Small open reading frames and cellular stress responses

Abstract

Small open reading frames (smORFs) encoding polypeptides of less than 100 amino acids in eukaryotes (50 amino acids in prokaryotes) were historically excluded from genome annotation. However, recent advances in genomics, ribosome footprinting, and proteomics have revealed thousands of translated smORFs in genomes spanning evolutionary space. These smORFs can encode functional polypeptides, or act as cis-translational regulators. Herein we review evidence that some smORF-encoded polypeptides (SEPs) participate in stress responses in both prokaryotes and eukaryotes, and that some upstream ORFs (uORFs) regulate stress-responsive translation of downstream cistrons in eukaryotic cells. These studies provide insight into a regulated subclass of smORFs and suggest that at least some SEPs may participate in maintenance of cellular homeostasis under stress.

Figure 1.. Locations of stress-response associated small open reading frames (smORFs) in the E. coli str. K-12 substr. MG1655 genome.

(A) Map of the E. coli str. K-12 substr. MG1655 genome. Tracks from the outside to inside represent: 1. Annotated coding sequences within the forward strand (dark blue). 2. Annotated coding sequences within the complement strand (light blue). 3. Stress-responsive smORFs discussed in this review, by color: cold shock (blue)^, , heat shock (red)^, , antibiotic stress-inducible (purple), and nutrient sensing (green)^–. (4) Percent GC plot with above average GC content in dark gray and below average GC content in light gray. Genome sequence, annotated coding sequences, and stress-responsive smORFs (tracks 1–3) were uploaded to DNAPlotter version 1.0 and selected to construct the map using NCBI RefSeq assembly accession: GCF_000005845.2. (B) Scale diagrams of the cspG and cspI genomic regions; previously annotated genes are depicted as gray arrows and recently discovered cold-inducible smORFs are depicted as blue arrows^, .

Figure 2.. Putative functions of selected membrane-bound bacterial stress-responsive microproteins.

a) AcrZ enhances export of specific antibiotics by the drug efflux pump AcrAB-TolC; b) SgrT expression is induced by high intracellular levels of glucose 6-phosphate to inhibit glucose uptake; c) MgrB regulates PhoPQ (green and orange circles) in low intracellular Mg²⁺, decreasing expression of PhoPQ-dependent genes; d) MgtS increases Mg²⁺ uptake and prevents Mg²⁺ export^, .

Figure 3.. Regulated translation of upstream open reading frame (uORF)-containing transcripts under cellular stress.

Under normal conditions, active eIF2 is abundant and, in the subset of transcripts that contain them, AUG-initiated uORFs are translated, downregulating expression of the downstream ORF. Stress induces phosphorylation of the eIF2α subunit and results in eIF2 inactivation. Limiting eIF2 concentrations cause ribosomes to bypass AUG-initiated uORFs and drive downstream ORF translation. Simultaneously, weak eIF2 competitor eIF2A can activate translation of non-AUG initiated uORFs in the transcripts that contain them.

Figure 4.. Microproteins influence muscle regeneration following injury.

a) In uninjured muscle, DWORF binds the SERCA calcium pump and increases calcium flow into the sarcoplasmic reticulum. b) In uninjured muscle, SPAR binds the Ragulator v-ATPase and prevents mTORC1 activation. C) Following injury, Minion mediates myoblast fusion.