Neurons provide a rich setting for studying post-transcriptional control. Here, we investigate the landscape of translational control in neurons and search for mRNA features that explain differences in translational efficiency (TE), considering the interplay between TE, mRNA poly(A)-tail lengths, microRNAs, and neuronal activation. In neurons and brain tissues, TE correlates with tail length, and a few dozen mRNAs appear to undergo cytoplasmic polyadenylation upon light or chemical stimulation. However, the correlation between TE and tail length is modest, explaining <5% of TE variance, and even this modest relationship diminishes when accounting for other mRNA features. Thus, tail length appears to affect TE only minimally. Accordingly, miRNAs, which accelerate deadenylation of their mRNA targets, primarily influence target mRNA levels, with no detectable effect on either steady-state tail lengths or TE. Larger correlates with TE include codon composition and predicted mRNA folding energy. When combined in a model, the identified correlates explain 38%-45% of TE variance. These results provide a framework for considering the relative impact of factors that contribute to translational control in neurons. They indicate that when examined in bulk, translational control in neurons largely resembles that of other types of post-embryonic cells. Thus, detection of more specialized control might require analyses that can distinguish translation occurring in neuronal processes from that occurring in cell bodies.

MicroRNAs (miRNAs) are endogenous approximately 22-nucleotide RNAs that mediate important gene-regulatory events by pairing to the mRNAs of protein-coding genes to direct their repression. Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels. Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels. For both ectopic and endogenous miRNA regulatory interactions, lowered mRNA levels account for most (>/=84%) of the decreased protein production. These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output.

Using the yeast Cryptococcus neoformans, we describe a mechanism by which transposons are initially targeted for RNAi-mediated genome defense. We show that intron-containing mRNA precursors template siRNA synthesis. We identify a Spliceosome-Coupled And Nuclear RNAi (SCANR) complex required for siRNA synthesis and demonstrate that it physically associates with the spliceosome. We find that RNAi target transcripts are distinguished by suboptimal introns and abnormally high occupancy on spliceosomes. Functional investigations demonstrate that the stalling of mRNA precursors on spliceosomes is required for siRNA accumulation. Lariat debranching enzyme is also necessary for siRNA production, suggesting a requirement for processing of stalled splicing intermediates. We propose that recognition of mRNA precursors by the SCANR complex is in kinetic competition with splicing, thereby promoting siRNA production from transposon transcripts stalled on spliceosomes. Disparity in the strength of expression signals encoded by transposons versus host genes offers an avenue for the evolution of genome defense.

The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian orthologue DDX3 are critical for the initiation of translation¹. Mutations in DDX3 are linked to tumorigenesis^2-4 and intellectual disability⁵, and the enzyme is targeted by a range of viruses⁶. How Ded1p and its orthologues engage RNAs during the initiation of translation is unknown. Here we show, by integrating transcriptome-wide analyses of translation, RNA structure and Ded1p-RNA binding, that the effects of Ded1p on the initiation of translation are connected to near-cognate initiation codons in 5' untranslated regions. Ded1p associates with the translation pre-initiation complex at the mRNA entry channel and repressing the activity of Ded1p leads to the accumulation of RNA structure in 5' untranslated regions, the initiation of translation from near-cognate start codons immediately upstream of these structures and decreased protein synthesis from the corresponding main open reading frames. The data reveal a program for the regulation of translation that links Ded1p, the activation of near-cognate start codons and mRNA structure. This program has a role in meiosis, in which a marked decrease in the levels of Ded1p is accompanied by the activation of the alternative translation initiation sites that are seen when the activity of Ded1p is repressed. Our observations indicate that Ded1p affects translation initiation by controlling the use of near-cognate initiation codons that are proximal to mRNA structure in 5' untranslated regions.