Ribosome biogenesis is a complex and energetically costly process for the cell. Eukaryotic cells exert precise temporal and stoichiometric control over ribosomal protein expression to ensure the production of functional ribosomes meets the growth demands of the cell. Multicellular organisms regulate ribosome biogenesis post-transcriptionally, at the level of mRNA translation (Meyuhas and Kahan, 2015; Perry, 2007). All transcripts encoding ribosomal proteins, most translation factors, and some RNA-binding proteins carry a cis-regulatory RNA element termed the 5’terminal oligopyrimidine (5’TOP) motif comprising an invariant 5’-cytidine followed by an uninterrupted tract of 4–14 pyrimidine nucleotides and preceded by the 7-methylguanosine triphosphate (m⁷Gppp) cap (Fonseca et al., 2014; Iadevaia et al., 2008; Meyuhas and Kahan, 2015; Levy et al., 1991). This motif is essential for translation regulation of TOP mRNAs (Biberman and Meyuhas, 1999; Avni et al., 1994; Meyuhas and Kahan, 2015).

The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway plays a prominent role in the control of TOP mRNA translation (Fonseca et al., 2014; Hsieh et al., 2012; Thoreen et al., 2012). In response to provision of nutrients (such as amino acids, growth factors, glucose, or oxygen), the growth-associated kinase complex mTORC1 boosts the production of components of the translation machinery encoded by TOP mRNAs (Meyuhas and Kahan, 2015; Chantranupong et al., 2015). Pharmacological inhibition of mTORC1 results in the pronounced repression of TOP mRNA translation (Terada et al., 1994; Jefferies et al., 1994; Thoreen et al., 2012; Hsieh et al., 2012).

Genome-wide ribosome profiling studies demonstrate that TOP mRNAs are the class of transcripts that are most sensitive to translation suppression by mTOR inhibitors (Thoreen et al., 2012; Hsieh et al., 2012). TOP mRNA translation has been the subject of intense study and several mechanisms have been proposed for its regulation by mTORC1 to date. Most recently, the mRNA cap-binding protein, eIF4E, and its repressor proteins, 4E-BP1 and 4E-BP2, have been linked to the control of TOP mRNA translation downstream of mTORC1 (Hsieh et al., 2012; Thoreen, 2017; Thoreen et al., 2012), but the exact contribution of 4E-BPs to the control of TOP mRNA translation remains a point of contention (Miloslavski et al., 2014). While 4E-BPs and many other candidates have been linked to the control of TOP mRNA translation (discussed in detail in (Meyuhas and Kahan, 2015)), the identity of the bona fide trans-acting factor that represses TOP mRNA translation downstream of mTORC1 remained enigmatic.

Recently, we and others (Fonseca et al., 2015; Tcherkezian et al., 2014) identified La-related protein 1 (LARP1) as a novel downstream target of mTORC1 and proposed that it plays an important role in the translation of TOP mRNAs. Specifically, we have shown that LARP1 associates with the regulatory-associated protein of mTOR (RAPTOR) when mTORC1 is active. Upon mTORC1 inhibition, a cellular state that is associated with TOP mRNA translation repression, we demonstrated that LARP1 dissociates from mTORC1 and binds to the 5’TOP motif of TOP mRNAs (Fonseca et al., 2015). We have also shown that LARP1 associates with the 5’TOP motif via a LARP1 family-specific ‘DM15 region’ located within its C-terminus (Lahr et al., 2015). In the present study, we confirm a direct association between the DM15 region and the 5’TOP motif.

Most importantly, our crystallographic data revealed an unexpected, but seminal role for the DM15 region of LARP1 in specialized cap-binding of TOP mRNAs. We show that the DM15 region of LARP1 specifically binds the 7-methylguanosine 5’−5’ triphosphate (m⁷Gppp) moiety and the invariant first cytidine of TOP mRNAs. Biochemical analyses reveal that LARP1 selectively prevents the binding of eIF4E to the m⁷Gppp cap to block the assembly of the eIF4F complex on TOP mRNAs. These important findings highlight a previously unrecognized dynamic interplay between LARP1 and eIF4F in the control of TOP mRNA translation and reconcile earlier, seemingly contradictory models of TOP mRNA translation control.

To better understand how LARP1 engages the 5’TOP motif and controls TOP mRNA translation, we determined the 2.6 Å resolution X-ray crystal structure of the DM15 region (DM15) of human LARP1 bound to an RNA oligonucleotide spanning a segment of the 5’TOP motif of ribosomal protein S6 (RPS6) mRNA. We selected nucleotides 4–11 of the 42-nucleotide TOP sequence of RPS6 for co-crystallization experiments (5’-CCUCUUUUCCG-3’; the sequence used in co-crystallization experiments is underlined). The sequence and length choice was informed by the dimensions of the identified RNA binding site in the structure of apo DM15 and the results of nuclease protection assays performed on a complex of DM15 with the first 42 nucleotides of the RPS6 mRNA (Lahr et al., 2015). Importantly, despite excluding the first three nucleotides of the biological RPS6 TOP sequence, the sequence chosen for crystallization fits the definition of a TOP motif: a short stretch of pyrimidines preceded by a cytidine and succeeded by a guanosine (Meyuhas and Kahan, 2015). As anticipated, based on the negatively-charged phosphate backbone of the RNA, the resulting RNA-bound structure of DM15 revealed that the 5’TOP sequence binds to the highly conserved, positively charged surface of the three tandem helix-turn-helix HEAT-like repeats of DM15, termed A, B, and C (Figure 1A, Figure 1—figure supplement 1, Table 1) .

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Fortuitously, the sequence of RNA chosen for co-crystallization experiments promoted additional crystal contacts, which revealed an unanticipated function for the DM15, discussed below. Co-crystallization of DM15 with part of the RPS6 5’TOP motif yielded a crystal lattice in which one protein molecule interacts with two molecules of RNA in the unit cell. A given protein molecule simultaneously binds nucleotides 1–5 of one RNA molecule and the 3’ guanosine (G8) of the RNA bound to the protein in the neighboring unit cell (Figure 1—figure supplement 2A). G8 sits ‘upstream’ or 5’ to nucleotides 1–5; we denote the 3’ terminal G derived from the RNA strand originating in the neighboring unit cell as G8* to indicate its discontinuity from the polypyrimidine motif in nucleotides 1 to 5.

Our observations of (1) a binding pocket complementary to the size and shape of a guanosine that (2) specifically recognizes its Watson-Crick face, and (3) sits upstream of the 5’ terminal cytidine of the bound TOP RNA led us to hypothesize that DM15 binds the cap of TOP mRNAs. To test this, we determined the co-crystal structure of DM15 bound to the cap analog 7-methylguanosine triphosphate (m⁷GTP) to 1.8 Å resolution. m⁷GTP binds the same pocket as G8* in the DM15-RNA co-crystal (Figure 1C, Figure 1—figure supplements 1B and 2B). Lysine-915 stabilizes the γ-phosphate of m⁷GTP (Figure 1F). Interestingly, the γ-phosphate of m⁷GTP aligns with the position of the bound sulfate ion that crystallized at the non-crystallographic dimer interface in the structure of the apo DM15 region (Lahr et al., 2015).

Residues from each of the conserved DM15 repeats (A, B, C) participate in the recognition of the invariant cytidine in the first position of TOP mRNAs (C1) and the guanosine (G8*) 5’ to it (Figure 1B–D). All of the amino acids that interact directly with the RNA are nearly 100% conserved from worms to mammals and plants (Figure 1—figure supplement 3) (Bousquet-Antonelli and Deragon, 2009; Lahr et al., 2015). Amino acids R840, R879, and H800 define the path of the RNA by aligning it on the surface of DM15 through ionic and hydrogen bonding interactions with the phosphate backbone. Amino acids Y922, Y883, and F844 stabilize the nucleobases of G8* and C1 through stacking interactions. Sequence-specific recognition of G8* through its Watson-Crick face is accomplished by hydrogen bonds with E886 and S882. Arginines 847 and 879 specifically recognize C1 (Bousquet-Antonelli and Deragon, 2009; Lahr et al., 2015). The position of R879 forces U2 to flip away from stacking with C1. Instead, U3 stacks on U2. The base of U4 flips back toward C1 to stack on H800, while the O2 on its Watson-Crick face hydrogen bonds with the backbone carbonyl of H797.

While the α-helical nature of DM15 is unique among cap-binding proteins, its cap recognition pocket exhibits the canonical architecture (Figure 1—figure supplement 4). The DM15 region of LARP1 stabilizes the nucleobase of m⁷GTP between two conserved aromatic amino acids, Y883 and Y922. In addition, E886 hydrogen bonds with the Watson-Crick face of the guanosine moiety, using acidic side chains for this recognition in a mechanism reminiscent to that of other cap-binding proteins (Figure 1—figure supplement 4, CBP20). The methyl group points away from the core of the DM15 region in a manner similar to that observed in the nuclear cap-binding complex; it is therefore likely that other regions of LARP1 outside of the crystallized construct interact with the methyl group at position 7 and the 5’−5’ triphosphate linkage connecting the m⁷Gppp moiety and the first cytidine nucleotide of the TOP motif.

The conservation of the residues interacting with m⁷GTP in addition to the nucleotide-specific recognition of G8* and C1 from the DM15-RNA and DM15-m⁷GTP co-crystal structures suggest the DM15 region of LARP1 specifically recognizes the m⁷GpppC motif. To test this hypothesis, we determined the crystal structure of DM15 bound to m⁷GpppC dinucleotide. Indeed, this dinucleotide binds the DM15 region in the cap- and C-binding pockets (Figure 1, Figure 1—figure supplements 1, 2 and 5).

In vitro biochemical RNA-binding assays analyzed by electrophoretic mobility shift corroborate our crystallographic observations. DM15 binds a 42-mer oligonucleotide of the 5’UTR of RPS6 containing an additional 5’G, effectively making it a non-5’TOP sequence, with an affinity of 7.5 μM; capping this substrate enhances affinity 3.8-fold (2.0 μM). Capping the TOP 42-mer RPS6 RNA substrate increases the affinity 360-fold (Figure 2; 21 nM) over the non-TOP substrate and 95-fold over the capped non-TOP substrate.

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Data for graphed EMSAs.

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Mutating the residues in DM15 that stabilize the cap, E886, Y922, or Y883, decreases the affinity for capped TOP RNA 380-fold (8.0 μM), 124-fold (2.6 μM), and greater than ~950 fold (N.D.,>20 μM), respectively (Figure 2—figure supplement 1). Capping another 5’TOP sequence comprised of the first 42 nucleotides of the 5’UTR of RPL32 mRNA enhances the interaction of DM15 with this sequence (Figure 2—figure supplement 2). Further, competition assays in which capped mRNAs better displace capped DM15-bound sequences than uncapped mRNAs do, suggest the interaction of DM15 with the cap moiety is specific (Figure 2—figure supplement 3). Finally, thermal shift stability assays demonstrate the interaction of DM15 with m⁷GTP and m⁷GpppC is increasingly stabilizing to the DM15 fold (Figure 2—figure supplement 4); notably, the m⁷GpppG dinucleotide does not impart additional stability to DM15 over m⁷GTP. Together with the conservation of the residues interacting with m⁷GTP, the base-specific hydrogen bonding of the Watson-Crick faces of nucleobases of G8* and C1 in the DM15-RNA co-crystal structure and the recognition of the m⁷GpppC dinucleotide in the DM15-m⁷GpppC co-crystal structure, these data suggest the DM15 region of LARP1 specifically recognizes the m⁷GpppC motif characteristic of TOP mRNAs. This observation is particularly remarkable because the first cytdine is indispensable for repression of TOP mRNA translation (Levy et al., 1991). Interestingly, the first cytidine is also essential for LARP1 association with RPS6 and RPL32 mRNAs (Fonseca et al., 2015). Higher-order complexes and nonspecific binding of DM15 to the RNA oligonucleotide are greatly reduced in the capped-TOP substrate binding reaction (Figure 2), indicating that the m⁷GpppC motif might lock the register of DM15 binding. Furthermore, these data provide a molecular mechanism by which LARP1 differentiates TOP mRNAs from all other cellular mRNAs, most of which have a purine in the +1 position (Schibler et al., 1977; Schibler and Perry, 1977).

We hypothesized that if DM15 recognizes the caps of TOP mRNAs, it might compete for cap binding with eIF4E, the eukaryotic initiation factor required for canonical cap-dependent translation initiation (Sonenberg et al., 1978, 1979). To test this, we conducted competition assays between the human DM15 region and recombinant human eIF4E for capped oligonucleotides (Figure 2E). While displacement of DM15 from capped TOP RNA requires high-micromolar concentrations of eIF4E, low-nanomolar concentrations of DM15 are sufficient to displace eIF4E from this substrate. Interestingly, the opposite is true for capped non-TOP mRNA: eIF4E outcompetes DM15 for the non-TOP substrate as the pre-bound or competitor protein (Figure 2—figure supplement 3). These results are consistent with the preferred binding of eIF4E for m⁷GpppG (Kiraga-Motoszko et al., 2011; Thoreen et al., 2012; et al., Tamarkin-Ben-Harush et al., 2017) and with studies suggesting that quiescent cells and translation systems contain a factor that represses TOP mRNA translation even in the presence of excess eIF4E (Shama et al., 1995; Biberman and Meyuhas, 1999). Since most mRNAs have a purine in the +1 position (Schibler et al., 1977; Schibler and Perry, 1977), eIF4E is anticipated to stimulate their translation; by contrast, TOP mRNAs have an invariant C in the first position, suggesting that LARP1 would sequester them, thereby preventing cap-dependent translation initiation until a signaling event releases this repression. Of note, we show that the affinity of the DM15 region of LARP1 for TOP mRNAs is considerably higher than that of full-length eIF4E protein for this class of mRNAs, likely explaining why excess eIF4E alone is insufficient to upregulate the translation of TOP mRNAs (Shama et al., 1995).

The eIF4F complex (formed by the cap-binding protein eIF4E, the scaffolding protein eIF4G and the ATP-dependent RNA helicase eIF4A) has been linked to the control of TOP mRNA translation (Thoreen et al., 2012; Hsieh et al., 2012). Moreover, eIF4G co-precipitates TOP mRNAs in an mTORC1–dependent manner (Fonseca et al., 2015). Notably, the ability of TOP mRNAs to associate with eIF4G is markedly inhibited upon overexpression of LARP1 in mammalian cells (Fonseca et al., 2015), consistent with the idea that LARP1 competes with eIF4E and eIF4G for binding to TOP mRNAs. This notion is substantiated by the present findings that LARP1 blocks the access of eIF4E to m⁷Gppp-TOP mRNAs.

To investigate the mechanism by which LARP1 hinders the assembly of the eIF4F complex on TOP mRNAs we generated mutations in two key amino acids (R840 and Y883) that play pivotal roles in TOP mRNA binding (R840 and Y883) and in recognizing the m⁷Gppp cap structure (Y883) (Lahr et al., 2015). We compared the effects of overexpression of full-length wild type LARP1 on the assembly of the eIF4F complex on TOP (RPS6 and RPL32) and non-TOP (β-actin) mRNAs with that of the full-length R840E/Y883A double mutant. Expression of wild type LARP1 hinders the binding of endogenous eIF4G to both RPS6 and RPL32 mRNAs, but not to β-actin mRNA, as determined by RNA-immunoprecipitation/RT-ddPCR (Figure 3). This finding is consistent with the model that LARP1 binds to the cap and 5’TOP sequence of TOP mRNAs, thus selectively inhibiting eIF4F assembly on this class of transcripts. Perhaps more importantly, expression of the LARP1 R840E/Y883A double mutant does not inhibit eIF4G association with TOP mRNAs (Figure 3), indicating that cap- and TOP-binding is essential for LARP1-mediated displacement of the eIF4F complex from TOP mRNAs. Taken together with data demonstrating mTORC1 inhibition decreases the association of eIF4G with TOP transcripts (Fonseca et al., 2015), these data suggest that mTORC1 blocks the inhibitory function of LARP1.

Figure 3

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Data analysis for eIF4G IPs.

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The data presented in this study provide important structural insights into the molecular mechanism of LARP1, a specific 5’TOP motif-binding protein. LARP1 plays a fundamental role in the specialized recognition of the m⁷GpppC motif, a unique feature of TOP mRNAs that encode all the core protein components of the ribosome and the translation apparatus. Cap-binding proteins display a variety of mRNA regulatory functions (Topisirovic et al., 2011). LARP1 regulates translation and stability of this class of mRNAs through specialized 5’TOP motif (Fonseca et al., 2015; Lahr et al., 2015; Aoki et al., 2013) and cap recognition. eIF4E and its binding partner, eIF4G, have been previously linked to the control of TOP mRNA translation (Thoreen et al., 2012; Hsieh et al., 2012). Our work demonstrates that LARP1 displaces eIF4E from capped TOP mRNAs, thereby preventing the assembly of the eIF4F complex required for translation initiation. These findings reveal a previously unrecognized mTORC1-regulated dynamic interplay between LARP1 and the eIF4F complex, whereby eIF4F stimulates TOP mRNA translation while LARP1 represses it. Notably, LARP1 exhibits higher specificity and affinity for TOP mRNAs than eIF4E does, suggesting it may function as the selective factor for TOP mRNA translation regulation. These findings corroborate our earlier report (Fonseca et al., 2015) that LARP1 functions as a repressor of TOP mRNA translation: overexpression of LARP1 in mammalian cells leads to reduced TOP mRNA translation, as inferred by the accumulation of TOP mRNAs in subpolysomal fractions. Conversely, depletion of LARP1 protein from mammalian cells leads to an accumulation TOP messages in heavy polysomal fractions (Fonseca et al., 2015), indicating that LARP1 represses TOP mRNA translation. Importantly, LARP1 is essential for the ability of allosteric (rapamycin) and active-site (Torin1) mTOR inhibitors to effectively suppress TOP mRNA translation (Fonseca et al., 2015), consistent with LARP1 functioning downstream of mTORC1.

It is noteworthy that whilst LARP1 is shown here to repress TOP translation, in cancer cells LARP1 has been shown to have a positive effect on overall protein synthesis (Burrows et al., 2010) and bind many mRNA targets, including those encoding oncogenes (Hopkins et al., 2016; Mura et al., 2015), in addition to TOP mRNAs (Aoki et al., 2013; Fonseca et al., 2015; Lahr et al., 2015; Tcherkezian et al., 2014). This suggests that LARP1, while repressing TOP translation, can activate the translation of other targets, although the mechanism for the latter has yet to be elucidated.

In conclusion, our findings elucidate a novel mechanism of translation control whereby LARP1 competes with eIF4E for binding the cap of TOP mRNAs, effectively preventing eIF4F complex assembly on TOP mRNAs and ultimately precluding the translation of this class of mRNAs. Our studies now establish LARP1 as a bona fide cap-binding protein and the long-sought regulator of TOP mRNA translation.

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Author details

1. Roni M Lahr

   Department of Biological Sciences, University of Pittsburgh, Pittsburgh, United States

   Contribution

   RML, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-7469-4565

2. Bruno D Fonseca

   Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Canada

   Contribution

   BDF, Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

3. Gabrielle E Ciotti

   Department of Biological Sciences, University of Pittsburgh, Pittsburgh, United States

   Contribution

   GEC, Data curation, Validation, Investigation, Visualization

   Competing interests

   The authors declare that no competing interests exist.

4. Hiba A Al-Ashtal

   Department of Biological Sciences, University of Pittsburgh, Pittsburgh, United States

   Contribution

   HAA-A, Data curation, Investigation, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

5. Jian-Jun Jia

   Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Canada

   Contribution

   J-JJ, Data curation, Validation, Investigation, Visualization

   Competing interests

   The authors declare that no competing interests exist.

6. Marius R Niklaus

   Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Canada

   Contribution

   MRN, Data curation, Validation, Investigation, Visualization

   Competing interests

   The authors declare that no competing interests exist.

7. Sarah P Blagden

   Department of Oncology, University of Oxford, Oxford, United Kingdom

   Contribution

   SPB, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

8. Tommy Alain

   Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Canada

   Contribution

   TA, Resources, Funding acquisition, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

9. Andrea J Berman

   Department of Biological Sciences, University of Pittsburgh, Pittsburgh, United States

   Contribution

   AJB, Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Validation, Writing—original draft, Writing—review and editing

   For correspondence

   ajb190@pitt.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0003-1217-7412

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