Exploiting the advantages of cationic copolymers and AgBr nanoparticles to optimize the antibacterial activity of chitosan.

Recently, the chitosan (CS)-based composites have attracted increasing attention for controlling and preventing the spread of pathogenic microorganisms. Herein, an amphiphilic copolymer containing epoxy and quaternary ammonium groups (PBGDBr) was synthesized via three common acrylate monomers. The epoxy groups of this copolymer were then crosslinked with the amino groups of CS to synthesize a natural/synthetic (PBGDBr-C) composite to increase the water solubility of CS under alkaline conditions and enhance its antibacterial activity based on chemical contact-type modes. Moreover, silver bromide nanoparticles (AgBr NPs)-decorated PBGDBr-C (AgBr@PBGDBr-C) composite was prepared, which aimed to endow the final AgBr@PBGDBr-C composite with a photodynamic antibacterial mode relying on the formation of Ag/AgBr nanostructures catalyzed by visible light on AgBr NPs. The results showed that the final composite possessed satisfactory bactericidal effects at concentrations higher than 64 and 128 µg/mL against Escherichia coli and Staphylococcus aureus, respectively. Additionally, The L929 cells treated with the final composite retained high cell viability (>80 %) at a concentration of 128 µg/mL, indicating its low toxicity to L929 cells. Overall, our synthetic strategy exploits a multi-modal system that enables chemical-photodynamic synergies to treat infections caused by pathogenic bacteria while delaying the development of bacterial resistance.

Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system.

We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S preinitiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach, we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5'-untranslated regions (5'UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5'UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5'UTRs.

Transcriptome-wide analysis of the function of Ded1 in translation preinitiation complex assembly in a reconstituted in vitro system.

We have developed a deep sequencing-based approach, Rec-Seq, that allows simultaneous monitoring of ribosomal 48S pre-initiation complex (PIC) formation on every mRNA in the translatome in an in vitro reconstituted system. Rec-Seq isolates key early steps in translation initiation in the absence of all other cellular components and processes. Using this approach we show that the DEAD-box ATPase Ded1 promotes 48S PIC formation on the start codons of >1000 native mRNAs, most of which have long, structured 5'-untranslated regions (5'UTRs). Remarkably, initiation measured in Rec-Seq was enhanced by Ded1 for most mRNAs previously shown to be highly Ded1-dependent by ribosome profiling of ded1 mutants in vivo, demonstrating that the core translation functions of the factor are recapitulated in the purified system. Our data do not support a model in which Ded1acts by reducing initiation at alternative start codons in 5'UTRs and instead indicate it functions by directly promoting mRNA recruitment to the 43S PIC and scanning to locate the main start codon. We also provide evidence that eIF4A, another essential DEAD-box initiation factor, is required for efficient PIC assembly on almost all mRNAs, regardless of their structural complexity, in contrast to the preferential stimulation by Ded1 of initiation on mRNAs with long, structured 5'UTRs.

Myopia in elementary school students in Eastern China during the COVID-19 pandemic.

Background: Myopia is an increasingly serious public concern, particularly among primary school students. The prevalence of myopia and its influencing factors in primary school pupils in Eastern China during the COVID-19 pandemic had not been explored. Methods: A randomly clustered sampling method was performed, and selected pupils from grade 1 to grade 3 in 15 primary schools in the Fenghua District of Zhejiang Province were included and given myopia screening and uniform questionnaire survey 1 year later. Results: A total of 4,213 students completed the myopia screening and questionnaire survey. Myopia was diagnosed in 1,356 pupils, with a myopia incidence of 32.19%. The spherical equivalent (SE) refraction of the included pupils decreased on average by 0.50 ± 2.15 D 1 year later. The myopia rate was positively correlated with the increase of grade, in which the myopia rate among grade 3 students was the highest at 39.69%. The myopia rate among female students was higher than that among male students. Students residing in urban areas had a higher myopia rate than in rural areas. Maintaining an near work distance ≥33 cm was a significant protective factor (OR = 0.84, 95% CI: 0.74-0.96). Students with two myopic parents had a higher risk of myopia (OR = 1.61, 95% CI: 1.34-1.92). Conclusion: During the COVID-19 pandemic, the myopia rate among early primary school students in Eastern China was high. More attention and implementation of interventions from health and education departments, such as training the development of good eye behavior, should be considered to strengthen the intervention of myopia in primary school students.

Generation paths of major production safety accidents - A fuzzy-set qualitative comparative analysis based on Chinese data.

Major production safety accidents have become the key obstacle to improve the overall safety production level. Analyzing the causal relationship of major production safety accidents is helpful to clarify its characteristics and laws. Based on the literature, the analytical framework of "individual - technology - management - environment" is proposed. Taking 37 production safety accidents as samples, fuzzy set qualitative comparative analysis (fsQCA) is used to analyze the occurrence path and mechanism of major production safety accidents. The results show that: (1) Major production safety accidents are the result of multiple factors coupling. Minor external supervision or abnormal production behaviors are more likely to cause major production safety accidents. (2) When the production behavior is abnormal and the safety management ability is insufficient, major production safety accidents are more likely to occur. (3) There are five ways and three mechanisms for major production safety accidents. This work enriches the cognition of causality of production safety accidents from the perspective of configuration, clearly shows which variable combinations lead to major accidents, and helps to prevent and reduce major production safety accidents and their risks.

Environmental collaborative governance of urban agglomeration in China: influencing factors and drivers.

This study attempts to examine how urban agglomerations establish sustainable environmental collaborative governance. To achieve this goal, the qualitative comparative analysis method is used to explore the conditions and models for urban agglomerations to establish environmental collaborative governance, with 12 urban agglomerations approved by the Chinese authorities as examples. Based on the collaborative governance framework, this paper proposes six starting conditions that affect the establishment of urban agglomeration collaboration: vertical intervention, horizontal cooperation, leadership attention, governance capacity, initial pollution, and economic governance. The interaction of these conditions was tested in the practice of environmental cooperation in urban agglomerations. The results show that horizontal cooperation, leadership attention, and economic governance are necessary conditions for the establishment of urban agglomeration cooperation. The authority-driven mode, capability-driven mode, and pressure-driven mode can promote cooperation. Vertical intervention, governance capacity, and initial pollution constitute the external and internal driving forces of urban agglomeration cooperation. These findings supplement the literature on urban agglomeration collaboration and provide policy makers with insight into sustainable urban agglomeration collaborative environmental governance.

Taxifolin attenuates inflammation via suppressing MAPK signal pathway in vitro and in silico analysis.

Objective: Taxifolin is a natural flavonoid compound that can be isolated from onions, grapes, oranges and grapefruit. It also acts as a medicine food homology with extraordinary antioxidant and anti-inflammatory activity. This study aims to explain the protective effects and potential mechanisms of taxifolin against inflammatory reaction. Methods: Levels of interleukin (IL)-6, IL-1ß and intracellular reactive oxygen species (ROS) were assessed in different time after the treatment of taxifolin in RAW264.7 cells induced by lipopolysaccharide (LPS). Subsequently, the mRNA and protein levels of inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α and the phosphorylation expression levels of the MAPK signal pathway were also evaluated. A silico analysis was used to explain the binding situation for the investigation of taxifolin and MAPK signal pathway. And then MAPK inhibitors were used to reveal the expression level of iNOS, VEGF, COX-2 and TNF-α in RAW264.7 cells. Results: It was demonstrated that cell inflammatory damage induced by LPS was significantly alleviated after the treatment of taxifolin. Then, the mRNA and protein levels of iNOS, VEGF, COX-2 and TNF-α were reduced and the phosphorylation expression levels of the MAPK signal pathway were down-regulated remarkably as well. In silico analysis, taxifolin could form a relatively stable combination with MAPK signal pathway. MAPK inhibitors showed increasing or decreasing effect in the mRNA levels of iNOS, VEGF, COX-2 and TNF-α, which suggesting that taxifolin down-regulated iNOS, VEGF, COX-2 and TNF-α expressions were not entirely through the MAPK pathway. Conclusion: This finding demonstrated that taxifolin improved the inflammatory responses that partly involved in the phosphorylation expression level of MAPK signal pathway in RAW264.7 cells exposed to acute stress.

Virtual reality technology enhances the cognitive and social communication of children with autism spectrum disorder.

Objective: We aimed to explore the impact of using virtual reality technology to intervene in and encourage the developmental behavior areas of cognition, imitation, and social interaction in children with autism spectrum disorder. Methods: Forty-four children with autism spectrum disorder were divided randomly into an intervention group and a control group, with each group consisting of 22 participants. Incorporating conventional rehabilitation strategies, virtual reality technology was used with the intervention group to conduct rehabilitation training in areas including cognition, imitation, and social interaction. The control group was provided conventional/routine clinical rehabilitation training. The children's cognitive development was evaluated before and 3 months after intervention. Results: After intervention, the developmental abilities of both groups of children in the areas of cognition, imitation, and social interaction were improved over their abilities measured before the intervention (P < 0.05). However, post-intervention score differences between the two groups demonstrated that the intervention group levels were better than the control group levels only in the areas of cognition and social interaction (P < 0.05). Conclusion: Combining virtual reality with conventional rehabilitation training improved the cognitive and social development of children with autism spectrum disorder and supported the goal of improving the rehabilitation effect.

Bibliometric analysis of research themes and trends in childhood autism spectrum disorders from 2012 to 2021.

Background: Autism spectrum disorders (ASD) are heterogeneous neurodevelopmental conditions that affect people worldwide. Early diagnosis and clinical support help achieve good outcomes. However, medical system structure and restricted resource availability create challenges that increase the risk of poor outcomes. Understanding the research progress of childhood ASD in recent years, based on clinical literature reports, can give relevant researchers and rehabilitation therapists more resonable research guides. Objective: This bibliometric study aimed to summarize themes and trends in research on childhood ASD and to suggest directions for future enquiry. Methods: Citations were downloaded from the Web of Science Core Collection database on childhood ASD published from 1 January 2012, to 31 December 2021. The retrieved information was analyzed using CiteSpace.5.8. R3, and VOS viewer. Results: A total of 7,611 papers were published across 103 areas. The United States was the leading source of publications. The clusters that have continued into 2020 include coronavirus disease 2019, gut microbiota, and physical activity, which represent key research topics. Keywords with frequency spikes during 2018-2021 were "disabilities monitoring network," "United States," and "caregiver." Conclusions: The Autism and Developmental Disabilities Monitoring Network in the United States can be used as a reference for relevant workers worldwide. An intelligent medical assistant system is being developed. Further studies are required to elucidate challenges associated with caring for a child with ASD.

Research progress of signaling pathways of the natural substances intervene dyslipidemia (Review).

Dyslipidemia is an umbrella term for a range of lipid metabolic disorders in the body. This condition has been widely reported to greatly increase the risk of cardiovascular diseases, threatening human health. In recent years, advances in molecular biology have deepened understanding of the dyslipidemia-related signaling pathways and specific mechanisms underlying dyslipidemia. Signaling pathways possess the ability to transmit an extracellular signal to the inside of the cell, leading to specific biological effects. Lipid metabolism disorders and lipid levels in the blood are frequently affected by aberrant alterations in the dyslipidemia-related signaling pathways. Therefore, further investigations into these pathways are required for the prevention and treatment of dyslipidemia. The present review summarizes the characteristics of six dyslipidemia-associated signaling pathways: Peroxisome proliferator-activated receptor, adenosine monophosphate-activated protein kinase, farnesoid X receptor, forkhead box O, adipocytokine and cyclic adenosine monophosphate signaling pathways. In particular, specific focus was placed on previous experimental studies and reports on the intervention effects of natural substances (compounds from animals, plants, marine organisms and microorganisms) on dyslipidemia.

Systematic Bibliometric Analysis of Research Hotspots and Trends on the Application of Virtual Reality in Nursing.

Background: With the emergence of the metaverse, virtual reality, as a digital technology, must be getting hotter. High quality virtual reality related nursing knowledge scene learning is gradually replacing traditional education and intervention skills. Objective: This systematic study aimed to gain insights into the overall application of virtual reality technology in the study of nursing. Methods: Citations downloaded from the Web of Science Core Collection database for use in VR in nursing publications published from January 1, 2012, to December 31, 2021, were considered in the research. Information retrieval was analyzed using https://bibliometric.com/app, CiteSpace.5.8. R3, and VOS viewer. Results: A total of 408 institutions from 95 areas contributed to relevant publications, of which the United States is the most influential country in this research field. The clustering labels of cited documents were obtained from the citing documents. Virtual simulation, virtual learning, clinical skills, and dementia are the clustering labels of co-cited documents. The burst keywords represented the research frontiers in 2020-2021, which were knowledge and simulation. Conclusion: Virtual nursing has had an impact on both nurses and clients. With the emergence of the concept of the metaverse, the research and application of virtual reality technology in nursing will gradually increase.

Interannual and seasonal variations of permafrost thaw depth on the Qinghai-Tibetan Plateau: A comparative study using long short-term memory, convolutional neural networks, and random forest.

An accurate estimation of thaw depth is critical to understanding permafrost changes due to climate warming on the Qinghai-Tibetan Plateau (QTP). However, previous studies mainly focused on the interannual changes of active layer thickness (ALT) across the QTP, and little is known about the changes in the seasonal thaw depth. Machine learning (ML) is a critical tool to accurately estimate the ALT of permafrost, but a direct comparison of ML with deep learning (DL) in ALT projection regarding the model performance is still lacking. Here, ML, namely random forest (RF), and DL algorithms like convolutional neural networks (CNN) and long short-term memory (LSTM) neural networks were compared to estimate the interannual changes of ALT and seasonal thaw depth on the QTP. Meteorological series, in-situ collected ALT observations, and geospatial information were used as predictors. The results show that both ML and DL methods are capable of estimating ALT and seasonal thaw depth in permafrost areas. The CNN and LSTM models developed using longer lagging times exhibit better performance in thaw depth prediction while the RF models are either mediocre or sometimes even worse as the lagging time increases. The results show that the ALT from 2003 to 2011 on the QTP exhibits an increasing trend, especially in the northern region. In addition, 68.8%, 88.7%, 52.5%, and 47.5% of the permafrost regions on the QTP have deepened seasonal thaw depth in spring, summer, autumn, and winter, respectively. The correlation between air temperature and permafrost thaw depth ranges from 0.65 to 1 with the time lag ranging from 1 to 32 days. This study shows that ML and DL can be effectively used in retrieving ALT and seasonal thaw depth of permafrost, and could present an efficient way to figure out the interannual and seasonal variations of permafrost conditions under climate warming.

Circ-HACE1 Aggravates Cigarette Smoke Extract-Induced Injury in Human Bronchial Epithelial Cells via Regulating Toll-Like Receptor 4 by Sponging miR-485-3p.

BACKGROUND: Smoking is the most common cause of chronic obstructive pulmonary disease (COPD), and the early diagnosis for COPD remains poor. Exploring the molecular mechanism and finding feasible biomarkers will be beneficial for clinical management of COPD. Circular RNAs (circRNAs) are noncoding RNAs that act as miRNA sponges to regulate the expression levels of genes, leading to the changes of cellular phenotypes and disease progression. CircRNA HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (circ-HACE1) was abnormally expressed after the induction of cigarette smoke extract (CSE) in cell model. This study was performed to explore its function and mechanism in COPD. METHODS: Circ-HACE1, microRNA-485-3p (miR-485-3p) and toll-like receptor 4 (TLR4) detection was performed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis/cell cycle were respectively examined using cell counting kit-8 (CCK-8) and flow cytometry. Inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Oxidative stress was evaluated through the measurement of malondialdehyde (MDA) and superoxide dismutase (SOD). The target binding analysis was conducted via dual-luciferase reporter assay. Western blot was employed for protein expression detection of TLR4. RESULTS: Circ-HACE1 was overexpressed in smokers or smokers with COPD and CSE upregulated circ-HACE1 expression in 16HBE cells. Knockdown of circ-HACE1 attenuated CSE-stimulated cell viability and cell cycle repression, as well as the enhancement of cell apoptosis, inflammatory response and oxidative stress. MiR-485-3p was a target of circ-HACE1. Circ-HACE1 regulated CSE-induced cell injury via targeting miR-485-3p. TLR4 was a downstream target of miR-485-3p, and miR-485-3p inhibited the CSE-induced cell damages by directly downregulating the level of TLR4. Circ-HACE1/miR-485-3p regulated TLR4 expression in CSE-treated 16HBE cells, and TLR4 overexpression also reversed all effects of si-circ-HACE1 on CSE-treated 16HBE cells. CONCLUSION: These findings elucidated that circ-HACE1 contributed to the CSE-induced cell damages in COPD cell models via regulating TLR4 by acting as the sponge of miR-485-3p.

Expansion of germline RPS20 mutation phenotype to include Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a ribosomopathy of variable expressivity and penetrance characterized by red cell aplasia, congenital anomalies, and predisposition to certain cancers, including early-onset colorectal cancer (CRC). DBA is primarily caused by a dominant mutation of a ribosomal protein (RP) gene, although approximately 20% of patients remain genetically uncharacterized despite exome sequencing and copy number analysis. Although somatic loss-of-function mutations in RP genes have been reported in sporadic cancers, with the exceptions of 5q-myelodysplastic syndrome (RPS14) and microsatellite unstable CRC (RPL22), these cancers are not enriched in DBA. Conversely, pathogenic variants in RPS20 were previously implicated in familial CRC; however, none of the reported individuals had classical DBA features. We describe two unrelated children with DBA lacking variants in known DBA genes who were found by exome sequencing to have de novo novel missense variants in RPS20. The variants affect the same amino acid but result in different substitutions and reduce the RPS20 protein level. Yeast models with mutation of the cognate residue resulted in defects in growth, ribosome biogenesis, and polysome formation. These findings expand the phenotypic spectrum of RPS20 mutation beyond familial CRC to include DBA, which itself is associated with increased risk of CRC.

eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide.

The translation preinitiation complex (PIC) scans the mRNA for an AUG codon in a favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with the reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at certain NCCs initiating amino-terminal extensions, including those that direct mitochondrial localization of the GRS1 and ALA1 products, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.

Temperature-dependent regulation of upstream open reading frame translation in S. cerevisiae.

BACKGROUND: Translation of an mRNA in eukaryotes starts at an AUG codon in most cases, but near-cognate codons (NCCs) such as UUG, ACG, and AUU can also be used as start sites at low levels in Saccharomyces cerevisiae. Initiation from NCCs or AUGs in the 5'-untranslated regions (UTRs) of mRNAs can lead to translation of upstream open reading frames (uORFs) that might regulate expression of the main ORF (mORF). Although there is some circumstantial evidence that the translation of uORFs can be affected by environmental conditions, little is known about how it is affected by changes in growth temperature. RESULTS: Using reporter assays, we found that changes in growth temperature can affect translation from NCC start sites in yeast cells, suggesting the possibility that gene expression could be regulated by temperature by altering use of different uORF start codons. Using ribosome profiling, we provide evidence that growth temperature regulates the efficiency of translation of nearly 200 uORFs in S. cerevisiae. Of these uORFs, most that start with an AUG codon have increased translational efficiency at 37 °C relative to 30 °C and decreased efficiency at 20 °C. For translationally regulated uORFs starting with NCCs, we did not observe a general trend for the direction of regulation as a function of temperature, suggesting mRNA-specific features can determine the mode of temperature-dependent regulation. Consistent with this conclusion, the position of the uORFs in the 5'-leader relative to the 5'-cap and the start codon of the main ORF correlates with the direction of temperature-dependent regulation of uORF translation. We have identified several novel cases in which changes in uORF translation are inversely correlated with changes in the translational efficiency of the downstream main ORF. Our data suggest that translation of these mRNAs is subject to temperature-dependent, uORF-mediated regulation. CONCLUSIONS: Our data suggest that alterations in the translation of specific uORFs by temperature can regulate gene expression in S. cerevisiae.

eIF1A residues implicated in cancer stabilize translation preinitiation complexes and favor suboptimal initiation sites in yeast.

The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context, and AUG recognition stabilizes a closed PIC conformation. The unstructured N-terminal tail (NTT) of yeast eIF1A deploys five basic residues to contact tRNAi, mRNA, or 18S rRNA exclusively in the closed state. Interestingly, EIF1AX mutations altering the human eIF1A NTT are associated with uveal melanoma (UM). We found that substituting all five basic residues, and seven UM-associated substitutions, in yeast eIF1A suppresses initiation at near-cognate UUG codons and AUGs in poor context. Ribosome profiling of NTT substitution R13P reveals heightened discrimination against unfavorable AUG context genome-wide. Both R13P and K16D substitutions destabilize the closed complex at UUG codons in reconstituted PICs. Thus, electrostatic interactions involving the eIF1A NTT stabilize the closed conformation and promote utilization of suboptimal start codons. We predict UM-associated mutations alter human gene expression by increasing discrimination against poor initiation sites.

Yeast eIF4A enhances recruitment of mRNAs regardless of their structural complexity.

eIF4A is a DEAD-box RNA-dependent ATPase thought to unwind RNA secondary structure in the 5'-untranslated regions (UTRs) of mRNAs to promote their recruitment to the eukaryotic translation pre-initiation complex (PIC). We show that eIF4A's ATPase activity is markedly stimulated in the presence of the PIC, independently of eIF4E•eIF4G, but dependent on subunits i and g of the heteromeric eIF3 complex. Surprisingly, eIF4A accelerated the rate of recruitment of all mRNAs tested, regardless of their degree of structural complexity. Structures in the 5'-UTR and 3' of the start codon synergistically inhibit mRNA recruitment in a manner relieved by eIF4A, indicating that the factor does not act solely to melt hairpins in 5'-UTRs. Our findings that eIF4A functionally interacts with the PIC and plays important roles beyond unwinding 5'-UTR structure is consistent with a recent proposal that eIF4A modulates the conformation of the 40S ribosomal subunit to promote mRNA recruitment.

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex.

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.

eIF4B stimulates translation of long mRNAs with structured 5' UTRs and low closed-loop potential but weak dependence on eIF4G.

DEAD-box RNA helicases eukaryotic translation initiation factor 4A (eIF4A) and Ded1 promote translation by resolving mRNA secondary structures that impede preinitiation complex (PIC) attachment to mRNA or scanning. Eukaryotic translation initiation factor 4B (eIF4B) is a cofactor for eIF4A but also might function independently of eIF4A. Ribosome profiling of mutants lacking eIF4B or with impaired eIF4A or Ded1 activity revealed that eliminating eIF4B reduces the relative translational efficiencies of many more genes than does inactivation of eIF4A, despite comparable reductions in bulk translation, and few genes display unusually strong requirements for both factors. However, either eliminating eIF4B or inactivating eIF4A preferentially impacts mRNAs with longer, more structured 5' untranslated regions (UTRs). These findings reveal an eIF4A-independent role for eIF4B in addition to its function as eIF4A cofactor in promoting PIC attachment or scanning on structured mRNAs. eIF4B, eIF4A, and Ded1 mutations also preferentially impair translation of longer mRNAs in a fashion mitigated by the ability to form closed-loop messenger ribonucleoprotein particles (mRNPs) via eIF4F-poly(A)-binding protein 1 (Pab1) association, suggesting cooperation between closed-loop assembly and eIF4B/helicase functions. Remarkably, depleting eukaryotic translation initiation factor 4G (eIF4G), the scaffold subunit of eukaryotic translation initiation factor 4F (eIF4F), preferentially impacts short mRNAs with strong closed-loop potential and unstructured 5' UTRs, exactly the opposite features associated with hyperdependence on the eIF4B/helicases. We propose that short, highly efficient mRNAs preferentially depend on the stimulatory effects of eIF4G-dependent closed-loop assembly.