Cap Recognition Research Articles

EIF4E-independent translation is largely eIF3d-dependent

Translation initiation is a highly regulated step needed for protein synthesis. Most cell-based mechanistic work on translation initiation has been done using non-stressed cells growing in medium with sufficient nutrients and oxygen. This has yielded our current understanding of ‘canonical’ translation initiation, involving recognition of the mRNA cap by eIF4E1 followed by successive recruitment of initiation factors and the ribosome. Many cells, however, such as tumor cells, are exposed to stresses such as hypoxia, low nutrients or proteotoxic stress. This leads to inactivation of mTORC1 and thereby inactivation of eIF4E1. Hence the question arises how cells translate mRNAs under such stress conditions. We study here how mRNAs are translated in an eIF4E1-independent manner by blocking eIF4E1 using a constitutively active version of eIF4E-binding protein (4E-BP). Via ribosome profiling we identify a subset of mRNAs that are still efficiently translated when eIF4E1 is inactive. We find that these mRNAs preferentially release eIF4E1 when eIF4E1 is inactive and bind instead to eIF3d via its cap-binding pocket. eIF3d then enables these mRNAs to be efficiently translated due to its cap-binding activity. In sum, our work identifies eIF3d-dependent translation as a major mechanism enabling mRNA translation in an eIF4E-independent manner.

The Odds of Protein Translation Control Under Stress.

Physical or chemical stress is commonly known to inhibit protein translation at the cellular level. Since the process of protein translation requires catalysis by a multi-component machinery containing eukaryotic initiation factors (eIFs) and ribosomes in a sequence of reactions, how the process fails to proceed and whether certain genes can escape such blockade have provoked research efforts. Lines of evidence have demonstrated that phosphorylation of eIF4E or dephosphorylation of 4E-binding proteins (4E-BPs) prevents the formation of the eukaryotic translation initiation factor 4F (eIF4F) complex, whereas phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) due to activation of heme-regulated inhibitor (HRI), general control nonderepressible 2 (GCN2), protein kinase RNA-like endoplasmic reticulum kinase (PERK), or protein kinase R (PKR) by a diverse array of stressors prevents eIF2-GTP-tRNAiMet ternary complex assembly. These signal the abandonment of translation initiation via 5'-7-methylguanine (m7G) cap recognition by eIF4E. Stress can promote cleavage of tRNAs, impediment of rRNA processing, changes in the epitranscriptomic landscape, ribosome stalling or collision, activation of ribosomal surveillance systems, and assembly of the stress granules. Although these events contribute to the general inhibition of protein translation, a few proteins can bypass such negativity and become translated selectively. Such selective protein translation is primarily m7G cap independent through the integrated stress response or Internal Ribosomal Entry Site (IRES). The newly synthesized proteins often influence cell fate, facilitate cell survival, and build endogenous defense. Insights into the general inhibition of protein translation and selective translation of specific proteins will advance our understanding of the etiology or progression of human diseases involving cellular stress from viral infection or inflammation to myocardial infarction, stroke, or neurodegenerative disease. Antioxid. Redox Signal. 40, 943-947.

The arms race between bacteria CBASS and bacteriophages.

The Bacterial Cyclic oligonucleotide-Based Anti-phage Signaling System (CBASS) is an innate immune system that induces cell suicide to defend against phage infections. This system relies on cGAS/DncV-like nucleotidyltransferases (CD-NTase) to synthesize cyclic oligonucleotides (cOs) and CD-NTase-associated proteins (Caps) to execute cell death through DNA cleavage, membrane damage, and NAD depletion, thereby inhibiting phage replication. Ancillary proteins expressed in CBASS, in combination with CD-NTase, ensure the normal synthesis of cOs and prepare CD-NTase for full activation by binding to phage genomes, proteins, or other unknown products. To counteract cell death induced by CBASS, phage genes encode immune evasion proteins that curb Cap recognition of cOs, allowing for phage replication, assembly, and propagation in bacterial cells. This review provides a comprehensive understanding of CBASS immunity, comparing it with different bacterial immune systems and highlighting the interplay between CBASS and phage. Additionally, it explores similar immune escape methods based on shared proteins and action mechanisms between prokaryotic and eukaryotic viruses.

METTL16 promotes translation and lung tumorigenesis by sequestering cytoplasmic eIF4E2

N6-methyladenosine (m6A) plays crucial roles in regulating RNA metabolisms. METTL16 identified as a single-component methyltransferase catalyzes m6A formation in the nucleus; whether it regulates cytoplasmic RNA fate remains unknown. Here, we detected the dual localization of METTL16 in the nucleus and cytoplasm. METTL16 depletion attenuates protein synthesis, but the methyltransferase activity is not required for its translation-promoting function. Mechanistically, we identified an interactor of METTL16, eIF4E2, which represses translation by acting as a competitor of eIF4E. The METTL16-eIF4E2 interaction impedes the recruitment of eIF4E2 to 5' cap structure, promoting the cap recognition by eIF4E and selective protein synthesis. Depletion of METTL16 suppresses lung tumorigenesis by downregulating the translation of key oncogenes. Collectively, our study reports a role of METTL16 in modulating translation and provides a therapeutic target for lung cancer treatment.

MRNA- and factor-driven dynamic variability controls eIF4F-cap recognition for translation initiation.

mRNA 5′ cap recognition by eIF4F is a key element of eukaryotic translational control. Kinetic differences in eIF4F–mRNA interactions have long been proposed to mediate translation-efficiency differences between mRNAs, and recent transcriptome-wide studies have revealed significant heterogeneity in eIF4F engagement with differentially-translated mRNAs. However, detailed kinetic information exists only for eIF4F interactions with short model RNAs. We developed and applied single-molecule fluorescence approaches to directly observe real-time Saccharomyces cerevisiae eIF4F subunit interactions with full-length polyadenylated mRNAs. We found that eIF4E–mRNA association rates linearly anticorrelate with mRNA length. eIF4G–mRNA interaction accelerates eIF4E–mRNA association in proportion to mRNA length, as does an eIF4F-independent activity of eIF4A, though cap-proximal secondary structure still plays an important role in defining the final association rates. eIF4F–mRNA interactions remained dominated by effects of eIF4G, but were modulated to different extents for different mRNAs by the presence of eIF4A and ATP. We also found that eIF4A-catalyzed ATP hydrolysis ejects eIF4E, and likely eIF4E•eIF4G from the mRNA after initial eIF4F•mRNA complex formation, suggesting a mechanism to prepare the mRNA 5′ end for ribosome recruitment. Our results support a role for mRNA-specific, factor-driven eIF4F association rates in kinetically controlling translation.

Structure of the poxvirus decapping enzyme D9 reveals its mechanism of cap recognition and catalysis

Poxviruses encode decapping enzymes that remove the protective 5' cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral double-stranded RNA (dsRNA), a trigger for antiviral responses. Here we present a high-resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a domain organization different from other decapping enzymes in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5' mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap-binding proteins VP39, eIF4E, and CBP20, and distinct from eukaryotic decapping enzyme Dcp2.

Structural characterization of a new subclass of panicum mosaic virus-like 3' cap-independent translation enhancer.

Canonical eukaryotic mRNA translation requires 5′cap recognition by initiation factor 4E (eIF4E). In contrast, many positive-strand RNA virus genomes lack a 5′cap and promote translation by non-canonical mechanisms. Among plant viruses, PTEs are a major class of cap-independent translation enhancers located in/near the 3′UTR that recruit eIF4E to greatly enhance viral translation. Previous work proposed a single form of PTE characterized by a Y-shaped secondary structure with two terminal stem-loops (SL1 and SL2) atop a supporting stem containing a large, G-rich asymmetric loop that forms an essential pseudoknot (PK) involving C/U residues located between SL1 and SL2. We found that PTEs with less than three consecutive cytidylates available for PK formation have an upstream stem-loop that forms a kissing loop interaction with the apical loop of SL2, important for formation/stabilization of PK. PKs found in both subclasses of PTE assume a specific conformation with a hyperreactive guanylate (G*) in SHAPE structure probing, previously found critical for binding eIF4E. While PTE PKs were proposed to be formed by Watson–Crick base-pairing, alternative chemical probing and 3D modeling indicate that the Watson–Crick faces of G* and an adjacent guanylate have high solvent accessibilities. Thus, PTE PKs are likely composed primarily of non-canonical interactions.

Single-molecule dynamics of eIF4E-mediated messenger RNA recognition

Recognition of the mRNA 5' cap by the heterotrimeric eIF4F complex is a critical step in eukaryotic translation initiation. As the final point of regulation prior to protein synthesis, regulation of steps of initiation is critical for maintaining cellular homeostasis. Despite efforts to characterize steps of translation initiation in real time, a detailed understanding of how cap recognition may vary between transcripts, and the contributions of individual initiation factors, is lacking due to an absence of appropriate methods.

Targeting cap-dependent translation to inhibit Chikungunya virus replication: selectivity of p38 MAPK inhibitors to virus-infected cells due to autophagy-mediated down regulation of phospho-ERK.

The 5' capped, message-sense RNA genome of Chikungunya virus (CHIKV) utilizes the host cell machinery for translation. Translation is regulated by eIF2 alpha at the initiation phase and by eIF4F at cap recognition. Translational suppression by eIF2 alpha phosphorylation occurs as an early event in many alphavirus infections. We observe that in CHIKV-infected HEK293 cells, this occurs as a late event, by which time the viral replication has reached an exponential phase, implying its minimal role in virus restriction. The regulation by eIF4F is mediated through the PI3K-Akt-mTOR, p38 MAPK and RAS-RAF-MEK-ERK pathways. A kinetic analysis revealed that CHIKV infection did not modulate AKT phosphorylation, but caused a significant reduction in p38 MAPK phosphorylation. It caused degradation of phospho-ERK 1/2 by increased autophagy, leaving the PI3K-Akt-mTOR and p38 MAPK pathways for pharmacological targeting. mTOR inhibition resulted in moderate reduction in viral titre, but had no effect on CHIKV E2 protein expression, indicating a minimal role of the mTOR complex in virus replication. Inhibition of p38 MAPK using SB202190 caused a significant reduction in viral titre and CHIKV E2 and nsP3 protein expression. Furthermore, inhibiting the two pathways together did not offer any synergism, indicating that inhibiting the p38 MAPK pathway alone is sufficient to cause restriction of CHIKV replication. Meanwhile, in uninfected cells the fully functional RAS-RAF-MEK-ERK pathway can circumvent the effect of p38 MAPK inhibition on cap-dependent translation. Thus, our results show that host-directed antiviral strategies targeting cellular p38 MAPK are worth exploring against Chikungunya as they could be selective against CHIKV-infected cells with minimal effects on uninfected host cells.

Research on Vision Perception Technology of Auto Fueling Robot on opencv-based gas tank cap recognition

Designing a suitable algorithm to recognize the fuel tank cap and perform automatic refueling by manipulating a robotic arm. In exploring a new recognition algorithm for automatic vehicle refueling technology, for motor vehicle fuel tank lid, through the difference of images taken by binocular camera, combined with the basic calculation principles such as the imaging principle of binocular camera and the conversion relationship between coordinate systems, the recognition of ROI area is carried out automatically, then a closed-loop system is formed for real-time tracking, and finally the accurate positioning of fuel tank lid position is completed. The matching recognition and tracking technology of the target combines the comprehensive discipline research of mathematical analysis, image processing, object kinematics, artificial intelligence, automation control and other related fields, and has a very important status and research value in military aerospace, industrial production, intelligent medical care, security monitoring, intelligent driving, human-computer interaction and other fields.

Single‐Molecule Dynamics of Eukaryotic Translation Initiation

Translation initiation is a critical control point for eukaryotic gene expression. The molecular mechanism of initiation requires a highly dynamic and exquisitely coordinated interplay of mRNA with the translation machinery. Recognition of the mRNA 5ʹ cap structure by initiation factor eIF4F is a key early initiation step, and plays important roles in translational control. We have developed single-molecule fluorescence approaches to directly observe eIF4F–mRNA interaction in real time, both on individual mRNAs and at transcriptome scale with single-molecule resolution. Our data reveal order-of-magnitude variability across mRNAs for interaction of the cap-binding eIF4F subunit, eIF4E, and provide insights into how the eIF4F subunits eIF4G and eIF4A dynamically modulate cap recognition. The data implicate features throughout the mRNA as controlling cap recognition at the 5ʹ end, including a relationship between coding-sequence length and the accessibility of the cap for recognition. We have also developed a single-molecule assay to directly observe the initiation steps that follow eIF4F–mRNA recognition, i.e. 43S ribosomal pre-initiation complex recruitment and pre-initiation complex scanning to locate the start codon. Taken together, our results evoke a multi-layered, mRNA-centric mechanism to kinetically control translation initiation and confer differential sensitivity to distinct regulatory mechanisms.

Manganese oxide anchored on carbon modified halloysite nanotubes: An electrochemical platform for the determination of chloramphenicol

Halloysite, a naturally occurring aluminosilicate clay mineral similar to kaolinite, displays high surface area, high stability, and tunable surface chemistry. In this study, preparation, and the electrochemical behavior of manganese oxide (Mn2O3) supported on carbon modified halloysite nanotube (CCH) composite for the selective recognition of CAP was successfully described. The carbon modified halloysite nanotubes were achieved via a glucose dehydration process using sulphuric acid and the Mn2O3 particles were prepared using a wet chemical process. Over and above, the wet impregnation method was engaged to anchor Mn2O3 particles on the CCH matrix. The structural and morphological details of the as-prepared Mn2O3@CCH composite were studied in detail. The CCH attached to the clump of Mn2O3 particles (9.69 m2 g–1) revealed an increase in surface area than pristine Mn2O3 particles (0.41 m2 g–1) using Brunauer–Emmett–Teller analysis. The electron transfer ability and their electrochemical performance on Mn2O3@CCH composite were scrutinized using electrochemical impedance spectroscopy, cyclic voltammetry, and differential pulse voltammetry studies. Under optimized conditions, DPV analysis shown a wide linear range of 0.005–91.94 μM with a detection limit of 0.03 μM. As proof of concept, the real-time viability was verified in water samples with satisfactory recovery results. In terms of sustainability, HNT and HNT modified composites are highly beneficial and it has remarkable potential as a low-cost catalyst.

Single-Molecule Dynamics of eIF4F-Mediated mRNA Recognition

Recognition of the mRNA 5’ cap is by the heterotrimeric eIF4F complex is a critical step in eukaryotic translation initiation. As the final point of regulation prior to protein synthesis, regulation of steps of initiation is critical for maintaining cellular homeostasis. Despite efforts to characterize steps of translation initiation in real time, a detailed understanding of how cap recognition may vary between transcripts, and the contributions of individual initiation factors, is lacking due to an absence of appropriate methods. We recently developed a single molecule assay for examining this process on yeast mRNAs. Using single-molecule FRET, we in vitro reconstituted cap recognition by the mRNA cap binding protein eIF4E, characterizing this process in real-time. We apply this method to individual transcripts as well as heterogeneous pools of mRNA extracted from S.cerevisiae, characterizing the extent at which cap recognition by the cap binding subunit eIF4E varies throughout populations in the transcriptome. Additionally, we looked at the contributions of other eIF4F subunits and ATP to the rate of formation and stability of the eIF4E-cap complex, characterizing the kinetics of cap recognition. Our results suggest that mRNAs are inherently recognized at different rates by eIF4F, which can contribute to the wide range of translational efficiencies observed in vivo. Specifically, our findings highlight how factor composition and variations in mRNA features could tune the dynamics of protein synthesis by altering the rates of events during early initiation. Our work lays a foundation for isolating and studying the early steps of eukaryotic translation initiation, which is a multi-step, multi-factor process difficult to dissect in vivo due to its complexity.

Computational study on the allosteric mechanism of Leishmania major IF4E-1 by 4E-interacting protein-1: Unravelling the determinants of m7GTP cap recognition

During their life cycle, Leishmania parasites display a fine-tuned regulation of the mRNA translation through the differential expression of isoforms of eukaryotic translation initiation factor 4E (LeishIF4Es). The interaction between allosteric modulators such as 4E-interacting proteins (4E-IPs) and LeishIF4E affects the affinity of this initiation factor for the mRNA cap. Here, several computational approaches were employed to elucidate the molecular bases of the previously-reported allosteric modulation in L. major exerted by 4E-IP1 (Lm4E-IP1) on eukaryotic translation initiation factor 4E 1 (LmIF4E-1). Molecular dynamics (MD) simulations and accurate binding free energy calculations (ΔGbind) were combined with network-based modeling of residue-residue correlations. We also describe the differences in internal motions of LmIF4E-1 apo form, cap-bound, and Lm4E-IP1-bound systems. Through community network calculations, the differences in the allosteric pathways of allosterically-inhibited and active forms of LmIF4E-1 were revealed. The ΔGbind values show significant differences between the active and inhibited systems, which are in agreement with the available experimental data. Our study thoroughly describes the dynamical perturbations of LmIF4E-1 cap-binding site triggered by Lm4E-IP1. These findings are not only essential for the understanding of a critical process of trypanosomatids’ gene expression but also for gaining insight into the allostery of eukaryotic IF4Es, which could be useful for structure-based design of drugs against this protein family.

Research on Recognition Method of Car Fuel Tank Cap Based on Residual Network

Aiming at the problem that traditional algorithms have low recognition accuracy for different car fuel tank caps, a car fuel tank cap recognition algorithm based on improved Faster-RCNN is proposed. Firstly, the VGG16 network in the feature extraction network link is improved to the ResNet-101 residual network to improve the recognition accuracy and system speed. Secondly, the ROI pooling of the original pooling layer is changed to ROI align regional feature aggregation method makes the target rectangular frame more accurate. Finally, the fully connected network is used to perform the classification of the car fuel tank cap and the accurate regression of the frame. Through the detection of 200 actual images of car fuel tank caps, the experimental results show that the algorithm can achieve 98.0% accuracy and a detection speed of 0.139 seconds per image. Compared with traditional image algorithms, it can obtain faster speed and higher accuracy, and can provide technical support for unmanned gas stations, which has certain engineering application value.

The multifaceted eukaryotic cap structure.

The 5' cap structure is added onto RNA polymerase II transcripts soon after initiation of transcription and modulates several post-transcriptional regulatory events involved in RNA maturation. It is also required for stimulating translation initiation of many cytoplasmic mRNAs and serves to protect mRNAs from degradation. These functional properties of the cap are mediated by several cap binding proteins (CBPs) involved in nuclear and cytoplasmic gene expression steps. The role that CBPs play in gene regulation, as well as the biophysical nature by which they recognize the cap, is quite intricate. Differences in mechanisms of capping as well as nuances in cap recognition speak to the potential of targeting these processes for drug development. In this review, we focus on recent findings concerning the cap epitranscriptome, our understanding of cap binding by different CBPs, and explore therapeutic targeting of CBP-cap interaction. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Recognition RNA Processing > Capping and 5' End Modifications Translation > Translation Mechanisms.

Abstract B32: A specialized post-transcriptional program in chemoresistant, quiescent cancer cells

Abstract Quiescent (G0) cells are a clinically relevant fraction in cancers, which include dormant cancer stem cells, and resist clinical therapy. G0 cells reveal extensive changes in gene expression at the protein and translation levels. We previously identified that the translation mechanism is altered in G0 cancer cells. MicroRNAs, noncoding RNAs that target distinct mRNAs to alter gene expression, were found to associate with a key RNA-binding protein and enable specialized functions in G0, where they recruit noncanonical translation factors to regulate specific mRNA translation. We find that G0 leukemic cells show similar proteome and translatome to cells isolated post-chemotherapy. These data suggest that specialized post-transcriptional mechanisms in G0 leukemic cells regulate a distinct translatome to mediate chemoresistance. To understand the role of post-transcriptional regulation in chemoresistance, we compared global transcriptome, translatome and proteome profiling in chemoresistant G0 acute monocytic leukemic (AML) cells. We find that chemotherapy or G0 induction leads to DNA damage responsive ATM and stress signaling, which alter post-transcriptional and translational mechanisms. ATM and stress-activated p38 MAPK/MK2 increase AU-rich-element (ARE) bearing proinflammatory cytokine and immune gene mRNAs, by regulating a key ARE RNA binding protein and modifying canonical translation. AREs are present on 3'UTRs of tightly regulated oncogenes and cytokines, to post-transcriptionally control their expression. Both rate-limiting steps—mRNA cap recognition and tRNA recruitment—in canonical translation are altered. These signaling pathways lead to low mTOR activity in G0, which activates the cap complex inhibitor, eIF4EBP to impair canonical translation, leading to noncanonical translation of specific mRNAs with specialized cap binding and ribosome recruitment factors. In addition, stress and STAT1/interferon signaling are activated to reduce the canonical tRNA recruitment mechanism, enabling noncanonical translation of specific mRNAs. These changes permit translation of ARE bearing proinflammatory cytokine TNFa, and immune and cell-migration modulators that promote survival. Co-inhibiting p38 MAPK and TNFa that promote antiapoptosis—prior to or along with chemotherapy—decreases chemoresistance in AML cells, in vivo, and in patient samples without affecting normal cells. Our studies reveal a proinflammatory subpopulation in AML that mediates resistance, enabled by DNA damage- and stress-regulated post-transcriptional and translational mechanisms that are mediated by AU-rich-elements and a critical ARE RNA binding protein. Disrupting ARE regulation reduces TNFα and chemoresistance, revealing AREs, an important ARE RNA binding protein and noncanonical translation as regulators of chemoresistance. These studies reveal the significance of post-transcriptional regulation of proinflammatory and immune gene-mediated chemoresistance. Citation Format: Sooncheol Lee, Syed I.A. Bukhari, Samuel S. Truesdell, Swapna Kollu, Richard D. Mortensen, Myriam Boukhali, Esha Jain, Dongjun Lee, Maria Mazzola, Radhika Raheja, Adam Langenbucher, Nicholas Haradhwala, Akiko Yanagiya, Michael Lawrence, Roopali Gandhi, Ruslan Sadreyev, David Sweetser, Wilhelm Haas, Shobha Vasudevan. A specialized post-transcriptional program in chemoresistant, quiescent cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B32.

RNA-tethering assay and eIF4G:eIF4A obligate dimer design uncovers multiple eIF4F functional complexes.

Eukaryotic cellular mRNAs possess a 5′ cap structure (m7GpppN) which plays a critical role in translation initiation mediated by eukaryotic initiation factor (eIF) 4F. The heterotrimeric eIF4F complex possesses several activities imparted by its subunits that include cap recognition (by eIF4E), RNA unwinding (eIF4A), and factor/ribosome recruitment (eIF4G). Mammalian cells have paralogs of all three eIF4F subunits and it remains an open question as to whether these all can participate in the process of ribosome recruitment. To query the activities of the eIF4F subunits in translation initiation, we adopted an RNA-tethering assay in which select subunits are recruited to a specific address on a reporter mRNA template. We find that all eIF4F subunits can participate in the initiation process. Based on eIF4G:eIF4A structural information, we also designed obligate dimer pairs to probe the activity of all combinations of eIF4G and eIF4A paralogs. We demonstrate that both eIF4GI and eIF4GII can associate with either eIF4A1 or eIF4A2 to recruit ribosomes to mRNA templates. In combination with eIF4E and eIF4E3, our results indicate the presence of up to eight eIF4F complexes that can operate in translation initiation.

Structure of the RNA Specialized Translation Initiation Element that Recruits eIF3 to the 5′-UTR of c-Jun

Specialized translation initiation is a novel form of regulation of protein synthesis, whereby RNA structures within the 5′-UTR regulate translation rates of specific mRNAs. Similar to internal ribosome entry sites (IRESs), specialized translation initiation requires the recruitment of eukaryotic initiation factor 3 (eIF3), but also requires cap recognition by eIF3d, a new 5′-m7GTP recognizing protein. How these RNA structures mediate eIF3 recruitment to affect translation of specific mRNAs remains unclear. Here, we report the nuclear magnetic resonance (NMR) structure of a stem-loop within the c-JUN 5′ UTR recognized by eIF3 and essential for specialized translation initiation of this well-known oncogene. The structure exhibits similarity to eIF3 recognizing motifs found in hepatitis C virus (HCV)–like IRESs, suggesting mechanistic similarities. This work establishes the RNA structural features involved in c-JUN specialized translation initiation and provides a basis to search for small molecule inhibitors of aberrant expression of the proto-oncogenic c-JUN.

Capturing the Mechanism Underlying TOP mRNA Binding to LARP1

The RNA-binding protein La-related protein 1 (LARP1) plays a central role in ribosome biosynthesis. Its C-terminal DM15 region binds the 7-methylguanosine (m7G) cap and 5' terminal oligopyrimidine (TOP) motif characteristic of transcripts encoding ribosomal proteins and translation factors. Under the control of mammalian target of rapamycin complex 1 (mTORC1), LARP1 regulates translation of these transcripts. Characterizing the dynamics of DM15-TOP recognition is essential to understanding this fundamental biological process. We use molecular dynamics simulations, biophysical assays, and X-ray crystallography to reveal the mechanism of DM15 binding to TOP transcripts. Residues C-terminal to the m7G-binding site play important roles in cap recognition. Furthermore, we show that the unusually static pocket that recognizes the+1 cytosine characteristic of TOP transcripts drives binding specificity. Finally, we demonstrate that the DM15 pockets involved in TOP-specific m7GpppC-motif recognition are likely druggable. Collectively, these studies suggest unique opportunities for further pharmacological development.