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NanoCAGE reveals 5’ UTR features that define specific modes of translation of functionally related mTOR-sensitive mRNAs

ABSTRACT: mTOR regulates mRNA translation. Whereas ribosome-profiling suggested that mTOR exclusively stimulates translation of TOP (containing a 5’-terminal oligopyrimidine [5’TOP] motif) and TOP-like mRNAs, polysome-profiling implied that mTOR also modulates translation of non-TOP mRNAs. We show that ribosome-, but not polysome-profiling, is biased towards identification of TOP mRNAs as differentially translated while obscuring detection of changes in non-TOP mRNA translation. Transcription start site profiling by Nano-Cap Analysis of Gene Expression (nanoCAGE) revealed that many mTOR-sensitive mRNAs do not have 5’TOP motifs. Moreover, nanoCAGE showed that 5’ UTR features distinguish two functionally and translationally distinct subsets of mTOR-sensitive mRNAs: i) those with short 5’ UTRs enriched for mitochondrial functions such as respiration, that are translated in an eIF4E, but not eIF4A1-dependent manner and ii) mRNAs encoding proliferation- and survival-promoting proteins, that harbor long 5’ UTRs, and require both eIF4E and eIF4A1 for their efficient translation. Selective inhibition of translation of mRNAs harboring long 5’ UTRs via suppression of eIF4A leads to uncoupling of expression of proteins involved in respiration (e.g. ATP5O) from those protecting mitochondrial integrity (e.g. BCL-2) ultimately resulting in apoptosis. Conversely, simultaneous translational downregulation of both long and short 5’ UTR mRNAs by mTOR inhibitors results in suppression of mitochondrial respiration and predominantly cytostatic effects. Therefore, 5’ UTR features define differential modes of translation of functionally distinct mTOR-sensitive mRNAs, which explains discrepancies between the effects of mTOR and eIF4A inhibitors on neoplastic cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE77033 | GEO | 2016/03/14

SECONDARY ACCESSION(S): PRJNA309302

REPOSITORIES: GEO

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Project description:DEAD-box RNA helicases eIF4A and Ded1 are believed to promote translation initiation by resolving mRNA secondary structures that impede ribosome attachment at the mRNA 5’ end or subsequent scanning of the 5’UTR, but whether they perform distinct functions or act redundantly in vivo is poorly understood. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling. Despite similar reductions in bulk translation, inactivation of a cold-sensitive Ded1 mutant substantially reduced the TEs of >600 mRNAs, whereas inactivation of a temperature-sensitive eIF4A mutant yielded <40 similarly impaired mRNAs. The broader requirement for Ded1 did not reflect more pervasive secondary structures at low temperature, as inactivation of temperature-sensitive and cold-sensitive ded1 mutants gave highly correlated results. Interestingly, Ded1-dependent mRNAs exhibit greater than average 5’UTR length and propensity for secondary structure, implicating Ded1 in scanning though structured 5' UTRs. Reporter assays confirmed that cap- distal stem-loop insertions increase dependence on Ded1 but not eIF4A for efficient translation. While only a small fraction of mRNAs is strongly dependent on eIF4A, this dependence is significantly correlated with requirements for Ded1 and 5’UTR features characteristic of Ded1- dependent mRNAs. Our findings suggest that Ded1 is critically required to promote scanning through secondary structures within 5’UTRs; and while eIF4A cooperates with Ded1 in this function, it also promotes a step of initiation common to virtually all yeast mRNAs. We compared the effects of mutations in Ded1 or eIF4A on global translational efficiencies (TEs) in yeast by ribosome footprint profiling.The study includes 32 samples, comprised of 16 mRNA-Seq samples and 16 ribosome footprint profiling samples, derived from biological replicates of 3 mutant strains, ded1-cs, ded1-ts and tif1-ts, and the corresponding wild-type strains. The tif1-ts mutant and its wild-type counterpart were analyzed at 30°C and 37°C.

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NanoCAGE reveals 5’ UTR features that define specific modes of translation of functionally related mTOR-sensitive mRNAs

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