Seán E. O’leary

The global impact of the COVID-19 pandemic highlights the need for a deeper understanding of SARS-CoV-2 replication. Although the essential roles of the RNA-dependent RNA polymerase (NSP12), primase (NSP8), and nucleocapsid (N) proteins are established, their contact points with the full-length viral RNA genome during active infection require further characterization. Here, we used enhanced crosslinking and immunoprecipitation (eCLIP) to map direct interactions of NSP8, NSP12, and N across the SARS-CoV-2 genome during live virus infection at early and late stages of replication. We mapped NSP8 and NSP12 binding to the 5′ and 3′ ends of positive- and negative-sense RNA, consistent with their roles in viral replication, transcription, and translation. We also observed an NSP12 binding site within the conserved Y1 domain of NSP3 that modulates RNA and protein abundance. In addition, N binds the 5′ untranslated region (UTR), supporting a role in translational regulation early in infection, and associates with the frameshift element, where it modulates ribosomal frameshifting. Together, these findings map SARS-CoV-2 RNA–protein interactions across the viral genome and delineate specific regulatory regions for potential antiviral intervention.

SUMMARY: Efficient eukaryotic messenger RNA translation requires dynamic collaboration between the three subunits of initiation factor 4F (eIF4F, eIF4E•G•A), which recognises and activates mRNA at its 5ʹ cap structure for ribosome recruitment. Despite its high biological and pharmacological importance, the dynamics of full human eIF4F–mRNA engagement remain largely uncharacterised, hindering mechanistic understanding of translation initiation and its regulation. Here we observed human eIF4F activity with single-molecule fluorescence assays that directly visualise mRNA cap recognition by its eIF4E subunit. Unexpectedly, we find that inherently transient eIF4E–cap binding is repressed by full-length human eIF4G, predominantly through its C-terminus, representing an unanticipated role for eIF4G as a central rate-limiting factor in the eIF4F complex. This repression is relieved by nucleotide-bound eIF4A in the eIF4F heterotrimer, placing eIF4A as a crucial determinant of efficient cap recognition for translation. Molecular dynamics simulations reveal that electrostatic modulation of eIF4E–mRNA interaction allows eIF4G to control the cap-recognition frequency. Our findings also indicate that intrinsic eIF4F– mRNA dynamics are insufficient to support cap-tethered ribosomal scanning to locate translation start sites. They illuminate fundamental design-principle differences for the overall mechanism and division of labour among eIF4F subunits during mRNA recognition in humans and yeast.

How eukaryotic ribosomes traverse messenger RNA (mRNA) leader sequences to search for protein-synthesis start sites remains one of the most mysterious aspects of translation and its regulation. While the search process is conventionally described by a linear “scanning” model, its exquisitely dynamic nature has restricted detailed mechanistic study. Here, we observed single Saccharomyces cerevisiae ribosomal scanning complexes in real time, finding that they scan diverse mRNA leaders at a rate of 10 to 20 nt s ⁻¹ . We show that specific binding of a protein to its mRNA leader sequence substantially arrests scanning. Conversely, impairing scanning-complex guanosine 5′-triphosphate hydrolysis results in native start-site bypass. Our results illustrate an mRNA-centric, kinetically controlled regulatory model where the ribosomal pre-initiation complex amplifies a nuanced energetic landscape to regulate scanning and start-site selection fidelity.

The parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 (∆eif1.2) markedly impeded bradyzoite cyst formation in vitro and in vivo. We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that ∆eif1.2 parasites are defective in upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in ∆eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

To initiate protein synthesis, the eukaryotic ribosomal pre-initiation complex must survey a messenger RNA leader sequence to identify the correct start codon. ¹ This pre-initiation complex motion through the leader, termed ‘scanning’, is coordinated by an intricate and highly-dynamic assemblage of translation factors, mRNA, initiator tRNA, and the small ribosomal subunit. ^2,3,4 Fundamental aspects of scanning dynamics remain poorly understood: estimates of its rate vary widely, and mechanisms that establish and regulate the motion remain largely unknown. Here we show, at the single-molecule level, that the Saccharomyces cerevisiae pre-initiation complex scans a diverse set of mRNA leaders at a rate of 10 – 20 nt s ⁻¹ . Our data quantitatively support a scanning mechanism in which the mRNA leader is inspected base by base, essentially unidirectionally, and with modest sensitivity to mRNA structure. Unexpectedly, scanning bypasses canonical start sites where the initiator tRNA is present but GTP hydrolysis in the pre-initiation complex is impaired. Conversely, binding of the S. cerevisiae poly(A)-binding protein Pab1p to its own mRNA leader hinders scanning in a concentration-dependent manner. At saturating, physiological concentrations, Pab1p prolongs scanning by more than four-fold, evoking an autoregulation mechanism for translation initiation. Our data provide a real-time mechanistic framework for scanning regulation and energetics.

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.

SUMMARY: mRNA 5ʹ cap recognition by eIF4F is a key step in eukaryotic translational control. While different mRNAs respond differently to eIF4F-directed regulation, the molecular basis for this variability remains unclear. We developed single-molecule fluorescence assays to directly observe eIF4F– mRNA interactions. We uncovered a complex interplay of mRNA features with factor activities that differentiates cap recognition between mRNAs. eIF4E–cap association rates are anticorrelated with mRNA length. eIF4A leverages ATP binding to differentially accelerate eIF4E– mRNA association; the extent of this acceleration correlates with translation efficiency in vivo . eIF4G lengthens eIF4E–cap binding to persist on the initiation timescale. The full eIF4F complex discriminates between mRNAs in an ATP-dependent manner. After eIF4F–mRNA binding, eIF4E is ejected from the cap by eIF4A ATP hydrolysis. Our results suggest features throughout mRNA coordinate in controlling cap recognition at the 5ʹ end, and suggest a model for how eIF4F–mRNA dynamics establish mRNA sensitivity to translational control processes.

Coronaviruses initiate translation through recognition of the viral RNA 5’ m ⁷ GpppA _m cap by translation factor eIF4F. eIF4F is a heterotrimeric protein complex with cap-binding, RNA-binding, and RNA helicase activities. Modulating eIF4F function through cellular regulation or small-molecule inhibition impacts coronavirus replication, including for SARS-CoV-2. Translation initiation involves highly coordinated dynamics of translation factors with messenger or viral RNA. However, how the eIF4F subunits coordinate on the initiation timescale to define cap-binding efficiency remains incompletely understood. Here we report that translation supported by the SARS-CoV-2 5’-UTR is highly sensitive to eIF4A inhibition by rocaglamide. Through a single-molecule fluorescence approach that reports on eIF4E–cap interaction, we dissect how eIF4F subunits contribute to cap-recognition efficiency on the SARS-CoV-2 5’ UTR. We find that free eIF4A enhances cap accessibility for eIF4E binding, but eIF4G alone does not change the kinetics of eIF4E–RNA interaction. Conversely, formation of the full eIF4F complex significantly alters eIF4E–cap interaction, suggesting that coordinated eIF4E and eIF4A activities establish the net eIF4F–cap recognition efficiency. Moreover, the eIF4F complex formed with phosphomimetic eIF4E(S209D) binds the viral UTR more efficiently than with wild-type eIF4E. These results highlight a dynamic interplay of eIF4F subunits and mRNA that determines cap-recognition efficiency.

Dynamic RNA–protein interactions underpin numerous molecular control mechanisms in biology. However, relatively little is known about the kinetic landscape of protein interactions with full-length RNAs. The extent to which interaction kinetics vary for the same RNA element across the transcriptome and the molecular determinants of variability therefore remain poorly defined. Moreover, it is unclear how one protein–RNA interaction might be transduced by RNA to kinetically impact a second. We report a parallelized, real-time single-molecule fluorescence assay for protein interaction kinetics on eukaryotic mRNA populations obtained from cells. We observed ∼100-fold heterogeneity for interactions of the translation initiation factor eIF4E with the universal mRNA 5′ cap structure, dominated by steric effects on barrier-height variability for association. We also found that an RNA helicase, eIF4A, independently accelerated eIF4E–cap association. These data support a kinetic mechanism for how mRNA can determine the sensitivity of its translation to reduction in cellular eIF4E concentrations. They also support the view that global RNA structure significantly modulates protein–RNA interaction dynamics and can facilitate real-time communication between protein interactions at distinct sites.

The microtubule cytoskeleton serves as a dynamic structural framework for mitosis in eukaryotic cells. TANGLED1 (TAN1) is a microtubule-binding protein that localizes to the division site and mitotic microtubules and plays a critical role in division plane orientation in plants. Here, in vitro experiments demonstrate that TAN1 directly binds microtubules, mediating microtubule zippering or end-on microtubule interactions, depending on their contact angle. Maize tan1 mutant cells improperly position the preprophase band (PPB), which predicts the future division site. However, cell shape–based modeling indicates that PPB positioning defects are likely a consequence of abnormal cell shapes and not due to TAN1 absence. In telophase, colocalization of growing microtubules ends from the phragmoplast with TAN1 at the division site suggests that TAN1 interacts with microtubule tips end-on. Together, our results suggest that TAN1 contributes to microtubule organization to ensure proper division plane orientation.