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. Author manuscript; available in PMC: 2018 Nov 2.
Published in final edited form as: Mol Cell. 2017 Oct 26;68(3):504–514.e7. doi: 10.1016/j.molcel.2017.10.002

m6A Facilitates eIF4F-Independent mRNA Translation

Ryan A Coots 1,2,3, Xiao-Min Liu 1,3, Yuanhui Mao 1, Leiming Dong 1, Jun Zhou 1, Ji Wan 1, Xingqian Zhang 1, Shu-Bing Qian 1,2,4,*
PMCID: PMC5913006  NIHMSID: NIHMS915694  PMID: 29107534

SUMMARY

In eukaryotic cells, protein synthesis typically begins with the binding of eIF4F to the 7-methylguanylate (m7G) cap found on the 5’ end of the majority of mRNAs. Surprisingly, overall translational output remains robust under eIF4F inhibition. The broad spectrum of eIF4F-resistant translatomes is incompatible with cap-independent translation mediated by internal ribosome entry sites (IRES). Here, we report that N6-methyladenosine (m6A) facilitates mRNA translation that is resistant to eIF4F inactivation. Depletion of the methyltransferase METTL3 selectively inhibits translation of mRNAs bearing 5’UTR methylation, but not mRNAs with 5’ terminal oligopyrimidine (TOP) elements. We identify ABCF1 as a critical mediator of m6A-promoted translation under both stress and physiological conditions. Supporting the role of ABCF1 in m6A-facilitated mRNA translation, ABCF1-sensitive transcripts largely overlap with METTL3-dependent mRNA targets. By illustrating the scope and mechanism of eIF4F-independent mRNA translation, these findings reshape our current perceptions of cellular translational pathways.

Keywords: translation, mRNA methylation, eIF4F, METTL3, ABCF1, stress response

In Brief

graphic file with name nihms915694u1.jpg

Coots et al. show that eIF4F inhibition partially represses global protein synthesis. m6A in the 5’UTR facilitates eIF4F-independent mRNA translation, and ABCF1 appears to be critical for m6A-facilitated mRNA translation. These differential translation modes are coordinated in response to environmental perturbations.

INTRODUCTION

Eukaryotic cells primarily employ a cap-dependent mechanism to initiate translation for the majority of mRNAs (Gebauer and Hentze, 2004; Hinnebusch, 2014; Jackson et al., 2010). The 5’ end of eukaryotic mRNAs is modified with a m7G cap structure, which is recognized by an eukaryotic initiation factor 4E (eIF4E). eIF4E forms the eIF4F complex by binding to eIF4G (a scaffold protein) and eIF4A (a helicase) (Gross et al., 2003; Marintchev et al., 2009; Schutz et al., 2008). The cap recognition determines which mRNAs are to be translated and is subject to regulation by eIF4E-binding proteins (4E-BPs). When hypo-phosphorylated, 4E-BPs outcompete eIF4G for a binding site on eIF4E and prevent eIF4F assembly at the 5’ end of transcripts (Pause et al., 1994). One major signaling pathway that phosphorylates 4E-BPs is the mammalian target of rapamycin complex 1 (mTORC1) (Ma and Blenis, 2009; Zoncu et al., 2011). By sensing extracellular signals as well as the intracellular energy status, activated mTORC1 phosphorylates 4E-BPs that dissociate from eIF4E, thereby promoting eIF4F complex assembly (Sonenberg and Hinnebusch, 2009). Despite this well-established regulatory mechanism, in many cell lines, mTORC1 inhibition has only modest effects on the rate of protein synthesis (Beretta et al., 1996; Choo et al., 2008). The simplest interpretation of this conundrum is that cells rely on a cap-independent mechanism for a substantial amount of mRNA translation.

Cap-independent translation occurs during normal cellular processes (e.g., mitosis and apoptosis) or when the cap-dependent translation machinery is compromised by either stress or disease (Sonenberg and Hinnebusch, 2007). The best characterized cap-independent initiation mechanisms involve internal ribosome entry sites (IRES) (Hellen and Sarnow, 2001). Discovered in picornavirus mRNAs, the IRES element in the 5’ untranslated region (5’UTR) forms a complex secondary structure capable of recruiting the translation machinery in the absence of some or even all initiation factors. A growing body of evidence suggests that certain cellular mRNAs may use the similar IRES mechanism for cap-independent translation initiation (Gilbert et al., 2007). Systematic approaches have been elaborated to identify putative IRES elements in human and viral genomes (Weingarten-Gabbay et al., 2016). Despite their capability of internal initiation, it is unclear whether these events truly occur within the original sequence context under physiological conditions. In fact, many cellular mRNAs that have been considered to contain IRESs failed to pass through stringent test for internal initiation (Gilbert et al., 2007).

It has been hypothesized that some cellular mRNAs exhibit a relaxed cap dependence because of the presence of so-called cap-independent translation enhancers (CITE) within the untranslated region (Terenin et al., 2013). CITEs are elements in the mRNA capable of recruiting key initiation factors, thereby promoting the assembly of translation initiation complexes. Despite years of speculation, the nature of CITE elements remains obscure. We recently discovered that mRNA methylation in the form of m6A enables cap-independent translation (Meyer et al., 2015; Zhou et al., 2015). As exemplified by selective translation of heat shock-induced Hsp70 mRNA, this finding suggests the existence of a translation initiation mechanism that is neither cap- nor IRES-dependent. This mode of translation initiation offers an attractive solution to the central puzzle that many cap-independent translation events do not follow the IRES mechanism. However, mechanistic details underlying m6A-mediated translation initiation are poorly understood. Several fundamental questions remain unanswered. First, since many transcripts bear 5’UTR methylation, how much does m6A-mediated translation contribute to cellular protein synthesis? Second, for capped mRNAs with 5’UTR methylation, are the canonical cap-dependent translation and the m6A-enabled cap-independent translation mutually exclusive? Third, what is the biological logic behind the selection for different modes of translation initiation? Here, we investigated the scope and mechanism of eIF4F-independent mRNA translation, which revealed a dynamic coordination between different translation modes in response to environmental and physiological stimuli.

RESULTS

The Scope of Physiological Cap-Independent Translation

In response to amino acid deprivation, global protein synthesis is suppressed via inhibition of mTORC1 and activation of the general control non-derepressible-2 kinase (GCN2) (Hinnebusch, 2005; Ma and Blenis, 2009; Wek et al., 2006; Zoncu et al., 2011). While the former regulates eIF4F-mediated 5’ end cap recognition, the latter controls the formation of a ternary complex (TC) comprised of eIF2, GTP and methionine-loaded initiator tRNA (Pisarev et al., 2007). To dissect the contribution of these two rate-limiting steps to the overall translational output, we took advantage of a mouse embryonic fibroblast (MEF) cell line harboring a non-phosphorylatable eIF2α in which the serine 51 (S/S) was mutated to an alanine (A/A) (Scheuner et al., 2001). As expected, wild type eIF2α (S/S) cells readily responded to amino acid deprivation by showing polysome disassembly and concomitant increase of monosome (Figure 1A). To our surprise, eIF2α (A/A) cells showed little changes in the polysome pattern upon amino acid starvation. Consistently, measurement of global protein synthesis revealed much less repression of translation in starved eIF2α (A/A) cells than the wild type (Figure 1B and Figure S1A). The striking resistance to amino acid limitation is also seen in cells lacking GCN2 kinase (Figure S1B). In addition, this phenomenon is highly reproducible under different types of stress, such as unfolded protein response in the endoplasmic reticulum (Figure S1C).

Figure 1. A Substantial Amount of Cellular Translation is Resistant to eIF4F Inhibition.

Figure 1

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(A) Sucrose gradient-based polysome profiling of eIF2α (S/S) and eIF2α (A/A) MEF cells before and after 1 h of amino acid starvation. The right panel shows the monosome/polysome ratio calculated using areas below the curve. Error bars, mean ± s.e.m.; n =3, biological replicates.

(B) Global protein synthesis in starved eIF2α(S/S) and eIF2α(A/A) MEF cells was quantified from puromycin labeling. Error bars, mean ± s.e.m.; n =3, biological replicates.

(C) Immunoblotting of mTORC1 downstream targets in eIF2α(S/S) and eIF2α(A/A) MEF cells before and after 1 h of amino acid starvation.

(D) Immunoblotting of m7GTP pulldown assay in eIF2α(S/S) and eIF2α(A/A) MEF cells before and after 1 h of amino acid starvation.

See also Figure S1 – S3.

Amino acid deprivation is expected to inhibit mTORC1 signaling and consequently suppress eIF4F complex formation at the 5’ end cap (Jewell et al., 2013). It is thus surprising to observe continuous translation in starved eIF2α (A/A) cells. Previous studies suggested that the sensitivity of mTORC1 to nutrient starvation is coupled with GCN2/eIF2α signaling (Ye et al., 2015). It is possible that, in the absence of eIF2α phosphorylation, mTORC1 remains active even under limited supply of amino acids. However, this is not the case. Similar to wild type cells, eIF2α (A/A) cells exhibited rapid dephosphorylation of mTORC1 downstream targets S6K1 and 4E-BP1 upon amino acid deprivation (Figure 1C). Notably, the phosphorylation status of the elongation factor eEF2 was comparable between S/S and A/A cells (Figure S1D). To directly measure the cap functionality in these cells, we conducted a m7G cap pull-down assay before and after nutrient starvation. It is clear that, upon amino acid deprivation, the m7G-associated scaffold protein eIF4G1 was largely replaced by 4E-BP1 in both wild type and eIF2α (A/A) cells (Figure 1D). Therefore, the cap-recognition machinery is inactive under amino acid starvation irrespective of eIF2α phosphorylation.

To independently assess the contribution of cap recognition to global protein synthesis, we took advantage of a chemical compound 4EGI-1 that inhibits eIF4F complex formation by destabilizing eIF4E-eIF4G interaction (Moerke et al., 2007). [35S] metabolic labeling revealed approximately 30% reduction in protein synthesis in both eIF2α (S/S) and (A/A) cells (Figure S2A). Notably, the majority of cellular translation sustained even after prolonged treatment of 4EGI-1 (5 h). We next examined the effect of Torin1, a potent active-site mTOR inhibitor (Thoreen et al., 2009). As expected, Torin1 treatment caused a rapid depletion of phosphorylation for mTORC1 downstream targets (Figure S2B). However, pre-exposure of cells to a high dose of Torin1 (1 µM) only led to less than 50% reduction in protein synthesis (Figure S2C). Therefore, a substantial amount of translation likely follows a mechanism independent of eIF4F.

Cap-Independent Translation Follows a Non-IRES Mechanism

It is possible that the translation maintained under eIF4F inactivation relies on a cap-independent mechanism like IRES or, as recently reported, an alternative cap-recognition mechanism mediated by eIF3d (Lee et al., 2016). In both cases, only a small subset of mRNAs undergo specialized translation. However, we found similar patterns of translational products resolved on the SDS-PAGE gel before and after eIF4F inactivation (Figure S1and S2). This result suggests that the same transcripts are capable of experiencing different modes of translation. We next examined the sensitivity of eIF4F-independent translation to hippuristanol, an eIF4A inhibitor that does not affect certain IRES-driven translation (Bordeleau et al., 2006). Nearly all the translation were repressed in the presence of hippuristanol irrespective of eIF2α signaling (Figure S3A). Therefore, eIF4F-independent translation still requires the scanning process. Further supporting its non-IRES feature, we observed minimal activation of IRES-driven translation in starved eIF2α(A/A) cells (Figure S3B). These results collectively suggest that, when the eIF4F-dependent translation is inactivated, cells readily employ a different mode of translation that is neither cap- nor IRES-dependent.

m6A Mediates Translation that is neither Cap nor IRES-dependent

We previously reported that m6A enables mRNA translation in a cap and IRES-independent manner (Meyer et al., 2015; Zhou et al., 2015). We next investigated whether the substantial amount of translation persisted under eIF4F inactivation follows the m6A-dependent mechanism. To test this possibility, we knocked down METTL3, a core subunit of methyltransferase complex, from MEF cells using shRNA. With more than 90% depletion of METTL3 (Figure 2A), we observed approximately 50% reduction of m6A levels on mRNAs (Figure S4A). METTL3 knockdown caused nearly 30% decrease of global protein synthesis (Figure S4B), which is consistent with the recent study that reported cytosolic function of METTL3 in translation (Lin et al., 2016). However, it is unresolved whether METTL3 plays a role in cap-dependent or cap-independent translation. We reasoned that if METTL3 mediates cap-dependent translation, the translational targets sensitive to METTL3 knockdown should overlap with mTORC1-sensitive targets. If so, METTL3 depletion is not expected to further decrease protein synthesis in the presence of mTORC1 inhibitors. In stark contrast, MEFs lacking METTL3 exhibited much greater sensitivity to Torin1 than the scramble control (Figure 2B), with nearly 80% reduction of protein synthesis after 2 h treatment of Torin1. To substantiate this finding further, we conducted METTL14 knockdown in MEF cells. Similar to METTL3 depletion, reducing METTL14 also sensitized MEF cells to Torin1 treatment (Figure S4C). The additive effect between methyltransferase knockdown and Torin1 treatment clearly indicates that m6A-responsible mRNA translation differs from eIF4F-controlled protein synthesis. To ensure that it is the m6A modification rather than the physical METTL3 binding that mediates eIF4F-independent translation, we complemented with either wild type METTL3 or an inactive D395A mutant to MEFs lacking endogenous METTL3. In the presence of Torin1, only the wild type METTL3, but not the mutant, restored the global protein synthesis (Figure S4D).

Figure 2. Physiological Cap-Independent Translation is Dependent on m6A Modification.

Figure 2

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(A) Immunoblotting of MEFs with or without METTL3 knockdown. The lower panel shows a schematic diagram distinguishing cap-dependent and cap-independent translation.

(B) Global protein synthesis in MEF cells with or without METTL3 knockdown was measured after pre-treatment of 1 µM Torin1 for various times. The right panel shows quantification of [35S] autoradiograph after Torin1 treatment. Error bars, mean ± s.e.m.; n =3, biological replicates.

See also Figure S4.

m6A-Mediated Translation Differs from eIF4F in mRNA Targets

To elucidate the scope of m6A-mediated translation, we examined the translation potential of endogenous transcripts in cells with either METTL3 knockdown or eIF4F inhibition. Previous genome-wide studies revealed that the mRNA subsets highly sensitive to mTORC1 signaling consist almost entirely of transcripts with 5’ terminal oligopyrimidine (TOP) elements (Hsieh et al., 2012; Thoreen et al., 2012). Supporting this notion, the translation of these TOP mRNAs was highly sensitive to Torin1 treatment as revealed by Ribo-seq in MEF cells (Figure S4E). Given the mutually exclusive nature between the TOP motif and m6A sequence context (Figure 3A), it is possible that TOP mRNAs in general have less m6A modification in the 5’UTR and thus heavily rely on the cap-dependent mechanism for translation. Indeed, sequence survey of mouse transcriptome revealed nearly absence of m6A consensus sequence in the 5’UTR of TOP mRNAs (Figure 3B). We further compared the methylation landscape between TOP and non-TOP mRNAs using m6A-seq data derived from MEF cells (Geula et al., 2015). Virtually no m6A modification occurs at the first half of TOP 5’UTR. Since both CDS and 3’UTR regions show comparable m6A distribution between TOP and non-TOP mRNAs, we argue that it is the 5’UTR sequence feature that influences the relative cap-dependency during translation.

Figure 3. m6A Mediates eIF4F-Independent Translation.

Figure 3

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(A) Sequence logo representing the consensus motif relative to m6A (upper panel) and TOP elements (lower panel, derived from Thoreen et al, 2012). For m6A motif, “0” indicates the position of m6A. For TOP motif, “1” represents the 5’ end position.

(B) The top panel shows the frequency of m6A motif across TOP-like (pink line) and non-TOP (blue line) mRNAs. The lower panel shows the distribution of m6A modification across TOP-like (pink line) and non-TOP (blue line) mRNAs using m6A-seq data sets obtained from MEFs.

(C) Scatter plot shows the TE fold change in MEF cells in response to Torin1 treatment or METTL3 knockdown. Blue dots refer to TOP-like transcripts. Both METTL3-sensitive (bottom 10%) and non-sensitive mRNAs (top 10%) are highlighted.

(D) m6A coverage obtained from m6A-seq was plotted for METTL3 non-sensitive (pink line) and METTL3 sensitive (blue line) mRNAs. Relative regions of 5’UTR, CDS, and 3’UTR are shown as the same size.

See also Figure S4.

To test this hypothesis, we examined ribosome profiling data sets obtained from MEF cells depleted of METTL3. Indeed, TOP mRNAs showed little response to METTL3 knockdown (Rho = 0.11) (Figure 3C). In coupling with m6A-seq data sets, we found that transcripts undergoing translational downregulation in the absence of METTL3 generally had higher 5’UTR methylation levels (Figure 3D). The similar observation was evident when we used different m6A-seq data sets derived from MEF cells (Schwartz et al., 2014) (Figure S4F). As an independent validation, we analyzed data sets obtained from HeLa cells with or without METTL3 depletion (Wang et al., 2015). Direct comparison of the translation efficiency (TE) revealed that translation of non-TOP mRNAs was more sensitive to METTL3 depletion than that of TOP mRNAs (P = 2.2 × 10−16) (Figure S4G). Taken together, these data suggest that m6A modification in the 5’UTR renders these transcripts insensitive to eIF4F inhibition by enabling cap-independent translation.

To search for potential m6A readers that mediate eIF4F-independent mRNA translation, we examined YTH domain family proteins residing in the cytoplasm. Interestingly, knocking down YTHDF3, but not YTHDF1 and YTHDF2, re-sensitized MEF cells to Torin1 treatment (Figure S5A). The putative role of YTHDF3 in eIF4F-independent translation is consistent with several recent studies reporting that YTHDF3 facilitated mRNA translation, including m6A-mediated circular RNA translation (Li et al., 2017; Shi et al., 2017; Yang et al., 2017).

METTL3 Recognizes Internal m6A but not the m7G Cap

Previous studies demonstrated that exogenously overexpressed METTL3 co-immunoprecipitated with cap-binding proteins such as CBP80/20 and eIF4E (Lin et al., 2016). These results have been interpreted as fitting a model in which METTL3 promotes cap-dependent translation. To test the cap-binding capability of METTL3, we employed a direct cap-binding assay using recombinant proteins purified from E. coli. The immobilized m7G cap readily precipitated eIF4E, as expected, but failed to pull down METTL3 (Figure 4A). We next examined the cap-binding feature of endogenous proteins by incubating the m7G beads with whole cell lysates. Although the m7G cap readily pull down a considerable amount of eIF4E, no detectable METTL3 was precipitated by the m7G cap (Figure 4B). Notably, the m7G cap failed to capture METTL3 upon mTORC1 inhibition, despite the fact that more eIF4E molecules were recovered from the same lysates. Therefore, endogenous METTL3 does not bind to the cap structure regardless of the functional status of eIF4F.

Figure 4. METTL3 Recognizes m6A but not the m7G Cap Structure.

Figure 4

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(A) Recombinant eIF4E and METTL3 proteins were purified from E. coli as GST fusion proteins followed by incubation with immobilized beads coated with m7GTP. Immunoblotting was conducted using an anti-GST antibody.

(B) Whole cell lysates from HEK293 cells with or without Torin1 treatment were incubated with immobilized beads coated with m7GTP followed by immunoblotting using antibodies indicated.

(C) Synthesized mRNA probes with or without single m6A were radiolabeled with 32P followed by incubation with an increasing dose of recombinant METTL3 (0, 0.3, 0.6, 1.2, 2.4 µg). The mRNA-protein complexes were resolved on a SDS-PAGE gel.

See also Figure S5.

Given the inherent AdoMet binding pocket of METTL3 (Wang et al., 2016a; Wang et al., 2016b), we next examined whether METTL3 directly interacts with methylated mRNA. Using a synthesized 30 nt mRNA with or without a single m6A site at the consensus sequence, we conducted a gel-shifting assay. Although the non-methylated probe weakly associated with METTL3, introducing a single m6A site significantly promoted METTL3 binding (Figure 4C). By contrast, METTL3 showed little preference in association with the capped mRNA synthesized via in vitro transcription (Figure S5B). This result indicates that METTL3 directly binds to the internal m6A, but not the 5’ end cap structure.

Cap-Independent Translation of Hsp70 Requires ABCF1

We previously demonstrated that m6A enables cap-independent translation especially under stress conditions. However, it is unclear how 5’UTR methylation promotes preinitiation complex assembly without forming the canonical eIF4F complex. In an attempt to identify potential mediators responsible for m6A-mediated cap-independent translation, we employed quantitative proteomic analysis to compare proteins associated with the same message experiencing cap-dependent or cap-independent translation. Hsp70 mRNA undergoes translational switch in response to heat shock stress (Zhou et al., 2015), and represents an ideal endogenous transcript to address this question. We adopted a method based on endogenous mRNA pull down without chemical crosslinking using biotinylated probes (Figure 5A). To identify and quantify the associated protein components, we employed an unbiased quantitative approach where digested peptides were labeled with 4-plex isobaric mass tags (iTRAQ) and subjected to liquid chromatography-tandem mass spectrometry analysis (LS-MS/MS). In addition to Hsp70 mRNA, we included β-actin mRNA as a control. HeLa was chosen because of the relatively high basal level of Hsp70 mRNA prior to heat shock stress, therefore permitting direct comparison of components associated with the same transcript before and after stress.

Figure 5. ABCF1 is Essential in Cap-Independent Translation of Hsp70.

Figure 5

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(A) The left panel shows the schematic of quantitative mass spectrometry using iTRAQ. Proteins enriched on β-actin mRNA (middle) or Hsp70 mRNA (right) purified from HeLa cells after heat shock stress are presented as scatter plots. The original peptide score (log2) and stress-induced fold changes (log2) are shown in the x-axis and the y-axis, respectively.

(B) Whole cell lysates from heat shocked HeLa cells were subjected to immunoprecipitation using antibodies indicated followed by immunoblotting.

(C) MEF cells with or without ABCF1 knockdown (Scramble) were collected at indicated times after heat shock stress (43°C, 1 h) followed by immunoblotting. N: no heat shock.

See also Figure S6.

As expected, nearly all the ribosomal proteins showed reduced enrichment after heat shock stress, forming a distinct cluster with high peptide scores (Figure 5A and Table S1). Interestingly, several translation initiation factors were selectively enriched on the Hsp70 transcript, but not the β-actin mRNA, upon heat shock stress (Figure 5A, right panel). Among the most prominent factors are α, β, and γ subunits of eIF2 (> 9 fold). Since eIF2 controls the ternary complex formation, it is clear that the stress-induced Hsp70 mRNA is capable of recruiting the initiator tRNA even when the cap recognition machinery is inactive under heat shock stress. A close inspection of the quantitative proteomic data revealed that eIF2A, eIF5, and ABCF1 were also associated with the stress-induced Hsp70 mRNA. In particular, ABCF1 showed a similar stoichiometry as eIF2 as judged by their comparable peptide scores (Figure 5A, right panel).

We validated the proteomic result by conducting immunoprecipitation (IP) of various endogenous proteins from cell lysates before and after stress. The non-canonical initiation factor eIF2A failed to pull down typical initiation factors such as eIF4G1 and eIF2β (Figure 5B). By contrast, eIF5 constitutively associated with these initiation factors irrespective of the stress condition. Only ABCF1 was able to precipitate eIF4G1 and eIF2β in a stress-dependent manner (Figure 5B). These results suggest that ABCF1 facilitates initiation complex assembly on stress messages in response to heat shock stress. To resolve the physiological role of ABCF1 in stress-induced Hsp70 synthesis, we knocked down ABCF1 in MEF cells using shRNA-expressing lentiviruses. Remarkably, after heat shock stress, the Hsp70 synthesis was severely abolished in cells lacking ABCF1 (Figure 5C). The critical role of ABCF1 in Hsp70 synthesis was also seen in HeLa cells, despite the different basal levels of these proteins (Figure S6A and S6B). Notably, the cellular Hsp70 mRNA levels were even higher in cells with ABCF1 knockdown (Figure S6C), further supporting the translational deficiency of Hsp70 synthesis in the absence of ABCF1.

ABCF1 Facilitates m6A-Mediated Translation

Having found that a substantial amount of cellular translation follows the eIF4F-independent mechanism under the normal growth condition, we asked whether ABCF1 is also responsible for physiological cap-independent translation. ABCF1 knockdown in non-stressed MEFs resulted in about 20% reduction of global protein synthesis (Figure S6D), which is consistent with the previous report (Paytubi et al., 2009). To confirm that ABCF1-responsible translation indeed follows cap-independent initiation mode, we suppressed eIF4F-dependent translation by Torin1 treatment. Similar to the cells lacking METTL3, ABCF1 knockdown rendered MEF cells highly sensitive to Torin1 treatment by showing >80% reduction of protein synthesis (Figure 6A). The increased sensitivity to mTOR1 inhibition in the absence of ABCF1 supports the notion that ABCF1-responsible translation differs from eIF4F-mediated cap-dependent translation.

Figure 6. ABCF1 Mediates eIF4F-Independent Translation.

Figure 6

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(A) Global protein synthesis in MEF cells with or without ABCF1 knockdown was measured after pre-treatment of 1 µM Torin1 for various times. The right panel shows quantification of [35S] autoradiograph after Torin1 treatment. Error bars, mean ± s.e.m.; n =3, biological replicates.

(B) Ribosome profiling data from cells with ABCF1 or METTL3 knockdown were used to determine the fold changes of TE. A scatter plot is presented to show positive correlation.

(C) m6A coverage obtained from m6A-seq was plotted for ABCF1-sensitive (blue line) and non-responsive (pink line) mRNAs. Relative regions of 5’UTR, CDS, and 3’UTR are shown as the same size.

See also Figure S6.

Since ABCF1 resembles METTL3 in mediating eIF4F-independent translation, we predicted that the ABCF1-sensitive transcripts should overlap with METTL3-responsible mRNA targets. This is indeed the case. Ribosome profiling of MEF cells lacking either ABCF1 or METTL3 showed a strong correlation in the changes of TE (r = 0.57, Figure 6B). Further supporting the notion that ABCF1 facilitates m6A-mediated translation, transcripts experiencing decreased translation in the absence of ABCF1 have higher 5’UTR methylation than the one resistant to ABCF1 depletion (Figure 6C). Taken together with the crucial role of ABCF1 and METTL3 in the cap-independent translation of Hsp70 (Figure S6E), these results indicate a functional coordination between ABCF1 and METTL3 in m6A-facilitated translation.

ABCF1 Controls METTL3 Translation

ABCF1 (also termed ABC50) is an ATP-binding cassette protein that, unlike most ABC proteins, lacks membrane-spanning domains (Paytubi et al., 2009; Tyzack et al., 2000). Previous studies demonstrated that ABCF1 promotes translation initiation by interacting with eIF2 and ribosomes (Paytubi et al., 2009; Tyzack et al., 2000). Curiously, in MEF cells lacking ABCF1, there was a decreased steady state level of endogenous METTL3 (Figure 7A). In METTL3-depleted cells, however, the level of ABCF1 was not affected. The reduced METTL3 in the absence of ABCF1 was not due to altered mRNA levels as qPCR revealed little changes of Mettl3 in cells lacking ABCF1 (Figure 7B). This unexpected finding is reminiscent of reduced METTL3 stability in cells lacking METTL14 (Liu et al., 2014), suggesting that ABCF1 could serve as a binding partner for METTL3. However, proteasome inhibition by MG132 treatment did not restore the level of METTL3 (Figure 7A). In addition, we failed to detect the mutual interaction between METTL3 and ABCF1 from either endogenous proteins or transfected genes bearing affinity tags (Figure S7A and S7B). This result is nevertheless consistent with the distinct cellular localization of these proteins: while METTL3 is predominantly a nuclear protein, ABCF1 is mainly localized in the cytoplasm (Figure S7C).

Figure 7. ABCF1 Mediates Translational Control of METTL3.

Figure 7

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(A) MEF cells with ABCF1 or METTL3 knockdown were treated with 5 µM MG132 for 16 hr. Whole cell lysates were collected for immunoblotting using antibodies indicated.

(B) Total RNAs were purified from MEF cells with ABCF1 or METTL3 knockdown followed by qPCR. Error bars, mean ± s.e.m.; n =3, biological replicates.

(C) m6A coverage of METTL3 mRNA using m6A-seq data sets obtained from HeLa and HEK293 cells. The transcript architecture is shown above.

(D) MEF cells were transfected with Fluc plasmids shown in the left and Fluc levels were recorded by real time luminometry. Error bars, mean ± s.e.m.; n =3, biological replicates. *, p < 0.05 (t-test).

See also Figure S7.

Given the critical role of ABCF1 in mRNA translation under stress, we asked whether ABCF1 controls the translation of METTL3. Interestingly, m6A-seq data sets from human cells revealed that the METTL3 mRNA is heavily methylated in the 5’UTR but not 3’UTR (Figure 7C). This unique feature is suggestive of relaxed cap-dependency in METTL3 translation. To test this possibility, we constructed a reporter by placing the 5’UTR of Mettl3 before the firefly luciferase (Fluc) coding region. While the Fluc control showed about 50% reduction of translation in the presence of Torin1, Mettl3-Fluc showed little response to mTOR inhibition (Figure 7D). However, depleting ABCF1 significantly decreased METTL3-Fluc translation (Figure 7D, right panel). The m6A-facilitated translation of m6A “writer” METTL3 suggests a self-regulatory mechanism that offers an alternative translation mode when the cap machinery is inhibited.

DISCUSSION

For years, researchers have been fixated on the idea that eukaryotic mRNA translation relies on two mutually exclusive mechanisms: cap-dependent ribosome scanning and cap-independent internal ribosome entry. Despite the predominant belief that eIF4F-mediated cap-dependent translation contributes to the majority of protein synthesis in eukaryotic cells, it is puzzling that inhibiting cap recognition by chemical inhibitors or genetic ablation only has modest effect on protein synthesis (Beretta et al., 1996; Choo et al., 2008; Yanagiya et al., 2012). The simplest interpretation of this conundrum is that cells rely on cap-independent initiation mechanism for a substantial amount of mRNA translation. The IRES-driven translation has become essentially synonymous with 5’ cap-independent mRNA translation. However, the extent of cellular mRNAs that have been considered to contain IRESs remains controversial (Gilbert, 2010). Although certain mRNAs use the IRES mechanism to achieve the ribosome specificity (Xue et al., 2015), additional concepts are needed to explain how cells maintain robust translation during episodes of eIF4F inhibition. Here we report that m6A-mediated translation initiation follows a cap- and IRES-independent mechanism. Unlike IRES-driven or eIF3d-mediated specialized translation, the m6A-promoted translation co-exists with eIF4F-mediated translation initiation for a great deal of transcripts. The scope of cap-independent translation is therefore much broader than previously appreciated.

METTL3 is an essential enzyme for m6A modification of mRNAs, which primarily occurs in the nucleus (Liu et al., 2014). A recent study adds a new twist to its functionality by demonstrating a cytosolic role of METTL3 in translation (Lin et al., 2016). It surprisingly acts as a “reader” rather than a “writer” of methylated transcripts because the m6A catalytic activity is dispensable in METTL3-promoted translation (Lin et al., 2016). However, it is unclear why only a subset of mRNAs is subjected to translational regulation by METTL3 in this manner. Although METTL3 appears to promote translation in cancer cells, it is not essential in embryonic stem cells (Geula et al., 2015). These puzzling observations call into questions the exact role of METTL3 in translational regulation. One of the key questions is whether METTL3-promoted translation follows cap-dependent or cap-independent mechanisms. We provided evidence that METTL3 in fact primarily facilitates translation independent of eIF4F. In addition, METTL3 does not seem to have the m7G cap-binding capacity, although it readily associates with internal m6A sites. The finding that m6A-mediated translation occurs on fully capped mRNAs suggests that the same transcript undergoes multiple translational modes, which explains the incomplete inhibition of translation by eIF4F inactivation. Indeed, only under the inactivation of mTORC1 signaling, does the remaining translation become highly sensitive to METTL3 depletion. Intriguingly, a recent study reported that m6Am at the 5’ end of mRNAs stabilizes mRNAs and likely promotes translation (Mauer et al., 2017). Since m6Am is part of the 5’ cap structure, it is of importance to demonstrate whether m6Am functions as internal m6A or coordinates with the m7G cap in translational control.

What advantage might m6A-mediated translation confer when the cap machinery is fully functional inside cells? The answer to this question has two distinct but interwoven parts. The first lies in the selectivity of mRNA translation and the second lies in the redistribution of cellular resources. Although mTORC1 primarily controls cap-dependent mRNA translation, it preferentially regulates the translation of TOP mRNAs via poorly understood mechanisms (Hamilton et al., 2006; Thoreen et al., 2012). Notably, the TOP mRNAs are among the most abundant messages in the cell, comprising up to 30% of cellular mRNAs during rapid growth in rich media (Warner, 1999). It is not surprising that the translation of TOP mRNAs must be quickly attenuated in response to limited supply of amino acids. It is conceivable that a diverse group of mRNAs must maintain their translation irrespective of the nutrient signaling. The m6A-mediated translation permits translation of some “privileged” mRNAs to produce proteins important for cell maintenance as well as cell survival. We propose that different modes of translation are coordinated to produce adaptive translatomes in response to environmental and physiological stimuli.

Under stress conditions like amino acid starvation, the amount of ternary complex becomes limited as a result of GCN2-triggered eIF2α phosphorylation (Liu and Qian, 2014; Wek et al., 2006). How does m6A-mediated translation initiation acquire the ternary complex to ensure productive translation? We found that ABCF1 serves as alternative recruiter for the ternary complex during non-canonical translation. ABCF1 is a close relative of the yeast protein GCN20, which is presumed to cooperate with GCN1 in starvation-induced translational response (Marton et al., 1997). Although GCN20 and ABCF1 are similar in their ABC domains, they differ markedly in their N termini. Intriguingly, it is the N terminal region of ABCF1 that interacts with eIF2 in mammalian cells (Paytubi et al., 2008). In cells lacking ABCF1, heat shock-induced Hsp70 translation was severely impaired. Importantly, ABCF1 also plays a role in m6A-facilitated translation under the normal growth condition. Not surprisingly, cells with ABCF1 knockdown exhibits similar translational phenotypes as cells lacking METTL3. Perhaps the most interesting finding is the self-regulation of METTL3 translation that is not only dependent on m6A but also subjected to ABCF1 regulation. This positive feedback loop provides a mechanism by which cells activates m6A-mediated translation upon inhibition of cap-dependent translation. Since the cap machinery evolved at a late stage during eukaryogenesis after the emergence of the nucleus and mRNA cap structure (Hernandez, 2009), it is conceivable that a cap-independent initiation mechanism exists for capped mRNAs in early eukaryotes. The dynamic coordination between different translation modes encourages us to reconsider our traditional view of eIF4F as the primary driver of protein synthesis.

STAR*METHODS

Cell lines and reagents

HeLa cells and MEF cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). Starvation media was based on Hank’s Balanced Salt Solution (HBSS) supplemented with 10% dialyzed FBS. Cycloheximide (#C7698-5G) and puromycin (#P7255-250MG) were purchased from Sigma Aldrich. Torin1 (#4247) was purchased from Tocris Bioscience and dissolved in DMSO. [35S]-methionine was purchased from PerkinElmer (#NEG772007MC). m7GTP beads for cap pulldown experiments were purchased from Jena Biosciences (AC-155).

Lentiviral shRNAs

All shRNA targeting sequences were cloned into DECIPHER™ pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro (Cellecta, CA). shRNA targeting sequences listed below were based on RNAi consortium at Broad Institute (http://www.broad.mit.edu/rnai/trc). Lentiviral particles were packaged using Lenti-X 293T cells (Clontech). Virus-containing supernatants were collected at 48-h after transfection and filtered to eliminate cells. MEF cells were infected by the lentivirus for 48 hr before selection by 2 µg ml−1 puromycin.

Puromycin Labeling

Cells were treated with HBSS + dFBS for 50 minutes before media was changed to HBSS + dFBS supplemented with 10 µM puromycin for an additional ten minutes. Cells were washed twice with ice-cold PBS and lysed with 1× SDS before proteins were separated using SDS-PAGE.

[35S] Radiolabeling

MEF cells were briefly incubated in methionine- and cysteine-free media before addition of 50 µCi of [35S]-methionine. Labeling was stopped by ice-cold DMEM containing 100 µM of cycloheximide. Cells were washed with PBS containing 100 µM of cycloheximide, and lysed with polysome lysis buffer. For the quantitation of [35S]-Met-labeled proteins, cell lysates were resolved on a 10% Tris-Glycine SDS-PAGE and radiography captured by Typhoon 9400. Quantification of [35S] methionine incorporation was done using ImageJ software. For scintillation counting, samples were precipitated by 10% trichloroacetic acid (TCA). The mixture was heated for 10 min at 90°C and then chilled on ice for 10 min. The precipitates were collected on GF/C filter membrane (Watman) and the [35S] incorporation was measured by scintillation counting (Beckman).

Cap pulldown

One 10 cm dish of cells was seeded at 70% confluency and incubated overnight at 37°C. Cells were then washed with ice-cold PBS and lysed with 500 µL polysome buffer supplemented with 1% NP-40 (v/v) and protease inhibitors. Dishes were scraped and cell lysates were clarified at 4°C for 10 minutes at 10,000g. 750 µL of lysate supernatant was added to m7GTP beads that had been washed three times with 1 mL polysome buffer supplemented with 0.1% NP-40 (v/v) followed by incubation at 4°C for 1 hour. Beads were then washed three times with polysome buffer at 4°C. 4× SDS buffer was added directly to beads after the final wash followed by immunoblotting.

Immunoblotting

Cells were lysed on ice in TBS buffer (50 mM Tris, pH7.5, 150 mM NaCl, 1 mM EDTA) containing protease inhibitor cocktail tablet, 1% Triton X-100, and 2 U ml−1 DNase. After incubating on ice for 30 min, the lysates were heated for 10 min in SDS/PAGE sample buffer [50 mM Tris (pH6.8), 100 mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, 10% glycerol). Proteins were separated on SDS-PAGE and transferred to Immobilon-P membranes (Millipore). Membranes were blocked for 1-h in TBS containing 5% non-fat milk and 0.1 % Tween-20, followed by incubation with primary antibodies overnight at 4°C. After incubation with horseradish peroxidase-coupled secondary antibodies at room temperature for 1 h, immunoblots were visualized using enhanced chemiluminescence (ECLPlus, GE Healthcare).

mRNA pulldown using DNA probes

To isolate β-actin and Hsp70 mRNAs, we adapted a previously published method (Starck et al., 2012). In brief, DNA oligos were designed to complement to the 3' ends of β-actin or Hsp70 mRNAs and synthesized by conjugating with biotin at the 5’ end. Cells were lysed using lysis buffer (20 mM Tris-Cl (pH 8.0), 5 mM MgCl2, 100 mM NaCl, 2 mM DTT, 8% sucrose, and 1% NP-40 (v/v)) followed by incubation with DNA oligos at 4°C for 1 hour. Streptavidin beads were added to capture endogenous mRNAs and the associated proteins.

Mass spectrometry

Samples were processed for mass spectrometry by Cornell's Proteomics & Mass Spectrometry Facility. Briefly, samples were desalted and normalized for protein content using a gel-based method. Proteins were digested using trypsin and the generated peptides were analyzed using an Orbitrap nanoLC-MS/MS 90–120 minute gradient. Mascot software was used to map identified peptides to proteins can calculate relative peptide scores.

Real-time PCR

Total RNA was isolated by TRIzol reagent (Invitrogen) and reverse transcription was performed using High Capacity cDNA Reverse Transcription Kit (Invitrogen). Real-time PCR analysis was conducted using Power SYBR Green PCR Master Mix (Applied Biosystems) and carried on a LightCycler 480 Real-Time PCR System (Roche Applied Science).

In vitro transcription

Plasmids containing the corresponding 5’-untranslated region sequences of mouse HSPA1A and full length firefly luciferase were used as templates. Transcripts with normal m7G cap were generated using the mMessage mMachine T7 Ultra kit (Ambion) and transcripts with non-functional cap analog GpppA were synthesized using MEGAscript® T7 Transcription Kit (Ambion). To obtain mRNAs with the adenosine replaced with m6A, in vitro transcription was conducted in a reaction in which 5% of the adenosine was replaced with N6-methyladenosine. All mRNA products were purified using the MEGAclear kit (Ambion) according to the manufacturer’s instruction.

Real-time luciferase assay

Cells grown in 35 mm dishes were transfected with in vitro synthesized mRNA containing the luciferase gene. Luciferase substrate D-luciferin (1 mM, Regis Tech) was added into the culture medium immediately after transfection. Luciferase activity was monitored and recorded using Kronos Dio Luminometer (Atto).

Electrophoretic mobility shift assay

Electrophoretic mobility shift assay was performed as previously described (Alarcón et al., 2015). The m6A-modified or -unmodified RNA probe was synthesized by Thermo Scientific with the sequence of 5'-CGAUCCUCGGCCAGGXCCAGCCUUCCCCA-3' (X=A or m6A). The capped or non-capped mRNA (Hsp70-5'UTR) was synthesized using the mMessage mMachine T7 Ultra kit (Ambion). The probe was labeled in a 50ul reaction mixture containing 2 µl RNA probe (1 µM), 5 µl 10×T4 PNK buffer (NEB), 1 µl T4 PNK (NEB), 40 U ml−1 RNaseOUT (Thermo Scientific), 1 µl [32P]ATP and 40 µl RNase-free water at 37°C for 1 h. The labeled probe was then purified by RNase-free micro bio-spin columns with bio-gel P30 (Bio-Rad 732-6250) according to manufacturer's protocols. After adding 2.5 µl 20×SSC (Promega) buffer, the RNA was denatured at 65 °C for 10 min and slowly cooled down. The purified probe (20 fmol) was incubated with increasing amount of GST-METTL3 at 4 °C for 1 h in binding buffer containing 10 mM HEPES, pH 8.0, 50 mM KCl, 1 mM EDTA, 0.05% Triton-X-100, 5% glycerol, 10 µg ml−1 salmon DNA, 1 mM DTT and 40 U ml-1 RNaseOUT (Thermo Scientific). The RNA-protein mixture was loaded on Novex 8% TBE gel and run at 100 V at 4 °C. The signal was recoded via autoradiography.

Recombinant protein purification and in vitro cap-binding assay

Mettl3 and Eif4e coding sequences were cloned into pGEX-6P-1 vector using the primers as follows: METTL3-F, 5'-ACGCGTCGACTCATGTCGGACACGTGGAGCTC-3'; METTL3-R, 5'-ATAAGAATGCGGCCGCCTATAAATTCTTAGGTTTAG-3'; eIF4E-F, 5'-GCGAATTCATGGCGACTGTCGAACCGGA-3'; eIF4E-R, 5'-CCGCTCGAGTTAAACAACAAACCTATTTTTAG-3'. The constructs were transformed into the E. coli bacteria BL21. After inducing the fusion protein expression at 20 °C for 3–4 h in the presence of 0.5 mM IPTG, cells were collected and lysed in PBS lysis buffer supplemented with 0.5 mM PMSF, 1 mM DTT, protease inhibitor cocktail (Roche), 0.1% (v/v) Triton X-100 and sonicated for 10 min. Cell debris was removed by centrifugation at 12,000 r.p.m. for 30 min, the supernatant was mixed with 2 ml equilibrated Pierce glutathione agarose and incubated for 2–3 h at 4 °C. The resin was washed five times and eluted in GST elution buffer (5mM glutathione, 50mM Tris-HCl (pH 8.0)).

Purified GST-METTL3 or GST-eIF4E protein was incubated with m7GTP agarose beads (Jena Bioscience) in binding buffer (20 mM Tris-HCl, 100mM NaCl, 25mM MgCl2, 0.5% Nonidet P-40, and protease inhibitors) at 4 °C for 2 h. Pelleted beads were washed four times with 0.5 ml of binding buffer and re-suspended in 0.6 ml of binding buffer supplemented with 1 mM GTP for another 1 h at 4 °C. After washing with lysis buffer for four times, the beads were re-suspended in sample buffer and boiled for 5 min. m7GTP-bound proteins, GTP wash and input (5% purified proteins) were loaded on 8% SDS-PAGE gels and subjected to western blot using anti-GST antibody (1:1000).

Immunofluorescence staining

Cells grown on glass coverslips were fixed in 4% paraformaldehyde for 10 min at room temperature. After permeabilization in 0.2% Triton X-100 for 5 min at room temperature, the cover slips were washed by PBS for three times and then blocked with 1% BSA for 30 min. Cells were stained with indicated primary antibody overnight at 4 °C, followed by incubation with Alexa Fluor 546 donkey anti-mouse secondary antibody or Alexa Fluor 546 donkey anti-rabbit secondary antibody for 1 h at room temperature. The nuclei were counter-stained with Hechest (1:1000) for 10 min. Cover slips were mounted onto slides and visualized using a Zeiss LSM710 confocal microscope.

Co-immunoprecipitation assay

HEK293 cells (one 10-cm plate) transfected with SBP-tagged METTL3 only or SBP-tagged METTL3 with ABCF1-myc plasmids for 24 h. The transfected cells were subjected to amino acid starvation for 2 h or with heat shock stress (42 °C) for 1 h followed by recovery at 37 °C for 2 h. Cells were collected by centrifuge at 1000 rpm for 5 min. The cell pellet was lysed in lysis buffer (150 mM KCl, 10 mM HEPES (pH 7.6), 1 mM EDTA, 0.5% NP-40, 0.5 mM DTT and protease inhibitor cocktail) and rotated at 4 °C for 30 min. The cell debris was removed by centrifugation at 14,000 rpm for 15 min. The supernatant was incubated with streptavidin magnetic beads in lysis buffer for 3–4 h at 4 °C. The beads were collected by magnetic stand, and the supernatant was saved as the fraction of flow through. Subsequently the beads were washed with 1 ml wash buffer (200 mM NaCl, 50 mM HEPES (pH 7.6), 1 mM EDTA and 0.05% NP-40, 0.5 mM DTT, protease inhibitor cocktail) for 6 times. SDS buffer was directly added in the beads and boiled for 5 min. The samples were loaded on 8% SDS-PAGE gels and subjected to western blot using indicated antibodies.

m6A dot blot

mRNA was purified from total RNA using Dynabeads® Oligo(dT)25 (Thermo Fisher). Equal amounts of mRNA were spotted to a nylon membrane (Fisher), followed by UV crosslinking at UV 254 nm, 0.12 J/cm2. After blocking in PBST containing 5% non-fat milk and 0.1 % Tween-20 for 1 hr, the membrane was incubated with 1:1000 diluted anti-m6A antibody overnight at 4 °C. The membrane was incubated with HRP-conjugated anti-rabbit IgG (1:5000 dilution) for 1 hr and visualized by using enhanced chemiluminescence (ECLPlus, GE Healthcare).

Polysome profiling analysis

Sucrose solutions were prepared in polysome buffer (10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2, 100 µg ml−1 cycloheximide and 2% Triton X-100). A 15%– 45% (w/v) Sucrose density gradients were freshly prepared in SW41 ultracentrifuge tubes (Backman) using a Gradient Master (BioComp Instruments). Cells were pre-treated with 100 µg ml−1 cycloheximide for 3 min at 37°C followed by washing using ice-cold PBS containing 100 µg ml−1 cycloheximide. Cells were then lysed in polysome lysis buffer. Cell debris were removed by centrifugation at14,000 rpm for 10 min at 4°C. 500 µl of supernatant was loaded onto sucrose gradients followed by centrifugation for 2 h 28 min at 38,000 rpm 4°C in a SW41 rotor. Separated samples were fractionated at 0.75 ml/min through an automated fractionation system (Isco) that continually monitores OD254 values. An aliquot of ribosome fraction were used to extract total RNA using Trizol LS reagent (Invitrogen) for real-time PCR analysis.

RNA-seq and m6A–seq

For m6A immunoprecipitation, total RNA was first isolated using Trizol reagent followed by fragmentation using freshly prepared RNA fragmentation buffer (10 mM Tris-HCl, pH 7.0, 10 mM ZnCl2). 5 µg fragmented RNA was saved as input control for RNA-seq. 1 mg fragmented RNA was incubated with 15 µg anti-m6A antibody (Millipore ABE572) in 1× IP buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 0.1% Igepal CA-630) for 2 hr at 4°C. The m6A-IP mixture was then incubated with Protein A beads for additional 2 hr at 4°C on a rotating wheel. After washing 3 times with IP buffer, bound RNA was eluted using 100 µl elution buffer (6.7 mM N6-Methyladenosine 5’-monophosphate sodium salt in 1× IP buffer), followed by ethanol precipitation. Precipitated RNA was used for cDNA library construction and high-throughput sequencing described below.

Ribo-seq

Ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop digestion. Total RNA was extracted using TRIzol reagent. Purified RNA was used for cDNA library construction and high-throughput sequencing described below.

cDNA library construction

Fragmented RNA input and m6A-IP elutes were dephosphorylated for 1 hr at 37°C in 15 µl reaction (1× T4 polynucleotide kinase buffer, 10 U SUPERase_In and 20 U T4 polynucleotide kinase). The products were separated on a 15 % polyacrylamide TBE-urea gel (Invitrogen) and visualized using SYBR Gold (Invitrogen). Selected regions of the gel corresponding to 40–60 nt (for RNA-seq and m6A-seq) or 25–35 nt (for Ribo-seq) were excised. The gel slices were disrupted by using centrifugation through the holes at the bottom of the tube. RNA fragments were dissolved by soaking overnight in 400 µl gel elution buffer (300 mM NaOAc, pH 5.5, 1 mM EDTA, 0.1 U/ml SUPERase_In). The gel debris was removed using a Spin-X column (Corning), followed by ethanol precipitation. Purified RNA fragments were re-suspended in nuclease-free water. Poly-(A) tailing reaction was carried out for 45 min at 37°C (1 × poly-(A) polymerase buffer, 1 mM ATP, 0.75 U/µl SUPERase_In and 3 U E. coli poly-(A) polymerase).

For reverse transcription, the following oligos containing barcodes were used: MCA02: 5’-pCAGATCGTCGGACTGTAGAACTCTCAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3’; LGT03: 5’-pGTGATCGTCGGACTGTAGAACTCTCAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3’; YAG04: 5’-pAGGATCGTCGGACTGTAGAACTCTCAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3’; HTC05: 5’-pTCGATCGTCGGACTGTAGAACTCTCAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN-3’.

In brief, the tailed-RNA sample was mixed with 0.5 mM dNTP and 2.5 mM synthesized primer and incubated at 65°C for 5 min, followed by incubation on ice for 5 min. The reaction mix was then added with 20 mM Tris (pH 8.4), 50 mM KCl, 5 mM MgCl2, 10 mM DTT, 40 U RNaseOUT and 200 U SuperScript III. Reverse transcription reaction was performed according to the manufacturer’s instruction. Reverse transcription products were separated on a 10% polyacrylamide TBE-urea gel as described earlier. The extended first-strand product band was expected to be approximately 100 nt, and the corresponding region was excised. The cDNA was recovered by using DNA gel elution buffer (300 mM NaCl, 1 mM EDTA). First-strand cDNA was circularized in 20 µl of reaction containing 1× CircLigase buffer, 2.5 mM MnCl2, 1M Betaine, and 100 U CircLigase II (Epicentre). Circularization was performed at 60 °C for 1 h, and the reaction was heat inactivated at 80 °C for 10 min. Circular single-strand DNA was re-linearized with 20 mM Tris-acetate, 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM DTT, and 7.5 U APE 1 (NEB). The reaction was carried out at 37 °C for 1 h. The linearized single-strand DNA was separated on a Novex 10% polyacrylamide TBE-urea gel (Invitrogen) as described earlier. The expected 100-nt product bands were excised and recovered as described earlier.

Deep sequencing

Single-stranded template was amplified by PCR by using the Phusion High-Fidelity enzyme (NEB) according to the manufacturer’s instructions. The oligonucleotide primers qNTI200 (5’-CAAGCAGAAGACGGCATA-3’) and qNTI201 (5’-AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACG-3’) were used to create DNA suitable for sequencing, i.e., DNA with Illumina cluster generation sequences on each end and a sequencing primer binding site. The PCR contains 1× HF buffer, 0.2 mM dNTP, 0.5 µM oligonucleotide primers, and 0.5 U Phusion polymerase. PCR was carried out with an initial 30 s denaturation at 98 °C, followed by 12 cycles of 10 s denaturation at 98 °C, 20 s annealing at 60 °C, and 10 s extension at 72 °C. PCR products were separated on a nondenaturing 8% polyacrylamide TBE gel as described earlier. Expected DNA at 120 bp (for Ribo-seq), or 140 bp (for RNA-seq and m6A-seq) was excised and recovered as described earlier. After quantification by Agilent BioAnalyzer DNA 1000 assay, equal amount of barcoded samples were pooled into one sample. Approximately 3–5 pM mixed DNA samples were used for cluster generation followed by deep sequencing by using sequencing primer 5’-CGACAGGTTCAGAGTTCTACAGTCCGACGATC-3’ (Illumina HiSeq).

Preprocessing of sequencing reads

For Ribo-seq, the 3’ adaptor and low quality bases were trimmed by Cutadapt(Martin, 2011). The trimmed reads with length < 15 nucleotides were excluded. The remaining reads were mapped to the mouse transcriptome (ENSEML gene, GRCm38, longest CDS was selected if multiple transcripts existing) using Bowtie (Langmead et al., 2009) with parameters: -a -m1 --best --strata. Two mismatches are permitted. Non-uniquely mapped reads were disregarded for further analysis due to ambiguity. The same mapping procedure was applied to RNA-seq and m6A-seq. For Ribo-seq, the 13th position (12nt offset from the 5’ end) of the uniquely mapped read was defined as the ribosome “P-site” position. Uniquely mapped reads of Ribo-seq in the mRNA coding region were used to calculate the RPKM values for translation levels. For RNAseq, uniquely mapped reads in the full length of mRNA were used to calculate the RPKM values. Transcripts with RPKM <1 were excluded. Translation efficiency (TE) was defined as the ratio of Ribo-seq over RNA-seq.

Identification of m6A sites

We used a similar scanning strategy reported previously to identify m6A peaks in the immunoprecipitation sample as compared to the input sample (Dominissini et al., 2012). In brief, for each ENSEMBL gene, a sliding window of 50 nucleotides with step size of 25 nucleotides was employed to scanning the longest isoform (based on CDS length; in the case of equal CDS, the isoform with longer UTRs was selected). Only transcripts in which the average coverage in immunoprecipitation is higher than 30 per kilo base were used in further analysis for reduction of background noise. For each window, a Peak-Over-Median score (POM) was derived by calculating the ratio of mean read coverage in the window to the median read coverage of the whole gene body. Windows scoring higher than 3 in the IP sample were obtained and all the resultant overlapping m6A peak windows in the IP sample were iteratively clustered to infer the boundary of m6A enriched region as well as peak position with maximal read coverage. Finally, a Peak-Over-Input (POI) score was assigned to each m6A enriched region by calculating the ratio of POM in the IP sample to that in the input sample. A putative m6A site was defined if the POI score was higher than 3. The peak position of each m6A site was classified into three mutually exclusive mRNA structural regions including 5’ UTR, CDS, and 3’ UTR. For whole-transcript coverage plots, coverage at individual site was normalized by mean coverage of the transcript firstly, then each mRNA region was divided into 100 bins, coverages in the same bins were aggregated.

Motif analysis

The 1000 m6A peaks with highest POI score were selected consensus motif finding. We used MEME Suite for motif analysis with parameters; -nmotifs3 -maxw6 –minw3 (Bailey et al., 2009). In brief, the flanking sequences of m6A peaks (± 40 nt) with POI scores were retrieved from mouse transcritpome and were used as MEME input.

Data and software availability

Statistical analysis was mainly done using GraphPad Prism and SigmaPlot (Systat Software). Unless otherwise noted, some analytical procedures were completed using custom Perl scripts. These scripts are available upon request. Deep sequencing data from Ribo-seq and RNA-seq have been deposited in the NCBI Gene Expression Omnibus (GEO) database with accession code: GSE101865. Original images can be found in Mendeley Data (doi:10.17632/9mrmpfb4y2.1).

Supplementary Material

1
2

Table S1. Quantitative proteomics of Hspa1a binding proteins, Related to Figure 5A

Highlights.

  • eIF4F inhibition partially represses global protein synthesis

  • Translation of non-TOP mRNAs depends on 5’UTR N6-methyladenosine

  • ABCF1 is critical for eIF4F-independent mRNA translation

  • ABCF1 coordinates with METTL3 in m6A-facilitated mRNA translation

Acknowledgments

We’d like to thank Dr. Jerry Pelletier for providing hippuristanol and Qian lab members for helpful discussion. We are grateful to Cornell University Life Sciences Core Laboratory Center for performing deep sequencing. This work was supported by grants to to S.-B.Q. from US National Institutes of Health (R01AG042400), US Department of Defense (W81XWH-14-1-0068), and HHMI Faculty Scholar (55108556).

Footnotes

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AUTHOR CONTRIBUTIONS

R.A.C. and S.-B.Q. conceived the project and designed the study. R.A.C. performed most of the experiments. X.-M.L. performed cap binding and protein interaction assays as well as Ribo-seq and RNA-seq. Y.M. and J.W. analyzed the sequencing data. J.Z. conducted m6A-seq. L.D. assisted Ribo-seq. X.Z. conducted MS experiment. S.-B.Q. wrote the manuscript. All authors discussed results and edited the manuscript.

References

  1. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37:W202–208. doi: 10.1093/nar/gkp335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beretta L, Gingras AC, Svitkin YV, Hall MN, Sonenberg N. Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation. EMBO J. 1996;15:658–664. [PMC free article] [PubMed] [Google Scholar]
  3. Bordeleau ME, Mori A, Oberer M, Lindqvist L, Chard LS, Higa T, Belsham GJ, Wagner G, Tanaka J, Pelletier J. Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A. Nat Chem Biol. 2006;2:213–220. doi: 10.1038/nchembio776. [DOI] [PubMed] [Google Scholar]
  4. Choo AY, Yoon SO, Kim SG, Roux PP, Blenis J. Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation. Proc Natl Acad Sci U S A. 2008;105:17414–17419. doi: 10.1073/pnas.0809136105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, et al. Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012;485:201–206. doi: 10.1038/nature11112. [DOI] [PubMed] [Google Scholar]
  6. Gebauer F, Hentze MW. Molecular mechanisms of translational control. Nat Rev Mol Cell Biol. 2004;5:827–835. doi: 10.1038/nrm1488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Geula S, Moshitch-Moshkovitz S, Dominissini D, Mansour AA, Kol N, Salmon-Divon M, Hershkovitz V, Peer E, Mor N, Manor YS, et al. Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science. 2015;347:1002–1006. doi: 10.1126/science.1261417. [DOI] [PubMed] [Google Scholar]
  8. Gilbert WV. Alternative ways to think about cellular internal ribosome entry. J Biol Chem. 2010;285:29033–29038. doi: 10.1074/jbc.R110.150532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gilbert WV, Zhou K, Butler TK, Doudna JA. Cap-independent translation is required for starvation-induced differentiation in yeast. Science. 2007;317:1224–1227. doi: 10.1126/science.1144467. [DOI] [PubMed] [Google Scholar]
  10. Gross JD, Moerke NJ, von der Haar T, Lugovskoy AA, Sachs AB, McCarthy JE, Wagner G. Ribosome loading onto the mRNA cap is driven by conformational coupling between eIF4G and eIF4E. Cell. 2003;115:739–750. doi: 10.1016/s0092-8674(03)00975-9. [DOI] [PubMed] [Google Scholar]
  11. Hamilton TL, Stoneley M, Spriggs KA, Bushell M. TOPs and their regulation. Biochem Soc Trans. 2006;34:12–16. doi: 10.1042/BST20060012. [DOI] [PubMed] [Google Scholar]
  12. Hellen CU, Sarnow P. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 2001;15:1593–1612. doi: 10.1101/gad.891101. [DOI] [PubMed] [Google Scholar]
  13. Hernandez G. On the origin of the cap-dependent initiation of translation in eukaryotes. Trends Biochem Sci. 2009;34:166–175. doi: 10.1016/j.tibs.2009.02.001. [DOI] [PubMed] [Google Scholar]
  14. Hinnebusch AG. Translational regulation of GCN4 and the general amino acid control of yeast. Annu Rev Microbiol. 2005;59:407–450. doi: 10.1146/annurev.micro.59.031805.133833. [DOI] [PubMed] [Google Scholar]
  15. Hinnebusch AG. The scanning mechanism of eukaryotic translation initiation. Annu Rev Biochem. 2014;83:779–812. doi: 10.1146/annurev-biochem-060713-035802. [DOI] [PubMed] [Google Scholar]
  16. Hsieh AC, Liu Y, Edlind MP, Ingolia NT, Janes MR, Sher A, Shi EY, Stumpf CR, Christensen C, Bonham MJ, et al. The translational landscape of mTOR signalling steers cancer initiation and metastasis. Nature. 2012;485:55–61. doi: 10.1038/nature10912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jackson RJ, Hellen CU, Pestova TV. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol. 2010;11:113–127. doi: 10.1038/nrm2838. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jewell JL, Russell RC, Guan KL. Amino acid signalling upstream of mTOR. Nat Rev Mol Cell Biol. 2013;14:133–139. doi: 10.1038/nrm3522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10:R25. doi: 10.1186/gb-2009-10-3-r25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee AS, Kranzusch PJ, Doudna JA, Cate JH. eIF3d is an mRNA cap-binding protein that is required for specialized translation initiation. Nature. 2016;536:96–99. doi: 10.1038/nature18954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Li A, Chen YS, Ping XL, Yang X, Xiao W, Yang Y, Sun HY, Zhu Q, Baidya P, Wang X, et al. Cytoplasmic m6A reader YTHDF3 promotes mRNA translation. Cell Res. 2017;27:444–447. doi: 10.1038/cr.2017.10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lin S, Choe J, Du P, Triboulet R, Gregory RI. The mA Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. Mol Cell. 2016 doi: 10.1016/j.molcel.2016.03.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Liu B, Qian SB. Translational reprogramming in cellular stress response. Wiley Interdiscip Rev RNA. 2014;5:301–315. doi: 10.1002/wrna.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, Jia G, Yu M, Lu Z, Deng X, et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol. 2014;10:93–95. doi: 10.1038/nchembio.1432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ma XM, Blenis J. Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol. 2009;10:307–318. doi: 10.1038/nrm2672. [DOI] [PubMed] [Google Scholar]
  26. Marintchev A, Edmonds KA, Marintcheva B, Hendrickson E, Oberer M, Suzuki C, Herdy B, Sonenberg N, Wagner G. Topology and regulation of the human eIF4A/4G/4H helicase complex in translation initiation. Cell. 2009;136:447–460. doi: 10.1016/j.cell.2009.01.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011;17:10–12. [Google Scholar]
  28. Marton MJ, Vazquez de Aldana CR, Qiu H, Chakraburtty K, Hinnebusch AG. Evidence that GCN1 and GCN20, translational regulators of GCN4, function on elongating ribosomes in activation of eIF2alpha kinase GCN2. Mol Cell Biol. 1997;17:4474–4489. doi: 10.1128/mcb.17.8.4474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mauer J, Luo X, Blanjoie A, Jiao X, Grozhik AV, Patil DP, Linder B, Pickering BF, Vasseur JJ, Chen Q, et al. Reversible methylation of m6Am in the 5' cap controls mRNA stability. Nature. 2017;541:371–375. doi: 10.1038/nature21022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Meyer KD, Patil DP, Zhou J, Zinoviev A, Skabkin MA, Elemento O, Pestova TV, Qian SB, Jaffrey SR. 5' UTR m(6)A Promotes Cap-Independent Translation. Cell. 2015;163:999–1010. doi: 10.1016/j.cell.2015.10.012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moerke NJ, Aktas H, Chen H, Cantel S, Reibarkh MY, Fahmy A, Gross JD, Degterev A, Yuan J, Chorev M, et al. Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G. Cell. 2007;128:257–267. doi: 10.1016/j.cell.2006.11.046. [DOI] [PubMed] [Google Scholar]
  32. Pause A, Belsham GJ, Gingras AC, Donze O, Lin TA, Lawrence JC, Jr, Sonenberg N. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature. 1994;371:762–767. doi: 10.1038/371762a0. [DOI] [PubMed] [Google Scholar]
  33. Paytubi S, Morrice NA, Boudeau J, Proud CG. The N-terminal region of ABC50 interacts with eukaryotic initiation factor eIF2 and is a target for regulatory phosphorylation by CK2. Biochem J. 2008;409:223–231. doi: 10.1042/BJ20070811. [DOI] [PubMed] [Google Scholar]
  34. Paytubi S, Wang X, Lam YW, Izquierdo L, Hunter MJ, Jan E, Hundal HS, Proud CG. ABC50 promotes translation initiation in mammalian cells. J Biol Chem. 2009;284:24061–24073. doi: 10.1074/jbc.M109.031625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pisarev AV, Hellen CU, Pestova TV. Recycling of eukaryotic posttermination ribosomal complexes. Cell. 2007;131:286–299. doi: 10.1016/j.cell.2007.08.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Scheuner D, Song B, McEwen E, Liu C, Laybutt R, Gillespie P, Saunders T, Bonner-Weir S, Kaufman RJ. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol Cell. 2001;7:1165–1176. doi: 10.1016/s1097-2765(01)00265-9. [DOI] [PubMed] [Google Scholar]
  37. Schutz P, Bumann M, Oberholzer AE, Bieniossek C, Trachsel H, Altmann M, Baumann U. Crystal structure of the yeast eIF4A-eIF4G complex: an RNA-helicase controlled by protein-protein interactions. Proc Natl Acad Sci U S A. 2008;105:9564–9569. doi: 10.1073/pnas.0800418105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schwartz S, Mumbach MR, Jovanovic M, Wang T, Maciag K, Bushkin GG, Mertins P, Ter-Ovanesyan D, Habib N, Cacchiarelli D, et al. Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5' sites. Cell Rep. 2014;8:284–296. doi: 10.1016/j.celrep.2014.05.048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shi H, Wang X, Lu Z, Zhao BS, Ma H, Hsu PJ, Liu C, He C. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA. Cell Res. 2017;27:315–328. doi: 10.1038/cr.2017.15. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sonenberg N, Hinnebusch AG. New modes of translational control in development, behavior, and disease. Mol Cell. 2007;28:721–729. doi: 10.1016/j.molcel.2007.11.018. [DOI] [PubMed] [Google Scholar]
  41. Sonenberg N, Hinnebusch AG. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell. 2009;136:731–745. doi: 10.1016/j.cell.2009.01.042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Starck SR, Jiang V, Pavon-Eternod M, Prasad S, McCarthy B, Pan T, Shastri N. Leucine-tRNA initiates at CUG start codons for protein synthesis and presentation by MHC class I. Science. 2012;336:1719–1723. doi: 10.1126/science.1220270. [DOI] [PubMed] [Google Scholar]
  43. Terenin IM, Andreev DE, Dmitriev SE, Shatsky IN. A novel mechanism of eukaryotic translation initiation that is neither m7G-cap-, nor IRES-dependent. Nucleic Acids Res. 2013;41:1807–1816. doi: 10.1093/nar/gks1282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Thoreen CC, Chantranupong L, Keys HR, Wang T, Gray NS, Sabatini DM. A unifying model for mTORC1-mediated regulation of mRNA translation. Nature. 2012;485:109–113. doi: 10.1038/nature11083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Thoreen CC, Kang SA, Chang JW, Liu Q, Zhang J, Gao Y, Reichling LJ, Sim T, Sabatini DM, Gray NS. An ATP-competitive mammalian target of rapamycin inhibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem. 2009;284:8023–8032. doi: 10.1074/jbc.M900301200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tyzack JK, Wang X, Belsham GJ, Proud CG. ABC50 interacts with eukaryotic initiation factor 2 and associates with the ribosome in an ATP-dependent manner. J Biol Chem. 2000;275:34131–34139. doi: 10.1074/jbc.M002868200. [DOI] [PubMed] [Google Scholar]
  47. Wang P, Doxtader KA, Nam Y. Structural Basis for Cooperative Function of Mettl3 and Mettl14 Methyltransferases. Mol Cell. 2016a;63:306–317. doi: 10.1016/j.molcel.2016.05.041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wang X, Feng J, Xue Y, Guan Z, Zhang D, Liu Z, Gong Z, Wang Q, Huang J, Tang C, et al. Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex. Nature. 2016b;534:575–578. doi: 10.1038/nature18298. [DOI] [PubMed] [Google Scholar]
  49. Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H, Weng X, Chen K, Shi H, He C. N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell. 2015;161:1388–1399. doi: 10.1016/j.cell.2015.05.014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Warner JR. The economics of ribosome biosynthesis in yeast. Trends Biochem Sci. 1999;24:437–440. doi: 10.1016/s0968-0004(99)01460-7. [DOI] [PubMed] [Google Scholar]
  51. Weingarten-Gabbay S, Elias-Kirma S, Nir R, Gritsenko AA, Stern-Ginossar N, Yakhini Z, Weinberger A, Segal E. Comparative genetics. Systematic discovery of cap-independent translation sequences in human and viral genomes. Science. 2016:351. doi: 10.1126/science.aad4939. [DOI] [PubMed] [Google Scholar]
  52. Wek RC, Jiang HY, Anthony TG. Coping with stress: eIF2 kinases and translational control. Biochem Soc Trans. 2006;34:7–11. doi: 10.1042/BST20060007. [DOI] [PubMed] [Google Scholar]
  53. Xue S, Tian S, Fujii K, Kladwang W, Das R, Barna M. RNA regulons in Hox 5' UTRs confer ribosome specificity to gene regulation. Nature. 2015;517:33–38. doi: 10.1038/nature14010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Yanagiya A, Suyama E, Adachi H, Svitkin YV, Aza-Blanc P, Imataka H, Mikami S, Martineau Y, Ronai ZA, Sonenberg N. Translational homeostasis via the mRNA cap-binding protein, eIF4E. Mol Cell. 2012;46:847–858. doi: 10.1016/j.molcel.2012.04.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yang Y, Fan X, Mao M, Song X, Wu P, Zhang Y, Jin Y, Yang Y, Chen LL, Wang Y, et al. Extensive translation of circular RNAs driven by N6-methyladenosine. Cell Res. 2017;27:626–641. doi: 10.1038/cr.2017.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Ye J, Palm W, Peng M, King B, Lindsten T, Li MO, Koumenis C, Thompson CB. GCN2 sustains mTORC1 suppression upon amino acid deprivation by inducing Sestrin2. Genes Dev. 2015;29:2331–2336. doi: 10.1101/gad.269324.115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zhou J, Wan J, Gao X, Zhang X, Jaffrey SR, Qian SB. Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature. 2015;526:591–594. doi: 10.1038/nature15377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol. 2011;12:21–35. doi: 10.1038/nrm3025. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS915694-supplement-1.pdf (1MB, pdf)
2

Table S1. Quantitative proteomics of Hspa1a binding proteins, Related to Figure 5A

NIHMS915694-supplement-2.xlsx (3.2MB, xlsx)

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