Biology subjects

Sokabe, M.

Publications and source records attributed to Sokabe, M..

9 recordsLinked to original sources

bioRxiv2026

Structural basis for repeat-associated non-AUG translation initiation on C9orf72 mRNA

A GGGGCC (G4C2) hexanucleotide repeat expansion in the first intron of C9orf72 is the most common monogenic cause of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). This element serves as a template for repeat-associated non-AUG (RAN) translation, which generates neurotoxic dipeptide repeat (DPR) proteins that aggregate in patient brains and contribute to neurodegeneration in model systems. To understand the mechanisms underlying RAN translation initiation, we used cryo-electron microscopy to determine the structure of an in vitro assembled human late-stage C9orf72 translation initiation complex at 3.2 A resolution and a near-native complex purified from human cell lysate at 3.5 A resolution. RAN translation on C9orf72 mRNA initiates predominantly at a near-cognate CUG codon that is located 24 nucleotides upstream of the G4C2 repeat and is flanked by a strong endogenous Kozak sequence. The structures further reveal a direct interaction between the G4C2 repeat and 18S ribosomal RNA expansion segment 9 (ES9S) on the 40S small ribosomal subunit through a kissing-loop interaction. Preventing this RNA-RNA interaction using complementary antisense oligonucleotides significantly reduces RAN translation in vitro and in patient-derived iNeurons, indicating that this structural interaction is functionally important. These findings provide mechanistic insights into RAN translation initiation and highlight a novel potential therapeutic approach to mitigate toxic DPR production in C9orf72-associated neurodegenerative disease.

molecular biology↗

bioRxiv2026

The eIF4B RNA recognition motif promotes higher-order organization of the translation initiation machinery during stress granule assembly.

Cells respond to environmental stress by rapidly remodeling translation and assembling stress granules (SGs), which are dynamic ribonucleoprotein condensates that contain untranslated mRNAs, translation initiation factors, and 40S ribosomal subunits. Although the translation initiation factor eIF4B has been implicated in SG biology, the contribution of its highly conserved RNA recognition motif (RRM) to SG assembly has remained unclear. Here, we developed a quantitative live-cell imaging framework that resolves distinct kinetic phases of SG assembly at single-cell resolution and combines these measurements with single-cell analysis of protein synthesis. Using this approach, we show that disruption of the eIF4B RRM delays SG nucleation, slows SG assembly, and reduces the number of SGs formed, while having little effect on mature SG size. Biochemical analyses revealed that the RRM mutant retained high-affinity binding to both RNA and the 40S ribosomal subunit and exhibited only a modest reduction in eIF4A helicase stimulation activity but displayed altered RNA engagement, consistent with impaired RNA-dependent organization of the translation initiation machinery. Coupling SG kinetics with single-cell measurements of protein synthesis further revealed that delayed SG nucleation is associated with reduced translational repression during oxidative stress. Together, our findings identify the conserved eIF4B RRM as a regulator of productive higher-order organization of the translation initiation machinery and establish a quantitative framework for investigating how SG assembly and translational remodeling are coordinated during cellular stress.

cell biology↗

bioRxiv2026

Remodeling of mRNA by eIF4F in human translation initiation

For the ribosome to load onto an mRNA during the early steps of translation initiation, the mRNA must be activated by the eIF4F complex. The mechanism of this activation step has remained elusive. Here we employ multi-perspective real-time single-molecule assays to observe directly mRNA-eIF4F binding near the 5' end, mRNA conformational remodeling, and 40S ribosomal subunit loading. eIFs 4E, 4G, and 4B play distinct roles in promoting eIF4F association and stabilizing eIF4A binding. Binding of eIF4F is the rate-limiting step in mRNA activation: once bound, mRNA conformation is rapidly extended in an ATP-dependent manner. The mRNA extended state is the necessary substrate for 43S PIC loading and perturbations to extension delay loading. Features of the mRNA, such as the 7-methylguanosine cap at the 5' end and secondary structures, modulate these steps and regulate ribosome loading. Our results establish a kinetic and mechanistic framework for the early steps in translation initiation.

biophysics↗

bioRxiv2026

A ribosomal kinetic checkpoint governs selective mRNA recruitment

Translation initiation begins with recruitment of mRNA to the ribosome, yet how mRNA engagement is converted into productive initiation remains unclear. Using real-time fluorescence assays with purified components, we show that mRNA recruitment proceeds through a branched kinetic pathway on the 40S subunit. Following rapid sampling, mRNAs partition into either a productive accommodated state or an arrested state that stabilizes ribosome binding before accommodation. mRNA structure, eIF3, and eIF3j bias recruitment toward arrest, whereas eIF4F promotes accommodation in an ATP-dependent manner coupled to displacement of eIF3j from the mRNA entry channel. Unstructured mRNAs accommodate independently of eIF4E, whereas structured mRNAs require an upstream eIF4E-dependent step, enabling selective recruitment under limiting eIF4E. Arrested complexes can convert directly into the accommodated state without dissociation, revealing a reversible standby intermediate poised for activation. Together, our findings establish mRNA accommodation as a ribosome-intrinsic checkpoint governing initiation and provide a framework for selective translation.

biochemistry↗

bioRxiv2026

Nanometer-scale RNA protein clusters (RPCs) Foster Helicase Activity of DEAD-box eIF4A

DEAD-box RNA helicases are central regulators of RNA metabolism, employing ATP-dependent mechanisms to remodel RNA structure and RNA-protein interactions, yet how helicase catalysis is coordinated with multi-subunit interactions between RNA and protein remains unresolved. Translation initiation helicase, eukaryotic initiation factor 4A (eIF4A), which acts as an intrinsically non-processive enzyme, is essential for unwinding structured mRNAs, relies on cofactors to achieve physiological activity. Here we uncover an unexpected RNA-helicase state of eIF4A, demonstrating that eIF4A forms nanometer-scale RNA-protein clusters (RPCs) of [~]2-5 MDa in presence of its physiological cofactors eIF4B and eIF4G, RNA and ATP under near-physiological concentrations. Using a single molecule approach, we directly resolve the formation of discrete clusters that recruit multiple copies of proteins with RNA upon ATP addition and show that RPC formation correlates with helicase activity in vitro. Further, we find eIF4B as a key determinant of this multi-subunit assembly. Its intrinsically disordered regions (IDRs) together with structured RNA-recognition motifs (RRMs) drive multivalent RNA-dependent clustering, critical for efficient helicase activity. Disrupting eIF4B-RNA interactions through a targeted point mutation (F139A) in the RRM reduces both the cluster size and the helicase activity, further establishing a functional link between cluster formation and catalytic activity. Consistent with these findings, in-cell diffusion measurements reveal markedly slower diffusion of wild-type eIF4B compared with the RNA-binding-deficient mutant, indicative of RPC formation within the cellular environment. Together, our results reveal regulated helicase clustering as a previously unrecognized characteristic of the translation initiation machinery, linking ATP-dependent DEAD-box helicase activity to nanometer-scale RNA-protein clusters and translation initiation regulation.

biophysics↗

bioRxiv2024

Human tumor suppressor protein Pdcd4 binds at the mRNA entry channel in 40S small ribosomal subunit

Translation is regulated mainly in the initiation step, and its dysregulation is implicated in many human diseases. Several proteins have been found to regulate translational initiation, including Pdcd4 (programmed cell death gene 4). Pdcd4 is a tumor suppressor protein that prevents cell growth, invasion, and metastasis. It is downregulated in most tumor cells, while global translation in the cell is upregulated. To understand the mechanisms underlying translational control by Pdcd4, we used single-particle cryo-electron microscopy to determine the structure of human Pdcd4 bound to 40S small ribosomal subunit, including Pdcd4-40S and Pdcd4-40S-eIF4A-eIF3-eIF1 complexes. The structures reveal the binding site of Pdcd4 at the mRNA entry site in the 40S, where the C-terminal domain (CTD) interacts with eIF4A at the mRNA entry site, while the N-terminal domain (NTD) is inserted into the mRNA channel and decoding site. The structures, together with quantitative binding and in vitro translation assays, shed light on the critical role of the NTD for the recruitment of Pdcd4 to the ribosomal complex and suggest a model whereby Pdcd4 blocks the eIF4F-independent role of eIF4A during recruitment and scanning of the 5' UTR of mRNA.

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↗

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↗

bioRxiv2021

Hypo-osmotic Stress Induces ATP Release via Volume-regulated Anion Channels in Undifferentiated Mammary Cells

The high interstitial ATP concentration in the cancer microenvironment is a major source of adenosine, which acts as a strong immune suppressor. However, the source of ATP release has not been elucidated. We measured the ATP release during hypotonic stress using a real-time ATP luminescence imaging system in primary cultured mammary cells and in breast cell lines. In primary cultured cells, ATP was intermittently released with transient-sharp peaks, while in breast cell lines ATP was released with a slowly rising diffuse pattern. The diffuse ATP release pattern was changed to a transient-sharp pattern by cholera toxin treatment and the reverse change was induced by transforming growth factor (TGF) {beta} treatment. DCPIB, an inhibitor of volume-regulated anion channels (VRACs), only suppressed the diffuse pattern. The inflammatory mediator sphingosine-1-phosphate (S1P) induced a diffuse ATP release pattern isovolumetrically. The knockdown of A isoform of leucine-rich repeat-containing protein 8 (LRRC8A), the essential molecular entity of VRACs, using shRNA suppressed the diffuse pattern. These results suggest that abundantly expressed VRACs are a conduit of ATP release in undifferentiated cells, including cancer cells.

physiology↗