Transcriptome-wide characterization of the eIF4A signature highlights plasticity in translation regulation.

BACKGROUND: Protein synthesis is tightly regulated and alterations to translation are characteristic of many cancers.Translation regulation is largely exerted at initiation through the eukaryotic translation initiation factor 4 F (eIF4F). eIF4F is pivotal for oncogenic signaling as it integrates mitogenic signals to amplify production of pro-growth and pro-survival factors. Convergence of these signals on eIF4F positions this factor as a gatekeeper of malignant fate. While the oncogenic properties of eIF4F have been characterized, genome-wide evaluation of eIF4F translational output is incomplete yet critical for developing novel translation-targeted therapies. RESULTS: To understand the impact of eIF4F on malignancy, we utilized a genome-wide ribosome profiling approach to identify eIF4F-driven mRNAs in MDA-MB-231 breast cancer cells. Using Silvestrol, a selective eIF4A inhibitor, we identify 284 genes that rely on eIF4A for efficient translation. Our screen confirmed several known eIF4F-dependent genes and identified many unrecognized targets of translation regulation. We show that 5'UTR complexity determines Silvestrol-sensitivity and altering 5'UTR structure modifies translational output. We highlight physiological implications of eIF4A inhibition, providing mechanistic insight into eIF4F pro-oncogenic activity. CONCLUSIONS: Here we describe the transcriptome-wide consequence of eIF4A inhibition in malignant cells, define mRNA features that confer eIF4A dependence, and provide genetic support for Silvestrol's anti-oncogenic properties. Importantly, our results show that eIF4A inhibition alters translation of an mRNA subset distinct from those affected by mTOR-mediated eIF4E inhibition. These results have significant implications for therapeutically targeting translation and underscore a dynamic role for eIF4F in remodeling the proteome toward malignancy.

Messenger RNA targeting to endoplasmic reticulum stress signalling sites.

Deficiencies in the protein-folding capacity of the endoplasmic reticulum (ER) in all eukaryotic cells lead to ER stress and trigger the unfolded protein response (UPR). ER stress is sensed by Ire1, a transmembrane kinase/endoribonuclease, which initiates the non-conventional splicing of the messenger RNA encoding a key transcription activator, Hac1 in yeast or XBP1 in metazoans. In the absence of ER stress, ribosomes are stalled on unspliced HAC1 mRNA. The translational control is imposed by a base-pairing interaction between the HAC1 intron and the HAC1 5' untranslated region. After excision of the intron, transfer RNA ligase joins the severed exons, lifting the translational block and allowing synthesis of Hac1 from the spliced HAC1 mRNA to ensue. Hac1 in turn drives the UPR gene expression program comprising 7-8% of the yeast genome to counteract ER stress. Here we show that, on activation, Ire1 molecules cluster in the ER membrane into discrete foci of higher-order oligomers, to which unspliced HAC1 mRNA is recruited by means of a conserved bipartite targeting element contained in the 3' untranslated region. Disruption of either Ire1 clustering or HAC1 mRNA recruitment impairs UPR signalling. The HAC1 3' untranslated region element is sufficient to target other mRNAs to Ire1 foci, as long as their translation is repressed. Translational repression afforded by the intron fulfils this requirement for HAC1 mRNA. Recruitment of mRNA to signalling centres provides a new paradigm for the control of eukaryotic gene expression.

A natural meiotic DNA break site in Schizosaccharomyces pombe is a hotspot of gene conversion, highly associated with crossing over.

In Schizosaccharomyces pombe, meiosis-specific DNA breaks that initiate recombination are observed at prominent but widely separated sites. We investigated the relationship between breakage and recombination at one of these sites, the mbs1 locus on chromosome I. Breaks corresponding to 10% of chromatids were mapped to four clusters spread over a 2.1-kb region. Gene conversion of markers within the clusters occurred in 11% of tetrads (3% of meiotic chromatids), making mbs1 a conversion hotspot when compared to other fission yeast markers. Approximately 80% of these conversions were associated with crossing over of flanking markers, suggesting a strong bias in meiotic break repair toward the generation of crossovers. This bias was observed in conversion events at three other loci, ade6, ade7, and ura1. A total of 50-80% of all crossovers seen in a 90-kb region flanking mbs1 occurred in a 4.8-kb interval containing the break sites. Thus, mbs1 is also a hotspot of crossing over, with breakage at mbs1 generating most of the crossovers in the 90-kb interval. Neither Rec12 (Spo11 ortholog) nor I-SceI-induced breakage at mbs1 was significantly associated with crossing over in an apparently break-free interval >25 kb away. Possible mechanisms for generating crossovers in such break-free intervals are discussed.