Thousands of small Open Reading Frames (smORFs) with the potential to encode small peptides of fewer than 100 amino acids exist in our genomes. However, the number of smORFs actually translated, and their molecular and functional roles are still unclear. In this study, we present a genome-wide assessment of smORF translation by ribosomal profiling of polysomal fractions in Drosophila. We detect two types of smORFs bound by multiple ribosomes and thus undergoing productive translation. The ‘longer’ smORFs of around 80 amino acids resemble canonical proteins in translational metrics and conservation, and display a propensity to contain transmembrane motifs. The ‘dwarf’ smORFs are in general shorter (around 20 amino-acid long), are mostly found in 5′-UTRs and non-coding RNAs, are less well conserved, and have no bioinformatic indicators of peptide function. Our findings indicate that thousands of smORFs are translated in metazoan genomes, reinforcing the idea that smORFs are an abundant and fundamental genome component.DOI: http://dx.doi.org/10.7554/eLife.03528.001

Translation of hundreds of small ORFs (smORFs) of less than 100 amino acids has recently been revealed in vertebrates and Drosophila. Some of these peptides have essential and conserved cellular functions. In Drosophila, we have predicted a particular smORF class encoding ~80 aa hydrophobic peptides, which may function in membranes and cell organelles. Here, we characterise hemotin, a gene encoding an 88aa transmembrane smORF peptide localised to early endosomes in Drosophila macrophages. hemotin regulates endosomal maturation during phagocytosis by repressing the cooperation of 14-3-3ζ with specific phosphatidylinositol (PI) enzymes. hemotin mutants accumulate undigested phagocytic material inside enlarged endo-lysosomes and as a result, hemotin mutants have reduced ability to fight bacteria, and hence, have severely reduced life span and resistance to infections. We identify Stannin, a peptide involved in organometallic toxicity, as the Hemotin functional homologue in vertebrates, showing that this novel regulator of phagocytic processing is widely conserved, emphasizing the significance of smORF peptides in cell biology and disease.

SummaryDrosophila hemocytes compose the cellular arm of the fly's innate immune system. Plasmatocytes, putative homologues to mammalian macrophages, represent ∼95% of the migratory hemocyte population in circulation and are responsible for the phagocytosis of bacteria and apoptotic tissues that arise during metamorphosis. It is not known as to how hemocytes become activated from a sessile state in response to such infectious and developmental cues, although the hormone ecdysone has been suggested as the signal that shifts hemocyte behaviour from quiescent to migratory at metamorphosis. Here, we corroborate this hypothesis by showing the activation of hemocyte motility by ecdysone. We induce motile behaviour in larval hemocytes by culturing them with 20-hydroxyecdysone ex vivo. Moreover, we also determine that motile cell behaviour requires the ecdysone receptor complex and leads to asymmetrical redistribution of both actin and tubulin cytoskeleton.

Soil salinity is one of the most challenging problems that restricts the normal growth and production of rice worldwide. It has therefore become very important to produce more saline tolerant rice varieties. This study shows constitutive over-expression of the vacuolar Na+/H+ antiporter gene (OsNHX1) from the rice landrace (Pokkali) and attainment of enhanced level of salinity tolerance in transgenic rice plants. It also shows that inclusion of the complete un-translated regions (UTRs) of the alternatively spliced OsNHX1 gene provides a higher level of tolerance to the transgenic rice. Two separate transformation events of the OsNHX1 gene, one with 1.9 kb region containing the 5′ UTR with CDS and the other of 2.3 kb, including 5′ UTR, CDS, and the 3′ UTR regions were performed. The transgenic plants with these two different constructs were advanced to the T3 generation and physiological and molecular screening of homozygous plants was conducted at seedling and reproductive stages under salinity (NaCl) stress. Both transgenic lines were observed to be tolerant compared to WT plants at both physiological stages. However, the transgenic lines containing the CDS with both the 5′ and 3′ UTR were significantly more tolerant compared to the transgenic lines containing OsNHX1 gene without the 3′ UTR. At the seedling stage at 12 dS/m stress, the chlorophyll content was significantly higher (P < 0.05) and the electrolyte leakage significantly lower (P < 0.05) in the order 2.3 kb > 1.9 kb > and WT lines. Yield in g/plant in the best line from the 2.3 kb plants was significantly more (P < 0.01) compared, respectively, to the best 1.9 kb line and WT plants at stress of 6 dS/m. Transformation with the complete transcripts rather than the CDS may therefore provide more durable level of tolerance.

Thousands of small Open Reading Frames (smORFs) encoding small peptides of fewer than 100 amino acids exist in our genomes. Examples of functional smORFs have been characterised in a few species but the actual number of translated smORFs, and their molecular, functional and evolutionary features are not known.Here we present a genome-wide assessment of smORF translation by ribosomal profiling of polysomal fractions. This 'polysomal ribo-Seq' suggests that smORFs are translated at the same level and in the same relative numbers (80%) as normal proteins. The smORF peptides appear widely conserved, show activity in cells, and display a putative amino acid signature. These findings reinforce the idea that smORFs are an abundant and fundamental genome component, displaying features usually attributed to canonical proteins, including high translation levels, biological function, amino acid sequence specificity and cross-species conservation.Small Open Reading Frames (smORFs) of fewer than 100 amino acids exist in genomes in the hundreds of thousands. Bioinformatic approaches predict that hundreds if not thousands of these are translated and functional in bacteria (1), yeast 2 (2, 3), plants (4) and in animals such as Drosophila (5), mouse (6) and humans (7). This is in contrast to their low rate of detection in biochemical (proteomic) and functional (genetic) screens. A handful of cases of translated smORFs encoding bioactive peptides have been described (8-11). However, translation and functionality of smORFs at a genomic scale has not been assessed in any species and therefore the actual number of translated smORFs is not known.The new technique of ribosomal profiling has corroborated and expanded the proteomes of yeast (12) mouse (13) and zebra fish (14). Thousands of new translated sequences have been described in each case, whether novel exons of annotated genes, alternative initiation sites, or entire ORFs. However, the application of ribosomal profiling outside canonical translated sequences can lead to differing conclusions (14,15). The problem remains that a ribosomal footprint cannot be equated with translation; non-productive binding of single ribosomes to mRNAs and 40S ribosomal subunit scanning can result in footprints yet do not constitute translation. It has been suggested that some smORFs associate with ribosomes in such a non-productive manner and do not undergo productive translation (16).Moreover, smORF mRNAs are short and present a small target for ribosomal binding and generation of footprints. To overcome these difficulties, we devised an improvement to ribosomal profiling. Instead of profiling all ribosomal-bound mRNAs, small and large polysome fractions were separated, RNase-digested, and ribosomeprotected mRNA fragments were deep sequenced ( Fig. 1A; methods). In this way, mRNAs bound by multiple ribosomes and hence actively translated can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single