Biology subjects

Fraser, C. S.

Publications and source records attributed to Fraser, C. S..

5 recordsLinked to original sources

bioRxiv2023

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 3 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.

biochemistry↗

bioRxiv2022

Monitoring RNA restructuring in a human cell-free extract reveals eIF4A-dependent and eIF4A-independent unwinding activity.

The canonical DEAD-box helicase, eIF4A, unwinds 5 UTR secondary structures to promote mRNA translation initiation. Growing evidence has indicated that other helicases, such as DHX29 and DDX3/ded1p, also function to promote the scanning of the 40S subunit on highly structured mRNAs. It is unknown how the relative contributions of eIF4A and other helicases regulate duplex unwinding on an mRNA to promote initiation. Here, we have adapted a real-time fluorescent duplex unwinding assay to precisely monitor helicase activity in the 5 UTR of a reporter mRNA that can be translated in a cell-free extract in parallel. We monitored the rate of 5 UTR-dependent duplex unwinding in the absence or presence of Hippuristanol, a dominant negative eIF4A (eIF4A-R362Q), or a mutant eIF4E (eIF4E-W73L) that can bind the m7G cap but not eIF4G. Our experiments reveal that roughly 50% of the duplex unwinding activity in the cell-free extract can be attributed to an eIF4A-dependent mechanism, while the remaining 50% of duplex unwinding activity is attributed to an eIF4A-independent mechanism. Importantly, we show that the robust eIF4A-independent duplex unwinding is not sufficient for translation. We also show that the m7G cap structure, and not the poly(A) tail, is the primary mRNA modification responsible for promoting duplex unwinding in our cell-free extract system. Overall, the fluorescent duplex unwinding assay provides a precise method to investigate how eIF4A-dependent and eIF4A-independent helicase activity regulates translation initiation in cell-free extracts. We anticipate that potential small molecule inhibitors could be tested for helicase inhibition using this duplex unwinding assay.

biochemistry↗

bioRxiv2022

The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A

SummaryInitiation of mRNA translation is a key regulatory step in gene expression in all eukaryotes. Canonical initiation of translation in eukaryotes involves recruitment of the 43S preinitiation complex to the 5' end of mRNA by the cap-binding complex eIF4F to form the 48S initiation complex (48S), followed by scanning along the mRNA until the start codon is selected.1-8 We have previously shown that eIF4F binds near the mRNA channel exit site of the 43S, leaving an open question about how mRNA secondary structure is removed as it enters the mRNA binding channel on the other side of the 40S subunit.4 Here we describe a human 48S positioned at the start codon that shows that in addition to the eIF4A that is part of eIF4F, there is a second eIF4A helicase bound to the mRNA entry site. The entry channel bound eIF4A is positioned through interactions with eIF3 and the 40S subunit to enable its ATP-dependent helicase activity to directly unwind secondary structure located downstream of the scanning 48S complex. The structure also reveals universally conserved interactions between eIF4F and the 48S, likely explaining how this complex can promote mRNA recruitment in all eukaryotes. mRNA translation has emerged as an important tool for developing innovative therapies, yet several fundamental aspects of its regulation remain unknown. This work sheds light on the critical regulatory roles of eIF4A and eIF4F during the recruitment and scanning of the 5' UTR of mRNA.

molecular biology↗

bioRxiv2022

5' Untranslated mRNA Regions Allow Bypass of Host Cell Translation Inhibition by Legionella pneumophila

Legionella pneumophila grows within membrane-bound vacuoles in alveolar macrophages during human disease. Pathogen manipulation of the host cell is driven by bacterial proteins translocated through a type IV secretion system (T4SS). Although host protein synthesis during infection is arrested by the action of several of these translocated effectors, translation of a subset of host proteins predicted to restrict the pathogen is maintained. To identify the spectrum of host proteins selectively synthesized after L. pneumophila challenge, macrophages infected with the pathogen were allowed to incorporate the amino acid analog azidohomoalanine (AHA) during a two-hour time window, and newly synthesized macrophage proteins were isolated by orthogonal chemistry followed by mass spectrometry. Among the proteins isolated were interferon-stimulated genes (ISGs) as well as proteins translated from highly abundant transcripts. Surprisingly, a large number of the identified proteins were from low abundance transcripts. These proteins were predicted to be among the most efficiently translated per unit transcript in the cell based on ribosome profiling datasets. To determine if high ribosome loading was a consequence of efficient translation initiation, the 5 untranslated regions (5UTR) of transcripts having the highest and lowest predicted loading levels were inserted upstream of a reporter, and translation efficiency was determined in response to L. pneumophila challenge. The efficiency of reporter expression largely correlated with predicted ribosome loading and lack of secondary structure. Therefore, determinants in the 5UTR allow selected host cell transcripts to overcome a pathogen-driven translation blockade.

microbiology↗

bioRxiv2021

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.

biophysics↗