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Cited by 1,260RNA modifications and cancerRNA Research and SplicingRNA regulation and disease

The DOI matches its Crossref record; 59 of the 59 references it cites were found in a registry or an index; none of the 32 sources its claims cite is checked for support yet.

YTHDC1 mediates nuclear export of N6-methyladenosine methylated mRNAs

Open access
Roundtree, Ian A, Luo, Guan-Zheng, Zhang, Zijie, Wang, Xiao, Zhou, Tao, Cui, Yiquang, Sha, Jiahao, Huang, Xingxu, Guerrero, Laura, Xie, Phil, He, Emily, Shen, Bin, He, Chuan·2017·eLife
DOI 10.7554/elife.31311PMC PMC5648532
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Abstract

N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic messenger RNA (mRNA) and plays critical roles in RNA biology. The function of this modification is mediated by m6A-selective ‘reader’ proteins of the YTH family, which incorporate m6A-modified mRNAs into pathways of RNA metabolism. Here, we show that the m6A-binding protein YTHDC1 mediates export of methylated mRNA from the nucleus to the cytoplasm in HeLa cells. Knockdown of YTHDC1 results in an extended residence time for nuclear m6A-containing mRNA, with an accumulation of transcripts in the nucleus and accompanying depletion within the cytoplasm. YTHDC1 interacts with the splicing factor and nuclear export adaptor protein SRSF3, and facilitates RNA binding to both SRSF3 and NXF1. This role for YTHDC1 expands the potential utility of chemical modification of mRNA, and supports an emerging paradigm of m6A as a distinct biochemical entity for selective processing and metabolism of mammalian mRNAs.

Overview

CoChat Summary

TL;DR

YTHDC1, a nuclear m6A reader, mediates nuclear-to-cytoplasmic export of methylated mRNAs in HeLa cells by linking m6A recognition to the export adaptor SRSF3 and the export receptor NXF1. Knockdown of YTHDC1 causes nuclear accumulation and cytoplasmic depletion of its target transcripts, independent of pre-mRNA splicing.

Key findings

  • Knockdown of YTHDC1 increased nuclear m6A levels and extended the apparent half-life of nuclear mRNA methylation more than 2-fold after transcription inhibition; YTHDF2 knockdown extended cytoplasmic m6A apparent half-life by ~4-fold.
  • YTHDC1 knockdown did not change total mRNA m6A/A but caused nuclear accumulation and cytoplasmic depletion of m6A and polyA RNA; overexpression of YTHDC1 after YTHDF2 knockdown increased cytoplasmic m6A.
  • YTHDC1 targets were defined as transcripts bound in PAR-CLIP and enriched at least 2-fold in RIP-seq, yielding over 700 targets; upon YTHDC1 knockdown, target abundance increased in nuclear RNA and decreased in cytoplasmic RNA, while total target abundance was unchanged.
  • YTHDC1 interacts with SRSF3, and SRSF3 interacts with NXF1; YTHDC1 knockdown reduced m6A enrichment by SRSF3 and abolished m6A enrichment by NXF1. Only transcripts bound by both YTHDC1 and SRSF3 showed significant nuclear accumulation upon YTHDC1 knockdown.
  • YTHDC1 knockdown affected alternative splicing, with 426 events at Δpsi ≥0.2; 85/737 (11.5%) occurred in YTHDC1 targets versus 108/4,848 (2.2%) in non-targets, but reporter assays showed export effects independent of splicing and m6A binding.

Methods

  • HeLa cells were used for siRNA knockdown, plasmid transfection, cellular fractionation, and RNA isolation; m6A/A was quantified by LC-MS/MS on polyA-selected, ribosomal RNA-depleted mRNA from total, nuclear, and cytoplasmic fractions.
  • RNA substrates were identified by YTHDC1 PAR-CLIP and RIP-seq, subcellular transcriptomes by RNA-seq after YTHDC1 knockdown, and binding by SRSF3/NXF1 by CLIP or RIP followed by LC-MS/MS and sequencing.
  • Mechanistic tests included endogenous and FLAG immunoprecipitation, western blotting, polyA FISH, nascent RNA labeling with 5-ethynyl uridine, RT-qPCR, and a Tet-inducible BoxB-luciferase reporter tethering assay with full-length, N-terminal, C-terminal, and W377A YTHDC1 constructs.

Limitations

  • The paper does not include a dedicated limitations section.
  • Evident from the text: most experiments are in HeLa cells, so generality to other cell types or organisms is not established.
  • The m6A/A measurements are ensemble bulk measurements; the paper notes this allows relative clearance comparisons without determining absolute half-lives of individual mRNAs.
  • Export conclusions rely heavily on knockdown/overexpression and tethering assays; the reporter system is splicing-independent and methylation-independent due to BoxB–lambda peptide binding, which the authors present as an advantage but which also means it does not test endogenous m6A-dependent export directly.

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Integrity signals

Registry

Crossref · 10.7554/elife.31311

Cites

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Statistics

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Figures

Figure 1. Methylated mRNAs are selectively cleared from the cytoplasm by YTHDF2 and from the nucleus by YTHDC1. (A) m6A methylation in cytoplasmic mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. Curves fit to exponential decay. (B) m6A methylation in nuclear mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. Curves fit to exponential decay. (C) m6A methylation in nuclear mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. Curves fit to exponential decay. (D) m6A methylation in cytoplasmic mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. Curves fit to exponential decay.
Figure 1. Methylated mRNAs are selectively cleared from the cytoplasm by YTHDF2 and from the nucleus by YTHDC1. (A) m6A methylation in cytoplasmic mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. Curves fit to exponential decay. (B) m6A methylation in nuclear mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. Curves fit to exponential decay. (C) m6A methylation in nuclear mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. Curves fit to exponential decay. (D) m6A methylation in cytoplasmic mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. Curves fit to exponential decay.CC BY · PubMed Central
Figure 1—figure supplement 1. Representative western blots for siRNA knockdown efficiency and northern blot nuclear and cytoplasmic RNA isolation. Calibration and unknown determination for LC-MS/MS quantification of m6A/A. (A) Representative western blot for YTHDF2 knockdown efficiency. (B) Representative Western blot for YTHDC1 knockdown efficiency. (C) Northern blot of nuclear and cytoplasmic RNA. (D,E) Representative data from LC-MS/MS for A and m6A. Standards curves (black) are generated, and unknown concentrations (red) are calculated.
Figure 1—figure supplement 1. Representative western blots for siRNA knockdown efficiency and northern blot nuclear and cytoplasmic RNA isolation. Calibration and unknown determination for LC-MS/MS quantification of m6A/A. (A) Representative western blot for YTHDF2 knockdown efficiency. (B) Representative Western blot for YTHDC1 knockdown efficiency. (C) Northern blot of nuclear and cytoplasmic RNA. (D,E) Representative data from LC-MS/MS for A and m6A. Standards curves (black) are generated, and unknown concentrations (red) are calculated.CC BY · PubMed Central
Figure 2. YTHDC1 mediates subcellular localization of methylated mRNAs. (A) Quantification of m6A methylation in total (T), nuclear (N), and cytoplasmic (C) mRNA. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (B) Representative western blot of YTHDF2 and members of the mRNA export pathway following knockdown of YTHDC1. (C) Representative analysis of polyA imaging. Nuclei were defined using DAPI signal. Cytoplasmic regions were defined by subtracting nuclear signal from total signal, as defined by DIC imaging. (D) Quantification of polyA signal density. n = 25, two-sided t-test. (E,F) Quantification of m6A methylation in nuclear and cytoplasmic mRNA. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance.
Figure 2. YTHDC1 mediates subcellular localization of methylated mRNAs. (A) Quantification of m6A methylation in total (T), nuclear (N), and cytoplasmic (C) mRNA. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (B) Representative western blot of YTHDF2 and members of the mRNA export pathway following knockdown of YTHDC1. (C) Representative analysis of polyA imaging. Nuclei were defined using DAPI signal. Cytoplasmic regions were defined by subtracting nuclear signal from total signal, as defined by DIC imaging. (D) Quantification of polyA signal density. n = 25, two-sided t-test. (E,F) Quantification of m6A methylation in nuclear and cytoplasmic mRNA. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance.CC BY · PubMed Central
Figure 2—figure supplement 1. YTHDC1 does not contribute to nuclear mRNA decay or transcription. (A) Representative western blot for YTHDC1 overexpression. (B) Representative western blot for YTHDC1 overexpression in a YTHDF2 knockdown background. (C) Interactions between YTHDC1 and components of the NEXT/PAXT machinery. (D) m6A/A ratios in pre-mRNA following knockdown of YTHDC1 and NEXT/PAXT components. (E) Representative western blots of knockdown samples for panel (D). (F) Representative immunofluorescence of YTHDC1–FLAG in HeLa cells. (G) Dot-blot of nascent RNA. HeLa cells were labeled for 1 hr with 4-thio-uridine (4SU), conjugated to biotin and exposed with Streptavidin–HRP. (H) Methylene blue staining of (G) (loading control).
Figure 2—figure supplement 1. YTHDC1 does not contribute to nuclear mRNA decay or transcription. (A) Representative western blot for YTHDC1 overexpression. (B) Representative western blot for YTHDC1 overexpression in a YTHDF2 knockdown background. (C) Interactions between YTHDC1 and components of the NEXT/PAXT machinery. (D) m6A/A ratios in pre-mRNA following knockdown of YTHDC1 and NEXT/PAXT components. (E) Representative western blots of knockdown samples for panel (D). (F) Representative immunofluorescence of YTHDC1–FLAG in HeLa cells. (G) Dot-blot of nascent RNA. HeLa cells were labeled for 1 hr with 4-thio-uridine (4SU), conjugated to biotin and exposed with Streptavidin–HRP. (H) Methylene blue staining of (G) (loading control).CC BY · PubMed Central
Figure 3. Knockdown of YTHDC1 affects the subcellular distribution of target mRNAs. (A) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in total mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. (B) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in nuclear mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. (C) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in cytoplasmic mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. Data represent biological replicates using unique siRNAs against YTHDC1.
Figure 3. Knockdown of YTHDC1 affects the subcellular distribution of target mRNAs. (A) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in total mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. (B) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in nuclear mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. (C) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in cytoplasmic mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. Data represent biological replicates using unique siRNAs against YTHDC1.CC BY · PubMed Central
Figure 3—figure supplement 1. Identification of YTHDC1 mRNA targets; target expression is unchanged following knockdown of YTHDC1. (A) Comparison of YTHDC1 RIP-seq data sets. (B) Selection of YTHDC1 target mRNAs from PAR-CLIP and RIP-seq data. (C) Expression of YTHDC1 target mRNAs under control and YTHDC1 knockdown conditions. Left: Cumulative fraction of RNA expression. P-values calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA expression. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test.
Figure 3—figure supplement 1. Identification of YTHDC1 mRNA targets; target expression is unchanged following knockdown of YTHDC1. (A) Comparison of YTHDC1 RIP-seq data sets. (B) Selection of YTHDC1 target mRNAs from PAR-CLIP and RIP-seq data. (C) Expression of YTHDC1 target mRNAs under control and YTHDC1 knockdown conditions. Left: Cumulative fraction of RNA expression. P-values calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA expression. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test.CC BY · PubMed Central
Figure 3—figure supplement 2. Subcellular abundances of YTHDC1 PAR-CLIP and RIP-seq targets are affected by YTHDC1. (A) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in nuclear mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. (B) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in cytoplasmic mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. (C) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in nuclear mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. (D) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in cytoplasmic mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test.
Figure 3—figure supplement 2. Subcellular abundances of YTHDC1 PAR-CLIP and RIP-seq targets are affected by YTHDC1. (A) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in nuclear mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. (B) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in cytoplasmic mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. (C) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in nuclear mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test. (D) Left: Cumulative distribution of log2 fold changes in RNA expression following knockdown of YTHDC1 in cytoplasmic mRNA. P-value calculated using the Mann-Whitney-Wilcoxon Test. Right: Boxplot representation of RNA-seq fold changes. Whiskers represent three times the interquartile range. P-value calculated using Welch’s T-test.CC BY · PubMed Central
Figure 4. YTHDC1 mediates nuclear to cytoplasmic transport of nascent mRNA. (A,B) RT-qPCR of nascent mRNA in the nucleus (A) and cytoplasm (B). Error bars represent mean ± standard deviation, n = 6 (three biological replicates x two technical replicates), two-sided t-test with equal variance. (C) Translation of reporter mRNA in response to binding by the full-length YTHDC1. Error bars represent mean ± standard deviation, n = 4. (D) Nuclear and cytoplasmic RT-qPCR of reporter mRNA 4 hr after induction of reporter transcript. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (E) Translation of reporter mRNA in response to binding by the YTHDC1 C-terminus. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (F) Nuclear and cytoplasmic RT-qPCR of reporter mRNA 4 hr after induction of reporter transcript. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance.
Figure 4. YTHDC1 mediates nuclear to cytoplasmic transport of nascent mRNA. (A,B) RT-qPCR of nascent mRNA in the nucleus (A) and cytoplasm (B). Error bars represent mean ± standard deviation, n = 6 (three biological replicates x two technical replicates), two-sided t-test with equal variance. (C) Translation of reporter mRNA in response to binding by the full-length YTHDC1. Error bars represent mean ± standard deviation, n = 4. (D) Nuclear and cytoplasmic RT-qPCR of reporter mRNA 4 hr after induction of reporter transcript. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (E) Translation of reporter mRNA in response to binding by the YTHDC1 C-terminus. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (F) Nuclear and cytoplasmic RT-qPCR of reporter mRNA 4 hr after induction of reporter transcript. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance.CC BY · PubMed Central
Figure 4—figure supplement 1. Nascent mRNA localization by metabolic labeling with 5-ethynyl-uridine (EU). (A) Schematic for nascent RNA labeling of HeLa cells. (B) Selection of YTHDC1 target transcripts based on PAR-CLIP and RIP-seq data. (C) Nuclear and cytoplasmic levels of nascent mRNA of non-target transcripts. Error bars represent mean ± standard deviation, n = 6 (three biological replicates x two technical replicates), two-sided t-test with equal variance. Differences between siControl and siYTHDC1 were not significant (p>0.05 in all cases).
Figure 4—figure supplement 1. Nascent mRNA localization by metabolic labeling with 5-ethynyl-uridine (EU). (A) Schematic for nascent RNA labeling of HeLa cells. (B) Selection of YTHDC1 target transcripts based on PAR-CLIP and RIP-seq data. (C) Nuclear and cytoplasmic levels of nascent mRNA of non-target transcripts. Error bars represent mean ± standard deviation, n = 6 (three biological replicates x two technical replicates), two-sided t-test with equal variance. Differences between siControl and siYTHDC1 were not significant (p>0.05 in all cases).CC BY · PubMed Central
Figure 4—figure supplement 2. YTHDC1 affects alternative pre-mRNA splicing independent of roles in mRNA binding and export. (A) Analysis of paired-end RNA-seq data using MISO (Katz et al., 2010). Example code can be found in the Materials and Methods section. (B) Comparison of YTHDC1-dependent skipped exons with METTL3-dependent skipped exons (data from Alarcón et al., 2015). (C) Comparison of YTHDC1 target mRNAs and mRNAs with alternative isoform usage upon YTHDC1 knockdown by RNA-seq. I-value calculated using Fisher’s Exact Test. (D) Comparison of YTHDC1 non-target mRNAs and mRNAs with alternative isoform usage upon YTHDC1 knockdown by RNA-seq. P-value calculated using Fisher’s Exact Test. (E) Sashimi plot and RT-PCR of YTHDC1 target mRNAs APC and MCL1 under control and YTHDC1-knockdown conditions.
Figure 4—figure supplement 2. YTHDC1 affects alternative pre-mRNA splicing independent of roles in mRNA binding and export. (A) Analysis of paired-end RNA-seq data using MISO (Katz et al., 2010). Example code can be found in the Materials and Methods section. (B) Comparison of YTHDC1-dependent skipped exons with METTL3-dependent skipped exons (data from Alarcón et al., 2015). (C) Comparison of YTHDC1 target mRNAs and mRNAs with alternative isoform usage upon YTHDC1 knockdown by RNA-seq. I-value calculated using Fisher’s Exact Test. (D) Comparison of YTHDC1 non-target mRNAs and mRNAs with alternative isoform usage upon YTHDC1 knockdown by RNA-seq. P-value calculated using Fisher’s Exact Test. (E) Sashimi plot and RT-PCR of YTHDC1 target mRNAs APC and MCL1 under control and YTHDC1-knockdown conditions.CC BY · PubMed Central
Figure 4—figure supplement 3. YTHDC1 reporter system. (A) Construction of the Luciferase reporter system, as previously reported (Wang et al., 2014a). (B) Modular structure of YTHDC1. Low complexity regions annotated according to SMART (Letunic et al., 2015). (C) Cellular localization of YTHDC1 effector constructs. (D) Translation of reporter mRNA in response to binding by YTHDC1–W377A. Error bars represent mean ± standard deviation, n = 4. (E) Translation of reporter mRNA in response to binding by the YTHDC1 N-terminus. Error bars represent mean ± standard deviation, n = 4. (F) Nuclear and cytoplasmic RT-qPCR of reporter mRNA 4 hr after induction of reporter transcript. Error bars represent mean ± standard deviation, n = 4, *p < 0.05.
Figure 4—figure supplement 3. YTHDC1 reporter system. (A) Construction of the Luciferase reporter system, as previously reported (Wang et al., 2014a). (B) Modular structure of YTHDC1. Low complexity regions annotated according to SMART (Letunic et al., 2015). (C) Cellular localization of YTHDC1 effector constructs. (D) Translation of reporter mRNA in response to binding by YTHDC1–W377A. Error bars represent mean ± standard deviation, n = 4. (E) Translation of reporter mRNA in response to binding by the YTHDC1 N-terminus. Error bars represent mean ± standard deviation, n = 4. (F) Nuclear and cytoplasmic RT-qPCR of reporter mRNA 4 hr after induction of reporter transcript. Error bars represent mean ± standard deviation, n = 4, *p < 0.05.CC BY · PubMed Central
Figure 5. YTHDC1 mediates export of m6A-modified mRNA through SRSF3 and NXF1. (A) Immunoprecipitation (IP) of endogenous YTHDC1 from HeLa cell lysate. (B) IP of endogenous SRSF3 and NXF1 from HeLa cell lysate. (C) LC-MS/MS of mRNA cross-linked to SRSF3 and NXF1. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (D) Quantification of m6A in nuclear mRNA following knockdown of SR proteins. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance.
Figure 5. YTHDC1 mediates export of m6A-modified mRNA through SRSF3 and NXF1. (A) Immunoprecipitation (IP) of endogenous YTHDC1 from HeLa cell lysate. (B) IP of endogenous SRSF3 and NXF1 from HeLa cell lysate. (C) LC-MS/MS of mRNA cross-linked to SRSF3 and NXF1. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (D) Quantification of m6A in nuclear mRNA following knockdown of SR proteins. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance.CC BY · PubMed Central
Figure 5—figure supplement 1. YTHDC1 interacts with export-competent SRSF3. (A) Immunoprecipitation (IP) of YTHDC1-FLAG with members of the SR protein family. (B) IP of YTHDC1 C- and N-terminal regions with phosphorylated SRSF3. (C) Coomassie stain of His-SRSF3-RRM purified from Escherichia coli. (D) In vitro IP of HeLa mRNA with His-SRSF3-RRM. Error bars represent mean ± standard deviation, n = 4 (two biological x two technical replicates), two-sided t-test of equal variance.
Figure 5—figure supplement 1. YTHDC1 interacts with export-competent SRSF3. (A) Immunoprecipitation (IP) of YTHDC1-FLAG with members of the SR protein family. (B) IP of YTHDC1 C- and N-terminal regions with phosphorylated SRSF3. (C) Coomassie stain of His-SRSF3-RRM purified from Escherichia coli. (D) In vitro IP of HeLa mRNA with His-SRSF3-RRM. Error bars represent mean ± standard deviation, n = 4 (two biological x two technical replicates), two-sided t-test of equal variance.CC BY · PubMed Central
Figure 5—figure supplement 2. Roles of SRSF3 and other SR proteins in the export of m6A-methylated mRNAs. (A,B) Full data from Figure 5D. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two sided t-test with equal variance. (C) Representative western blots for siRNA knockdown performed for Figure 5D. (D) PolyA imaging of HeLa cells following treatment with siControl, siYTHDC1, or siSRSF3. Yellow outlines the nuclear regions (defined by DAPI signal) and the cytoplasmic regions (defined by DIC). (E) m6A methylation in nuclear mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two-sided t-test with equal variance. Curves fit to exponential decay.
Figure 5—figure supplement 2. Roles of SRSF3 and other SR proteins in the export of m6A-methylated mRNAs. (A,B) Full data from Figure 5D. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two sided t-test with equal variance. (C) Representative western blots for siRNA knockdown performed for Figure 5D. (D) PolyA imaging of HeLa cells following treatment with siControl, siYTHDC1, or siSRSF3. Yellow outlines the nuclear regions (defined by DAPI signal) and the cytoplasmic regions (defined by DIC). (E) m6A methylation in nuclear mRNA analyzed at 0, 3, and 6 hr after transcription inhibition. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two-sided t-test with equal variance. Curves fit to exponential decay.CC BY · PubMed Central
Figure 5—figure supplement 3. Estimate of relative protein stoichiometry. (A) Fragments per kilobase of transcript per million (FPKM) from total RNA-seq (average of two biological replicates). (B) Estimate of protein copies per cell in HeLa cells (Nagaraj et al., 2011).
Figure 5—figure supplement 3. Estimate of relative protein stoichiometry. (A) Fragments per kilobase of transcript per million (FPKM) from total RNA-seq (average of two biological replicates). (B) Estimate of protein copies per cell in HeLa cells (Nagaraj et al., 2011).CC BY · PubMed Central
Figure 5—figure supplement 4. Subcellular distribution of YTHDC1 target mRNAs as a function of YTHDC1, SRSF3, and m6A. (A) Representative western blot for simultaneous siRNA knockdown of METTL3 and METTL14 in HeLa cells. (B) RT-PCR of nuclear RNA following knockdown of YTHDC1, SRSF3, and METTL3/14 in HeLa cells. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two-sided t-test with equal variance. (C) RT-PCR of cytoplasmic RNA following knockdown of YTHDC1, SRSF3, and METTL3/14 in HeLa cells. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two-sided t-test with equal variance.
Figure 5—figure supplement 4. Subcellular distribution of YTHDC1 target mRNAs as a function of YTHDC1, SRSF3, and m6A. (A) Representative western blot for simultaneous siRNA knockdown of METTL3 and METTL14 in HeLa cells. (B) RT-PCR of nuclear RNA following knockdown of YTHDC1, SRSF3, and METTL3/14 in HeLa cells. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two-sided t-test with equal variance. (C) RT-PCR of cytoplasmic RNA following knockdown of YTHDC1, SRSF3, and METTL3/14 in HeLa cells. Error bars represent mean ± standard deviation, n = 4, *p<0.05, **p<0.01, ***p<0.001, two-sided t-test with equal variance.CC BY · PubMed Central
Figure 6. YTHDC1 facilitates RNA binding by mRNA export machinery. (A) LC-MS/MS of mRNA cross-linked to SRSF3. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (B) Target enrichment by SRSF3. Whiskers represent three times the interquartile range. Data were analyzed across two independent experiments representing biological replicates. P-value calculated using Welch’s T-test. Whiskers represent three times the interquartile range. (C) LC-MS/MS of mRNA cross-linked to NXF1. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (D) Target enrichment by NXF1. Whiskers represent three times the interquartile range. Data were analyzed across two independent experiments representing biological replicates. P-value calculated using Welch’s T-test. Whiskers represent three times the interquartile range.
Figure 6. YTHDC1 facilitates RNA binding by mRNA export machinery. (A) LC-MS/MS of mRNA cross-linked to SRSF3. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (B) Target enrichment by SRSF3. Whiskers represent three times the interquartile range. Data were analyzed across two independent experiments representing biological replicates. P-value calculated using Welch’s T-test. Whiskers represent three times the interquartile range. (C) LC-MS/MS of mRNA cross-linked to NXF1. Error bars represent mean ± standard deviation, n = 4, two-sided t-test with equal variance. (D) Target enrichment by NXF1. Whiskers represent three times the interquartile range. Data were analyzed across two independent experiments representing biological replicates. P-value calculated using Welch’s T-test. Whiskers represent three times the interquartile range.CC BY · PubMed Central
Figure 7. Association with SRSF3 is required for facilitated export by YTHDC1. (A) Nuclear abundance of transcripts based on binding by YTHDC1 and/or SRSF3. Cumulative fractions: Mann-Whitney-Wilcoxon Test. Box plot: Welch’s T-Test. Whiskers represent three times the interquartile range. (B) Model of selective export of methylated mRNAs by YTHDC1.
Figure 7. Association with SRSF3 is required for facilitated export by YTHDC1. (A) Nuclear abundance of transcripts based on binding by YTHDC1 and/or SRSF3. Cumulative fractions: Mann-Whitney-Wilcoxon Test. Box plot: Welch’s T-Test. Whiskers represent three times the interquartile range. (B) Model of selective export of methylated mRNAs by YTHDC1.CC BY · PubMed Central
Figure 7—figure supplement 1. Definition of transcript groups based on YTHDC1 and SRSF3 binding, and effect on cytoplasmic abundance. (A) Definition of transcript groups based on YTHDC1 target binding by SRSF3. (B) Cytoplasmic abundance of transcripts based on binding by YTHDC1 and/or SRSF3. Cumulative fractions: Mann-Whitney-Wilcoxon Test. Box plot: Welch’s T-Test. Whiskers represent three times the interquartile range.
Figure 7—figure supplement 1. Definition of transcript groups based on YTHDC1 and SRSF3 binding, and effect on cytoplasmic abundance. (A) Definition of transcript groups based on YTHDC1 target binding by SRSF3. (B) Cytoplasmic abundance of transcripts based on binding by YTHDC1 and/or SRSF3. Cumulative fractions: Mann-Whitney-Wilcoxon Test. Box plot: Welch’s T-Test. Whiskers represent three times the interquartile range.CC BY · PubMed Central

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Metadata

Authors
Roundtree, I. A., Luo, G.-Z., Zhang, Z., Wang, X., Zhou, T., Cui, Y., Sha, J., Huang, X., Guerrero, L., Xie, P., He, E., Shen, B., & He, C.
Affiliations
  • Howard Hughes Medical Institute (US)
  • ShanghaiTech University (CN)
  • State Key Laboratory of Reproductive Medicine (CN)
  • University of Chicago (US)
Corresponding author
  • Shen, Bin
  • He, Chuan
ORCID
Venue
eLife
Publisher
eLife Sciences Publications, Ltd
Published
2017-10-06
Volume
6
ISSN
2050-084X (ISSN-L)
DOI
10.7554/elife.31311
PMC
PMC5648532
OpenAlex
W2764301500
Cited by
1,256
References
59
Citations by year
2026: 99 · 2025: 163 · 2024: 199 · 2023: 198 · 2022: 201 · 2021: 181 · 2020: 114 · 2019: 57 · 2018: 43
FWCI
20.75
Type
journal article
Language
en
Open access
Licence
Locations
  • eLife (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Knowledge@UChicago (University of Chicago) (submittedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • University of Chicago (cc-by)
Funders
  • National Institute of General Medical Sciences (F30GM117646, HG008688, GM113194)
  • Howard Hughes Medical Institute
  • National Science Foundation (CHE-1048528)
  • National Natural Science Foundation of China (31171377, 31471400)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA modifications and cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Keywords
YTHDC1, N6-methyladenosine, m6A reader proteins, mRNA export, mRNA splicing, SRSF3, NXF1, mRNA modification, RNA metabolism
MeSH terms
Serine-Arginine Splicing Factors, RNA Splicing Factors, Adenosine, HeLa Cells, Humans, Nerve Tissue Proteins, Protein Binding, RNA, Messenger, RNA-Binding Proteins, Active Transport, Cell Nucleus, Nucleocytoplasmic Transport Proteins, Gene Knockdown Techniques

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