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. 2018 Sep 14;8(1):13827.
doi: 10.1038/s41598-018-32310-8.

The m6A-methylase complex recruits TREX and regulates mRNA export

Simon Lesbirel  1 , Nicolas Viphakone  1 , Matthew Parker  1 , Jacob Parker  1 , Catherine Heath  1 , Ian Sudbery  1 , Stuart A Wilson  2
Affiliations

The m6A-methylase complex recruits TREX and regulates mRNA export

Simon Lesbirel et al. Sci Rep. .

Abstract

N6-methyladenosine (m6A) is the most abundant internal modification of eukaryotic mRNA. This modification has previously been shown to alter the export kinetics for mRNAs though the molecular details surrounding this phenomenon remain poorly understood. Recruitment of the TREX mRNA export complex to mRNA is driven by transcription, 5' capping and pre-mRNA splicing. Here we identify a fourth mechanism in human cells driving the association of TREX with mRNA involving the m6A methylase complex. We show that the m6A complex recruits TREX to m6A modified mRNAs and this process is essential for their efficient export. TREX also stimulates recruitment of the m6A reader protein YTHDC1 to the mRNA and the m6A complex influences the interaction of TREX with YTHDC1. Together our studies reveal a key role for TREX in the export of m6A modified mRNAs.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
The TREX complex associates with the m6A methyltransferase complex. (A) WTAP and KIAA1429 co-IP/Western analysis with TREX complex subunits. CONTROL antibody was anti-FLAG throughout the study. Antibodies used for IP are shown above the panels and antibodies used for detection on the right hand side of panels throughout the study. (B) METTL3/METTL14 Co-IP/Western with TREX subunits. (C) Western analysis showing efficient depletion of KIAA1429 by RNAi (upper panel). Combined KIAA1429 and WTAP siRNA treatment resulted in substantial knockdown of both proteins (lower panel). (D) WTAP Co-IP/Western analysis in a KIAA1429 siRNA background with the indicated TREX and m6A machinery subunits. (E) METTL3 co-IP/Western analysis with ALYREF. METTL3 antibody was used for the IP. All Co-IP analysis presented in the paper were carried out in the presence of RNase A. Where separate panels are shown for same the protein in Western blots, these are all taken from the same blot at the same exposure. Full size blots are displayed in Supplementary Information.
Figure 2
Figure 2
Co-knockdown of KIAA1429 and WTAP results in an export block for methylated transcripts. (A) qRT-qPCR analysis of m6A modified (TAF7 (intronless), DICER1, PTPN12 (spliced) and non-m6A modified mRNAs (GSTP1, SYMPK). The nuclear/cytoplasmic ratios normalised to control siRNA treated cells are shown. (B) ALYREF RNA immunoprecipitation (RIP) analysis by RT-qPCR are shown together with the positions of primers used on the long internal exon genes. Long internal exons with reported m6A sites have a black outline. (C) DDX39A/B RIP analysis by RT-qPCR. (D) RT-qPCR analysis the nuclear/cytoplasmic ratios for selected transcripts following THOC5/ALYREF combined RNAi. RT-qPCR results throughout the paper represent the averages from 3 independent experiments with standard deviations presented.
Figure 3
Figure 3
Global pattern of mRNA distribution following WTAP/KIAA1429 knockdown. (A) Volcano plot showing log2 fold change in nuclear/cytoplasmic ratio in WTAP/KIAA1429 knockdown vs. control RNAi, determined by RNA-seq (n = 3). A fold change of 0.5 and an FDR of 0.1 were used to identify significantly differentially localised genes. (B) Overlap of nuclear accumulating genes with methylated genes, as determined by m6A iCLIP (miCLIP). Genes were considered to be methylated if they contained one or more miCLIP peaks within an exonic region of the gene. CIMS (cross linking induced mutation site) and CITS (crosslinking-induced truncation sites) are two alternative ways of identifying m6A sites as described in. (C) Number of m6A sites in exonic regions per gene for nuclear accumulating (NA) methylated genes, vs. other methylated genes. (D) Length of the longest final exon per gene (log10 scale) for NA methylated genes vs. non-NA non-methylated genes.
Figure 4
Figure 4
Effect of WTAP/KIAA1429 knockdown on intron retention. (A) Introns were divided into three categories (lower panel) – intron that is already annotated as being retained (Annotated retained intron), sequence that is always intronic in the annotation (Constitutive intron) and sequence that is sometimes intronic, sometimes exonic (Alternative intron). We measured the number of gene products containing an intron with evidence of a significant increase in intron retention (adjusted p-value < 0.1; logFC > 1) for each intron type as a fraction of gene products that contained introns of that type (upper panel). (B) Overlap of gene products showed to be methylated (m6A) with gene products showing evidence of increased intron retention on WTAP/KIAA1429 knockdown. (C) Overlap of gene products that show significant evidence of increased intron retention with gene products that show significant nuclear accumulation upon WTAP/KIAA1429 knockdown.
Figure 5
Figure 5
YTHDC1 associates with TREX and is required for mRNA export. (A) RT-qPCR analysis of the nuclear:cytoplasmic ratios for m6A modified (TAF7, DICER1, PTPN12) and non-modified (MC1R) transcripts following knockdown of YTHDC1. (B) Co-IP/Western analysis of ALYREF and CHTOP TREX subunits with YTHDC1 and other TREX subunits. PM = A lane where a protein marker was loaded. (C) Co-IP/Western analysis of NXF1 and YTHDC1. (D) WTAP co-IP/Western analysis with the indicated proteins in Control and ALYREF/CHTOP RNAi backgrounds (E) CO-IP/Western analysis of ALYREF with YTHDC1 and UAP56 in a background where METLL3, WTAP and KIAA1429 were knocked down using RNAi as indicated. (F) RT-qPCR RIP analysis of ALYREF in control RNAi and YTHDC1 RNAi cells. (G) UV crosslinking-mRNP capture analysis for the indicated proteins following depletion of ALYREF/CHTOP combined or NXF1. “R” indicates treatment with RNase A prior to oligo (dT) capture, as a control. (H) RT-qPCR RIP analysis of YTHDC1 in control and ALYREF/CHTOP RNAi cells. Where separate panels are shown for same the protein in Western blots, these are all taken from the same blot at the same exposure. Full size blots are displayed in Supplementary Information.
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