Iron increases synthesis rates of proteins encoded in iron-responsive element (IRE)-mRNAs; metabolic iron ("free," "labile") is Fe 2þ . The noncoding IRE-RNA structure, approximately 30 nt, folds into a stem loop to control synthesis of proteins in iron trafficking, cell cycling, and nervous system function. IRE-RNA riboregulators bind specifically to iron-regulatory proteins (IRP) proteins, inhibiting ribosome binding. Deletion of the IRE-RNA from an mRNA decreases both IRP binding and IRP-independent protein synthesis, indicating effects of other "factors." Current models of IRE-mRNA regulation, emphasizing iron-dependent degradation/modification of IRP, lack answers about how iron increases IRE-RNA/IRP protein dissociation or how IRE-RNA, after IRP dissociation, influences protein synthesis rates. However, we observed Fe 2þ (anaerobic) or Mn 2þ selectively increase the IRE-RNA/IRP K D . Here we show: (i) Fe 2þ binds to the IRE-RNA, altering its conformation (by 2-aminopurine fluorescence and ethidium bromide displacement); (ii) metal ions increase translation of IRE-mRNA in vitro; (iii) eukaryotic initiation factor (eIF)4F binds specifically with high affinity to IRE-RNA; (iv) Fe 2þ increased eIF4F/IRE-RNA binding, which outcompetes IRP binding; (v) exogenous eIF4F rescued metal-dependent IRE-RNA translation in eIF4F-depeleted extracts. The regulation by metabolic iron binding to IRE-RNA to decrease inhibitor protein (IRP) binding and increase activator protein (eIF4F) binding identifies IRE-RNA as a riboregulator.ferrous ion regulation | metabolic riboregulator I ron increases rates of ferritin protein synthesis in animals by facilitating messenger RNA/ribosome binding; metabolic iron (i.e., "labile" or "free" iron in cells) is considered to be ferrous (1). The iron response requires a noncoding riboregulator called the "iron-responsive element" (IRE), which is approximately 30 nt, folded into a distorted, bulged helix loop (2-5). This riboregulatory structure is also found in mRNAs for proteins of iron traffic (6-9), cell cycling (10), and the nervous system (11). IRP proteins bind with different stabilities to IRE-RNAs of the IRE-RNA family (12, 13), creating a graded or hierarchal set of mRNA responses to iron in vivo. Deletion of the 30 nt IRE-RNA not only removes IRP regulation but also decreases the rate of IRP-independent protein synthesis (14). A number of current models of IRE-RNA/IRP regulation feature iron-dependent degradation/modification of the IRP proteins as the main control point (8,9,15,16). Such models do not answer two important questions: (i) How does iron increase release of IRP protein for [4Fe-4S]-modification and/or degradation? Overlap of the IRE-RNA and the Fe-S binding sites on IRP1 prevents Fe-S insertion in the IRP1/IRE-RNA complex (5). (ii) How does the IRE-RNA control rates of IRP-independent protein synthesis (14)? In an earlier study, we showed that Fe 2þ ions (anaerobic) selectively increased the dissociation constant for the IRE-RNA/IRP1 complex in solution (12). Here we r...

SUMMARY FMRP (fragile X mental retardation protein) is a poly-some-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-4EBP-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.

Ribosome profiling has emerged as a powerful tool for genome-wide measurements of translation, but library construction requires multiple ligation steps and remains cumbersome relative to more conventional deep-sequencing experiments. We report a new, ligation-free approach to ribosome profiling that does not require ligation. Library construction for ligation-free ribosome profiling can be completed in one day with as little as 1 ng of purified RNA footprints. We apply ligation-free ribosome profiling to mouse brain tissue to identify new patterns of cell type-specific translation and test its ability to identify translational targets of mTOR signaling in the brain.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-1005-1) contains supplementary material, which is available to authorized users.