Recruitment of the ribosomal 40S subunit to the 3\u27untranslated region of a viral mRNA, via the eIF4 complex, facilitates cap-independent translation

Translation of uncapped plant viral RNAs can be facilitated by either an internal ribosomal entry site (IRES) in the 5\u27 untranslated region (UTR) or a cap-independent translation element (CITE) in the 3\u27 UTR. Barley yellow dwarf virus (BYDV) mRNA, which lacks both cap and poly(A) tail, has a translation element (3\u27BTE) in its 3\u27 UTR that is essential for efficient translation initiation at the 5\u27-proximal AUG. This mechanism requires binding of the eukaryotic initiation factor 4G (eIF4G) subunit of the heterodimer eIF4F to the 3\u27BTE and base pairing between the 3\u27BTE and the 5\u27 UTR. Here we investigate how this interaction recruits the ribosome to the 5\u27 end of the mRNA. Using fluorescence anisotropy, SHAPE analysis and toe printing, we found that (i) 40S ribosomal subunits bind to the 3\u27BTE, (ii) the helicase complex eIF4F-eIF4A-eIF4B-ATP increases affinity of 40S subunit binding to the conserved SL-I of the 3\u27 BTE by exposing more unpaired bases of the 3\u27BTE and (iii) long-distance base pairing transfers this complex to the 5\u27 end of the mRNA where translation initiates. These results reveal an utterly novel mechanism of ribosome recruitment to an mRNA

Widespread Alterations in Translation Elongation in the Brain of Juvenile Fmr1 Knockout Mice

Summary: FMRP (fragile X mental retardation protein) is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we utilize ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24-day-old Fmr1 knockout mice. We find a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP and an increase for terminal oligopyrimidine (TOP) motif-containing genes canonically controlled by mammalian target of rapamycin-eIF4E-binding protein-eIF4E binding protein-eukaryotic initiation factor 4E (mTOR-4E-BP-eIF4E) signaling. Amino acid motif- and gene-level analyses both show a widespread reduction of translational pausing in Fmr1 knockout mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in fragile X syndrome. : Silencing of Fmr1, the gene that encodes FMRP, causes fragile X syndrome. Das Sharma et al. used ribosome profiling in the cortex of 24-day-old Fmr1 knockout mice to dissect FMRP-mediated translational regulation. Fmr1 loss leads to a relief of translational pausing across a large number of genes. Keywords: fragile X syndrome, translational regulation, ribosome profilin

Additional file 5: Table S1. of Ligation-free ribosome profiling of cell type-specific translation in the brain

List of cell type-specific gene ontologies and their median TEs. (XLSX 13 kb

Additional file 7: Table S2. of Ligation-free ribosome profiling of cell type-specific translation in the brain

Table of genes altered following AZD-8055 treatment with p adj values <0.05 and fold change >2. (XLSX 11 kb

Additional file 1: Figure S1. of Ligation-free ribosome profiling of cell type-specific translation in the brain

Sensitivity of conventional and ligation-free strategies. Ligation-free and conventional libraries were generated from a serially diluted 34-base RNA oligonucleotide and analyzed via Bioanalyzer following an equal number of PCR cycles for each library. All ligation-free library preparations except for the 0.01 ng sample were loaded onto the Bioanalyzer at a 1:10 dilution to avoid saturating the detector at high concentrations. Detectable libraries were successfully generated for all concentrations using the ligation-free method but could only be generated using conventional methods for the 100- and 10-ng inputs. (PDF 665 kb