eIF3 and Its mRNA-Entry-Channel Arm Contribute to the Recruitment of mRNAs With Long 5′-Untranslated Regions

Translation initiation in eukaryotes is a multi-step pathway and the most regulated phase of translation. Eukaryotic initiation factor 3 (eIF3) is the largest and most complex of the translation initiation factors, and it contributes to events throughout the initiation pathway. In particular, eIF3 appears to play critical roles in mRNA recruitment. More recently, eIF3 has been implicated in driving the selective translation of specific classes of mRNAs. However, unraveling the mechanism of these diverse contributions—and disentangling the roles of the individual subunits of the eIF3 complex—remains challenging. We employed ribosome profiling of budding yeast cells expressing two distinct mutations targeting the eIF3 complex. These mutations either disrupt the entire complex or subunits positioned near the mRNA-entry channel of the ribosome and which appear to relocate during or in response to mRNA binding and start-codon recognition. Disruption of either the entire eIF3 complex or specific targeting of these subunits affects mRNAs with long 5′-untranslated regions and whose translation is more dependent on eIF4A, eIF4B, and Ded1 but less dependent on eIF4G, eIF4E, and PABP. Disruption of the entire eIF3 complex further affects mRNAs involved in mitochondrial processes and with structured 5′-untranslated regions. Comparison of the suite of mRNAs most sensitive to both mutations with those uniquely sensitive to disruption of the entire complex sheds new light on the specific roles of individual subunits of the eIF3 complex.

Small uORFs favor translation re-initiation but do not protect mRNAs from nonsense-mediated decay

It is estimated that nearly 50% of mammalian transcripts contain at least one upstream open reading frame (uORF), which are typically one to two orders of magnitude smaller than the downstream main ORF. Most uORFs are thought to be inhibitory as they sequester the scanning ribosome, but in some cases allow for translation re-initiation. However, termination in the 5ʹ UTR at the end of uORFs resembles pre-mature termination that is normally sensed by the nonsense-mediated mRNA decay (NMD) pathway. Translation re-initiation has been proposed as a method for mRNAs to prevent NMD. Here we test how uORF length influences translation re-initiation and mRNA stability. Using custom 5ʹ UTRs and uORF sequences, we show that re-initiation can occur on heterologous mRNA sequences, favors small uORFs, and is supported when initiation occurs with more initiation factors. After determining reporter mRNA half-lives and mining available mRNA half-life datasets for cumulative uORF length, we conclude that translation re-initiation after uORFs is not a robust method for mRNAs to evade NMD. Together, these data support a model where uORFs have evolved to balance coding capacity, translational control, and mRNA stability.

eIF4A2 targets developmental potency and histone H3.3 transcripts for translational control of stem cell pluripotency

Translational control has emerged as a fundamental regulatory layer of proteome complexity that governs cellular identity and functions. As initiation is the rate-limiting step of translation, we carried out an RNAi screen for key translation initiation factors required to maintain embryonic stem cell (ESC) identity. We identified eIF4A2 and defined its mechanistic action through Rps26-independent and -dependent ribosomes in translation initiation activation of mRNAs encoding pluripotency factors and the histone variant H3.3 with demonstrated roles in maintaining stem cell pluripotency. eIF4A2 also mediates translation initiation activation of Ddx6, which acts together with eIF4A2 to restrict the totipotent 2-cell transcription program in ESCs through Zscan4 mRNA degradation and translation repression. Accordingly, knockdown of eIF4A2 disrupts ESC proteome causing the loss of ESC identity. Collectively, we establish a translational paradigm of the protein synthesis of pluripotency transcription factors and epigenetic regulators imposed on their established roles in controlling pluripotency.

Integrating Wheat Nucleolus Structure and Function: Variation in the Wheat Ribosomal RNA and Protein Genes

We review the coordinated production and integration of the RNA (ribosomal RNA, rRNA) and protein (ribosomal protein, RP) components of wheat cytoplasmic ribosomes in response to changes in genetic constitution, biotic and abiotic stresses. The components examined are highly conserved and identified with reference to model systems such as human, Arabidopsis, and rice, but have sufficient levels of differences in their DNA and amino acid sequences to form fingerprints or gene haplotypes that provide new markers to associate with phenotype variation. Specifically, it is argued that populations of ribosomes within a cell can comprise distinct complements of rRNA and RPs to form units with unique functionalities. The unique functionalities of ribosome populations within a cell can become central in situations of stress where they may preferentially translate mRNAs coding for proteins better suited to contributing to survival of the cell. In model systems where this concept has been developed, the engagement of initiation factors and elongation factors to account for variation in the translation machinery of the cell in response to stresses provided the precedents. The polyploid nature of wheat adds extra variation at each step of the synthesis and assembly of the rRNAs and RPs which can, as a result, potentially enhance its response to changing environments and disease threats.

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2–GTP–Met-tRNAiMet, and eIF3. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

Molecular architecture of 40S initiation complexes on the Hepatitis C virus IRES: from ribosomal attachment to eIF5B-mediated reorientation of initiator tRNA

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Direct binding of the IRES to the 40S subunit places the initiation codon into the P site, where it base-pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Then, eIF5 and eIF5B mediate subunit joining. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. Here, we present cryo-EM structures of IRES initiation complexes at resolutions up to 3.5 Å that cover all major stages from initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role for IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes that prepare them for subunit joining.

eIF5B and eIF1A remodel human translation initiation complexes to mediate ribosomal subunit joining

Joining of the ribosomal subunits at a translation start site on a messenger RNA during initiation commits the ribosome to synthesize a protein. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when universally-conserved eukaryotic initiation factors (eIFs) eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we examined initiation complexes that contained both eIF1A and eIF5B using single-particle electron cryo-microscopy. The resulting structure illuminated how eukaryote-specific contacts between eIF1A and eIF5B remodel the initiation complex to orient initiator tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during human translation initiation.

Cilia locally synthesize proteins to sustain their ultrastructure and functions

Cilia are microtubule-based hair-like organelles propelling locomotion and extracellular liquid flow or sensing environmental stimuli. As cilia are diffusion barrier-gated subcellular compartments, their protein components are thought to come from the cell body through intraflagellar transport or diffusion. Here we show that cilia locally synthesize proteins to maintain their structure and functions. Multicilia of mouse ependymal cells are abundant in ribosomal proteins, translation initiation factors, and RNA, including 18 S rRNA and tubulin mRNA. The cilia actively generate nascent peptides, including those of tubulin. mRNA-binding protein Fmrp localizes in ciliary central lumen and appears to function in mRNA delivery into the cilia. Its depletion by RNAi impairs ciliary local translation and induces multicilia degeneration. Expression of exogenous Fmrp, but not an isoform tethered to mitochondria, rescues the degeneration defects. Therefore, local translation defects in cilia might contribute to the pathology of ciliopathies and other diseases such as Fragile X syndrome.

The EIF4E1-4EIP cap-binding complex of Trypanosoma brucei interacts with the terminal uridylyl transferase TUT3

Most transcription in Trypanosoma brucei is constitutive and polycistronic. Consequently, the parasite relies on post-transcriptional mechanisms, especially affecting translation initiation and mRNA decay, to control gene expression both at steady-state and for adaptation to different environments. The parasite has six isoforms of the cap-binding protein EIF4E as well as five EIF4Gs. EIF4E1 does not bind to any EIF4G, instead being associated with a 4E-binding protein, 4EIP. 4EIP represses translation and reduces the stability of a reporter mRNA when artificially tethered to the 3’-UTR, whether or not EIF4E1 is present. 4EIP is essential during the transition from the mammalian bloodstream form to the procyclic form that lives in the Tsetse vector. In contrast, EIF4E1 is dispensable during differentiation, but is required for establishment of growing procyclic forms. In Leishmania, there is some evidence that EIF4E1 might be active in translation initiation, via direct recruitment of EIF3. However in T. brucei, EIF4E1 showed no detectable association with other translation initiation factors, even in the complete absence of 4EIP. There was some evidence for interactions with NOT complex components, but if these occur they must be weak and transient. We found that EIF4E1is less abundant in the absence of 4EIP, and RNA pull-down results suggested this might occur through co-translational complex assembly. We also report that 4EIP directly recruits the cytosolic terminal uridylyl transferase TUT3 to EIF4E1/4EIP complexes. There was, however, no evidence that TUT3 is essential for 4EIP function.

Dysregulation of Translation Factors EIF2S1, EIF5A and EIF6 in Intestinal-Type Adenocarcinoma (ITAC)

Intestinal-type adenocarcinoma (ITAC) is a rare cancer of the nasal cavity and paranasal sinuses that occurs sporadically or secondary to exposure to occupational hazards, such as wood dust and leather. Eukaryotic translation initiation factors have been described as promising targets for novel cancer treatments in many cancers, but hardly anything is known about these factors in ITAC. Here we performed in silico analyses, evaluated the protein levels of EIF2S1, EIF5A and EIF6 in tumour samples and non-neoplastic tissue controls obtained from 145 patients, and correlated these results with clinical outcome data, including tumour site, stage, adjuvant radiotherapy and survival. In silico analyses revealed significant upregulation of the translation factors EIF6 (ITGB4BP), EIF5, EIF2S1 and EIF2S2 (p < 0.05) with a higher arithmetic mean expression in ITAC compared to non-neoplastic tissue (NNT). Immunohistochemical analyses using antibodies against EIF2S1 and EIF6 confirmed a significantly different expression at the protein level (p < 0.05). In conclusion, this work identifies the eukaryotic translation initiation factors EIF2S1 and EIF6 to be significantly upregulated in ITAC. As these factors have been described as promising therapeutic targets in other cancers, this work identifies candidate therapeutic targets in this rare but often deadly cancer.