Cap-binding Protein Research Articles

Critical and differential roles of eIF4A1 and eIF4A2 in B-cell development and function.

Eukaryotic initiation factor 4 A (eIF4A) plays critical roles during translation initiation of cellular mRNAs by forming the cap-binding eIF4F complex, recruiting the 40S small ribosome subunit, and scanning the 5' untranslated region (5' UTR) for the start codon. eIF4A1 and eIF4A2, two isoforms of eIF4A, are highly conserved and exchange freely within eIF4F complexes. The understanding of their biological and molecular functions remains incomplete if not fragmentary. In this study, we showed that eIF4A1 and eIF4A2 exhibit different expression patterns during B-cell development and activation. Mouse genetic analyses showed that they play critical but differential roles during B-cell development and humoral immune responses. While eIF4A1 controls global protein synthesis, eIF4A2 regulates the biogenesis of 18S ribosomal RNA and the 40S ribosome subunit. This study demonstrates the distinct cellular and molecular functions of eIF4A1 and eIF4A2 and reveals a new role of eIF4A2 in controlling 40S ribosome biogenesis.

Cap-independent translation directs stress-induced differentiation of the protozoan parasite Toxoplasma gondii.

Translational control mechanisms modulate microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5'-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5'-cap is impaired by insertion of a 5'-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we determined that a trans-acting RNA-binding protein called BFD2/ROCY1 is necessary for cap-independent translation of BFD1 through its binding to the 5'-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress, but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.

RNA splicing controls organ-wide maturation of postnatal heart in mice.

Postnatal cardiac development requires the orchestrated maturation of diverse cellular components for which unifying control mechanisms are still lacking. Using full-length sequencing, we examined the transcriptomic landscape of the maturating mouse heart (E18.5-P28) at single-cell and transcript isoform resolution. We identified dynamically changing intercellular networks as a molecular basis of the maturing heart and alternative splicing (AS) as a common mechanism that distinguished developmental age. Manipulation of RNA-binding proteins (RBPs) remodeled the AS landscape, cardiac cell maturation, and intercellular communication through direct binding of splice targets, which were enriched for functions related to general, as well as cell-type-specific, maturation. Overexpression of an RBP nuclear cap-binding protein subunit 2 (NCBP2) in neonatal hearts repressed cardiac maturation. Together, our data suggest AS regulation by RBPs as an organ-level control mechanism in mammalian postnatal cardiac development and provide insight into the possibility of manipulating RBPs for therapeutic purposes.

Second-Generation Cap Analogue Prodrugs for Targeting Aberrant Eukaryotic Translation Initiation Factor 4E (eIF4E) Activity in Drug-Resistant Melanoma.

Melanoma is the deadliest form of skin cancer with a 5-year survival rate of less than 20%. While significant strides have been made in the field of kinase-targeted and immune-based therapies for melanoma, the development of resistance to these therapeutic agents has hindered the success of treatment. Drug-resistant melanoma is particularly reliant on enhanced cap-dependent translation to drive the production of oncoproteins that promote growth and survival. The m7GpppX cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) is the rate-limiting factor of cap-dependent translation initiation, and its overexpression in melanoma tumors has been shown to drive resistance to BRAFV600E kinase-targeted inhibitors. These findings point to eIF4E-targeted therapies as a promising strategy to overcome drug resistance in melanoma. Herein, we build upon our previous work of developing cell-permeable cap analogue inhibitors to design second-generation cap analogues that inhibit eIF4E-mediated cap-dependent translation in drug-resistant melanoma cells.

Cap-independent translation directs stress-induced differentiation of the protozoan parasite Toxoplasma gondii.

Translational control mechanisms modulate microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5'-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5'-cap is impaired by insertion of a 5'-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we show that a trans -acting RNA-binding protein called BFD2/ROCY1 is necessary for cap-independent translation of BFD1 through its binding to the 5'-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress, but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.

Cryo-EM structure of the CBC-ALYREF complex

In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5′ end with a 7-methylguanosine (m7G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5′ end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.

Cryo-EM structure of the CBC-ALYREF complex.

In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5' end with a 7-methylguanosine (m7G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism, including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5' end of mRNA. However, the molecular mechanism for CBC-mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.

Non-redundant roles for the human mRNA decapping cofactor paralogs DCP1a and DCP1b.

Eukaryotic gene expression is regulated at the transcriptional and post-transcriptional levels, with disruption of regulation contributing significantly to human diseases. The 5' m7G mRNA cap is a central node in post-transcriptional regulation, participating in both mRNA stabilization and translation efficiency. In mammals, DCP1a and DCP1b are paralogous cofactor proteins of the mRNA cap hydrolase DCP2. As lower eukaryotes have a single DCP1 cofactor, the functional advantages gained by this evolutionary divergence remain unclear. We report the first functional dissection of DCP1a and DCP1b, demonstrating that they are non-redundant cofactors of DCP2 with unique roles in decapping complex integrity and specificity. DCP1a is essential for decapping complex assembly and interactions between the decapping complex and mRNA cap-binding proteins. DCP1b is essential for decapping complex interactions with protein degradation and translational machinery. DCP1a and DCP1b impact the turnover of distinct mRNAs. The observation that different ontological groups of mRNA molecules are regulated by DCP1a and DCP1b, along with their non-redundant roles in decapping complex integrity, provides the first evidence that these paralogs have qualitatively distinct functions.

CircZMYM2 plays a pivotal role in osteosarcoma by regulating the translation of NACA and ARPC1B

Osteosarcoma (OS) is a primary malignant bone tumor in children and young adults because of its intertumoral heterogeneity and absence of molecular targets. This study aimed to elucidate the role of circular RNA in the pathogenesis of OS. Using bioinformatics analysis and OS clinical samples, we found that hsa_circ_0029634 formed during the splicing process of Zinc finger MYM-type containing 2 (circZMYM2) was highly expressed in OS tissues. Nuclear cap-binding protein subunit 2 regulated circZMYM2 bio-generation by binding to flanking sequences of ZMYM2 transcripts. In addition, knockdown circZMYM2 inhibited cell growth and metastasis in OS cell lines in vitro and inhibited tumor growth in vivo. Mechanistically, circZMYM2 competitively bound to FXR1 to regulate the translation of NACA and ARPC1B. CircZMYM2 may be a crucial regulator of OS tumorigenesis and a potential diagnostic and therapeutic biomarker for OS patients.

The cap-binding complex modulates ABA-responsive transcript splicing during germination in barley (Hordeum vulgare)

To decipher the molecular bases governing seed germination, this study presents the pivotal role of the cap-binding complex (CBC), comprising CBP20 and CBP80, in modulating the inhibitory effects of abscisic acid (ABA) in barley. Using both single and double barley mutants in genes encoding the CBC, we revealed that the double mutant hvcbp20.ab/hvcbp80.b displays ABA insensitivity, in stark contrast to the hypersensitivity observed in single mutants during germination. Our comprehensive transcriptome and metabolome analysis not only identified significant alterations in gene expression and splicing patterns but also underscored the regulatory nexus among CBC, ABA, and brassinosteroid (BR) signaling pathways.

Photoactivatable mRNA 5' Cap Analogs for RNA-Protein Crosslinking.

Chemical modification of messenger RNA (mRNA) has paved the way for advancing mRNA-based therapeutics. The intricate process of mRNA translation in eukaryotes is orchestrated by numerous proteins involved in complex interaction networks. Many of them bind specifically to a unique structure at the mRNA 5'-end, called 5'-cap. Depending on the 5'-terminal sequence and its methylation pattern, different proteins may be involved in the translation initiation and regulation, but a deeper understanding of these mechanisms requires specialized molecular tools to identify natural binders of mRNA 5'-end variants. Here, a series of 8 new synthetic 5'-cap analogs that allow the preparation of RNA molecules with photoreactive tags using a standard in vitro transcription reaction are reported. Two photoreactive tags and four different modification sites are selected to minimize potential interference with cap-protein contacts and to provide complementary properties regarding crosslinking chemistry and molecular interactions. The tailored modification strategy allows for the generation of specific crosslinks with model cap-binding proteins, such as eIF4E and Dcp2. The usefulness of the photoreactive cap analogs is also demonstrated for identifying the cap-binding subunit in a multi-protein complex, which makes them perfect candidates for further development of photoaffinity labeling probes to study more complex mRNA-related processes.

Cryo-EM structure of the CBC-ALYREF complex.

In eukaryotes, RNAs transcribed by RNA Pol II are modified at the 5' end with a 7-methylguanosine (m 7 G) cap, which is recognized by the nuclear cap binding complex (CBC). The CBC plays multiple important roles in mRNA metabolism including transcription, splicing, polyadenylation, and export. It promotes mRNA export through direct interaction with a key mRNA export factor, ALYREF, which in turn links the TRanscription and EXport (TREX) complex to the 5' end of mRNA. However, the molecular mechanism for CBC mediated recruitment of the mRNA export machinery is not well understood. Here, we present the first structure of the CBC in complex with an mRNA export factor, ALYREF. The cryo-EM structure of CBC-ALYREF reveals that the RRM domain of ALYREF makes direct contact with both the NCBP1 and NCBP2 subunits of the CBC. Comparing CBC-ALYREF with other cellular complexes containing CBC and/or ALYREF components provides insights into the coordinated events during mRNA transcription, splicing, and export.

IGF2BP3/NCBP1 complex inhibits renal tubular senescence through regulation of CDK6 mRNA stability

Renal aging and the subsequent rise in kidney-related diseases are attributed to senescence in renal tubular epithelial cells (RTECs). Our study revealed that the abnormal expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3), a reader of RNA N6-methyladenosine, is critically involved in cisplatin-induced renal tubular senescence. In cisplatin-induced senescence of RTECs, the promoter activity and transcription of IGF2BP3 is markedly suppressed. It was due to the down regulation of MYC proto-oncogene (MYC), which regulates IGF2BP3 transcription by binding to the putative site at 1852–1863 of the IGF2BP3 promoter. Overexpression of IGF2BP3 ameliorated cisplatin-induced renal tubular senescence in vitro. Mechanistic studies revealed that IGF2BP3 inhibits cellular senescence in RTECs by enhancing cyclin-dependent kinase 6 (CDK6) mRNA stability and increasing its expression. The inhibition effect of IGF2BP3 on tubular senescence is partially reversed by the knockdown of CDK6. Further, IGF2BP3 recruits nuclear cap binding protein subunit 1 (NCBP1) and inhibits CDK6 mRNA decay, by recognizing m6A modification. Specifically, IGF2BP3 recognizes m6A motif "GGACU" at nucleotides 110–114 in the 5′ untranslated region (UTR) field of CDK6 mRNA. The involvement of IGF2BP3/CDK6 in alleviating tubular senescence was confirmed in a cisplatin-induced acute kidney injury (AKI)-to-chronic kidney disease (CKD) model. Clinical data also suggests an age-related decrease in IGF2BP3 and CDK6 levels in renal tissue or serum samples from patients. These findings suggest that IGF2BP3/CDK6 may be a promising target in cisplatin-induced tubular senescence and renal failure.

Helicobacter Pylori-Enhanced hnRNPA2B1 Coordinates with PABPC1 to Promote Non-m6A Translation and Gastric Cancer Progression.

Helicobacter pylori (H. pylori) infection is the primary risk factor for the pathogenesis of gastric cancer (GC). N6-methyladenosine (m6A) plays pivotal roles in mRNA metabolism and hnRNPA2B1 as an m6A reader is shown to exert m6A-dependent mRNA stabilization in cancer. This study aims to explore the role of hnRNPA2B1 in H. pylori-associated GC and its novel molecular mechanism. Multiple datasets and tissue microarray are utilized for assessing hnRNPA2B1 expression in response to H. pylori infection and its clinical prognosis in patients with GC. The roles of hnRNPA2B1 are investigated through a variety of techniques including glucose metabolism analysis, m6A-epitranscriptomic microarray, Ribo-seq, polysome profiling, RIP-seq. In addition, hnRNPA2B1 interaction with poly(A) binding protein cytoplasmic 1 (PABPC1) is validated using mass spectrometry and co-IP. These results show that hnRNPA2B1 is upregulated in GC and correlated with poor prognosis. H. pylori infection induces hnRNPA2B1 upregulation through recruiting NF-κB to its promoter. Intriguingly, cytoplasm-anchored hnRNPA2B1 coordinated PABPC1 to stabilize its relationship with cap-binding eIF4F complex, which facilitated the translation of CIP2A, DLAT and GPX1 independent of m6A modification. In summary, hnRNPA2B1 facilitates the non-m6A translation of epigenetic mRNAs in GC progression by interacting with PABPC1-eIF4F complex and predicts poor prognosis for patients with GC.

Abstract PR011: Inhibiting eIF4E phosphorylation sensitizes triple-negative breast cancer to CDK4/6 inhibition

Abstract Despite significant progress in breast cancer treatment, Triple-Negative Breast Cancer (TNBC) presents a unique challenge due to the absence of three conventional drug targets found in other breast cancer subtypes, highlighting an urgent need for novel therapeutic strategies. A critical element of TNBC pathogenesis is the dysregulation of mRNA translation, a critical process required by rapidly dividing cancer cells to synthesize proteins as building blocks. This hallmark positions mRNA translation as a promising target for therapeutic intervention. At the forefront of regulating mRNA translation, is the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E). While its role in general mRNA translation initiation is crucial, eIF4E phosphorylation by the MNK1/2 [MAPK (Mitogen-Activated Protein Kinase)-Interacting Kinase1/2] has been implicated in tumorigenesis. Specifically, phosphorylated eIF4E (p-4E) selectively enhances the translation of mRNAs encoding proteins critical for cancer cell survival and metastasis, such as myeloid cell leukemia-1 (MCL1) and matrix metalloproteinase-3 (MMP3). Yet, inhibiting eIF4E phosphorylation markedly impairs TNBC metastasis without affecting the primary tumor’s growth rates in vivo, emphasizing its specific role in metastatic progression. To dissect the mechanism of p-4E in TNBC, we performed an in vitro shRNA synthetic lethal screen in TNBC cells to identify genes whose knockdown synergizes with p-4E inhibition to limit cell growth. A small-molecule inhibitor of MNK1/2, eFT508, was utilized to mitigate p-4E level during the screen. This screen uncovered that knockdown of cyclin-dependent kinase 4 (CDK4), known primarily for its role in cell cycle regulation, synergizes with p-4E inhibition. Through validation using various cell proliferation assays and three different FDA-approved CDK4/6 inhibitors (palbociclib, ribociclib, and abemaciclib), we demonstrated a strong synergy between CDK4/6 inhibition and eFT508 in reducing cell growth. The synergy between CDK4/6 and p-4E inhibition not only impaired TNBC cell proliferation, but also significantly reduced eIF4E activity and mRNA translation. Additionally, our findings reveal a novel role for CDK4/6 inhibitors in modulating mRNA translation via mTORC1 signaling pathways, extending beyond their canonical role in cell cycle regulation. This discovery suggests a potential combinatorial therapeutic approach for TNBC treatment involving MNK1/2 and CDK4/6 inhibitors. Such a strategy could exploit the vulnerabilities in TNBC’s dysregulated mRNA translation machinery, offering a promising direction in treating this aggressive breast cancer subtype. Citation Format: Qiyun Deng, Mehdi Amiri, Anastasija Ana Piric, Yasaman Bagherian, Zilan Li, Sidong Huang, Michael Pollak, Nahum Sonenberg. Inhibiting eIF4E phosphorylation sensitizes triple-negative breast cancer to CDK4/6 inhibition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR011.

CMTR-1 RNA methyltransferase mutations activate widespread expression of a dopaminergic neuron-specific mitochondrial complex I gene

The mitochondrial proteome is comprised of approximately 1,100 proteins,1 all but 12 of which are encoded by the nuclear genome in C.elegans. The expression of nuclear-encoded mitochondrial proteins varies widely across cell lineages and metabolic states,2,3,4 but the factors that specify these programs are not known. Here, we identify mutations in two nuclear-localized mRNA processing proteins, CMTR1/CMTR-1 and SRRT/ARS2/SRRT-1, which we show act via the same mechanism to rescue the mitochondrial complex I mutant NDUFS2/gas-1(fc21). CMTR-1 is an FtsJ-family RNA methyltransferase that, in mammals, 2'-O-methylates the first nucleotide 3' to the mRNA CAP to promote RNA stability and translation5,6,7,8. The mutations isolated in cmtr-1 are dominant and lie exclusively in the regulatory G-patch domain. SRRT-1 is an RNA binding partner of the nuclear cap-binding complex and determines mRNA transcript fate.9 We show that cmtr-1 and srrt-1 mutations activate embryonic expression of NDUFS2/nduf-2.2, a paralog of NDUFS2/gas-1 normally expressed only in dopaminergic neurons, and that nduf-2.2 is necessary for the complex I rescue by the cmtr-1 G-patch mutant. Additionally, we find that loss of the cmtr-1 G-patch domain cause ectopic localization of CMTR-1 protein to processing bodies (P bodies), phase-separated organelles involved in mRNA storage and decay.10 P-body localization of the G-patch mutant CMTR-1 contributes to the rescue of the hyperoxia sensitivity of the NDUFS2/gas-1 mutant. This study suggests that mRNA methylation at P bodies may control nduf-2.2 gene expression, with broader implications for how the mitochondrial proteome is translationally remodeled in the face of tissue-specific metabolic requirements and stress.

MRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency.

Toxoplasma gondii is an opportunistic pathogen important to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation is required. Here we show that the mRNA cap-binding protein, eIF4E1, regulates the encystation process. Encysted parasites reduce eIF4E1 levels, and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drives Toxoplasma encystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.

Non-canonical mRNA translation initiation in cell stress and cancer.

The now well described canonical mRNA translation initiation mechanism of m7G 'cap' recognition by cap-binding protein eIF4E and assembly of the canonical pre-initiation complex consisting of scaffolding protein eIF4G and RNA helicase eIF4A has historically been thought to describe all cellular mRNA translation. However, the past decade has seen the discovery of alternative mechanisms to canonical eIF4E mediated mRNA translation initiation. Studies have shown that non-canonical alternate mechanisms of cellular mRNA translation initiation, whether cap-dependent or independent, serve to provide selective translation of mRNAs under cell physiological and pathological stress conditions. These conditions typically involve the global downregulation of canonical eIF4E1/cap-mediated mRNA translation, and selective translational reprogramming of the cell proteome, as occurs in tumor development and malignant progression. Cancer cells must be able to maintain physiological plasticity to acquire a migratory phenotype, invade tissues, metastasize, survive and adapt to severe microenvironmental stress conditions that involve inhibition of canonical mRNA translation initiation. In this review we describe the emerging, important role of non-canonical, alternate mechanisms of mRNA translation initiation in cancer, particularly in adaptation to stresses and the phenotypic cell fate changes involved in malignant progression and metastasis. These alternate translation initiation mechanisms provide new targets for oncology therapeutics development.

Human eukaryotic initiation factor 4G directly binds the 40S ribosomal subunit to promote efficient translation

Messenger RNA (mRNA) recruitment to the 40S ribosomal subunit is mediated by eukaryotic initiation factor 4F (eIF4F). This complex includes three subunits: eIF4E (m7G cap-binding protein), eIF4A (DEAD-box helicase), and eIF4G. Mammalian eIF4G is a scaffold that coordinates the activities of eIF4E and eIF4A and provides a bridge to connect the mRNA and 40S ribosomal subunit through its interaction with eIF3. While the roles of many eIF4G binding domains are relatively clear, the precise function of RNA binding by eIF4G remains to be elucidated. In this work, we used an eIF4G-dependent translation assay to reveal that the RNA binding domain (eIF4G-RBD; amino acids 682-720) stimulates translation. This stimulating activity is observed when eIF4G is independently tethered to an internal region of the mRNA, suggesting that the eIF4G-RBD promotes translation by a mechanism that is independent of the m7G cap and mRNA tethering. Using a kinetic helicase assay, we show that the eIF4G-RBD has a minimal effect on eIF4A helicase activity, demonstrating that the eIF4G-RBD is not required to coordinate eIF4F-dependent duplex unwinding. Unexpectedly, native gel electrophoresis and fluorescence polarization assays reveal a previously unidentified direct interaction between eIF4G and the 40S subunit. Using binding assays, our data show that this 40S subunit interaction is separate from the previously characterized interaction between eIF4G and eIF3. Thus, our work reveals how eIF4F can bind to the 40S subunit using eIF3-dependent and eIF3-independent binding domains to promote translation initiation.

Nonsense-mediated mRNA decay of mRNAs encoding a signal peptide occurs primarily after mRNA targeting to the endoplasmic reticulum

Translation of mRNAs encoding integral membrane proteins or secreted proteins occurs on the surface of the endoplasmic reticulum (ER). When a nascent signal peptide is synthesized from the mRNAs, the ribosome-nascent chain complex (RNC) is recognized by the signal recognition particle (SRP) and then transported to the surface of the ER. The appropriate targeting of the RNC–SRP complex to the ER is monitored by a quality control pathway, a nuclear cap-binding complex (CBC)-ensured translational repression of RNC–SRP (CENTRE). In this study, using ribosome profiling of CBC-associated and eIF4E-associated mRNAs, we reveal that, at the transcriptomic level, CENTRE is in charge of the translational repression of the CBC–RNC–SRP until the complex is specifically transported to the ER. We also find that CENTRE inhibits the nonsense-mediated mRNA decay (NMD) of mRNAs within the CBC–RNC–SRP. The NMD occurs only after the CBC–RNC–SRP is targeted to the ER and after eIF4E replaces CBC. Our data indicate dual surveillance for properly targeting mRNAs encoding integral membrane or secretory proteins to the ER. CENTRE blocks gene expression at the translation level before the CBC–RNC–SRP delivery to the ER, and NMD monitors mRNA quality after its delivery to the ER.