by a red bar; data are from4. Not enough reads could be collected to make a plot for the DNAJC25-GNG10 m6A-switch region.
Mentions: To map the m6A sites around hnRNP C binding sites, we performed Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP)29 to isolate all hnRNP C bound RNA regions (Input control) followed by anti-m6A immunoprecipitation (MeRIP)13,14 to enrich m6A-containing hnRNP C bound RNA regions (IP). Both the Input control and IP samples from two biological replicates were sent for RNA-seq (Fig. 2c and Extended data Fig. 3b, c). This approach, termed PARCLIP-MeRIP, identified transcriptome-wide the m6A proximal hnRNP C binding site, such as the enriched peak around the MALAT1-2,577 site (Fig. 2d). Remarkably, hnRNP C PARCLIP-MeRIP peaks harbored two consensus motifs, the hnRNP C RBM (U-tracts) and the m6A consensus motif GRACH (a subset of RRACH13,14) (Fig. 2e). Both motifs were located mostly within 50 residues, suggesting transcriptome-wide RRACH-U-tract coupling events within the hnRNP C binding sites (Extended Data Fig. 4a, b). About 62% of all RRACH-U-tracts coupling events within hnRNP C binding sites are enriched at the RRACH motif (Fig. 2f). Our PARCLIP-MeRIP approach identified a total of 39,060 hnRNP C m6A-switches which corresponded to m6A-modified RRACH-U-tracts coupling events at FDR ≤ 5% (Extended Data Fig. 4c). These switches account for ~7% of 592,477 hnRNP C binding sites identified by PAR-CLIP. The majority (87%) of m6A-switches occur within introns (Extended Data Fig. 4d, e), consistent with the literature that hnRNP C is nuclear localized and primarily binds nascent transcripts20,23. We validated two intronic m6A-switches in hairpin structures where m6A residues increase the U-tract accessibility, and enhance hnRNP C binding by ~3-4 fold (Fig. 2g, h and Extended data Fig. 5).

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