SmORFing for Calcium: Genomes contain thousands of small open reading frames (smORFs), short DNA sequences coding for peptides of less than 100 amino acids. Magny et al. (p. 1116 , published on 22 August) describe two smORF-encoded peptides of less than 30 amino acids regulating calcium transport and, hence, regular heart contraction, in the fruit fly Drosophila . These peptides seem to have been conserved for more than 550 million years in a range of species from flies to humans, where they have been implicated in severe heart diseases. Such conservation suggests that smORFs might be an ancient part of our functional genome.

Background: The relationship between DNA sequence and encoded information is still an unsolved puzzle. The number of protein-coding genes in higher eukaryotes identified by genome projects is lower than was expected, while a considerable amount of putatively non-coding transcription has been detected. Functional small open reading frames (smORFs) are known to exist in several organisms. However, coding sequence detection methods are biased against detecting such very short open reading frames. Thus, a substantial number of non-canonical coding regions encoding short peptides might await characterization. Results: Using bio-informatics methods, we have searched for smORFs of less than 100 amino acids in the putatively non-coding euchromatic DNA of Drosophila melanogaster, and initially identified nearly 600,000 of them. We have studied the pattern of conservation of these smORFs as coding entities between D. melanogaster and Drosophila pseudoobscura, their presence in syntenic and in transcribed regions of the genome, and their ratio of conservative versus non-conservative nucleotide changes. For negative controls, we compared the results with those obtained using random short sequences, while a positive control was provided by smORFs validated by proteomics data. Conclusions: The combination of these analyses led us to postulate the existence of at least 401 functional smORFs in Drosophila, with the possibility that as many as 4,561 such functional smORFs may exist.

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. In our genomes there are millions of short open reading frames that could produce small peptides of less than 100 amino-acids if translated. These sequences have been so far disregarded, but increasing evidence supports the notion that a subset of these–termed smORFs–are translated; however the function of most of the resulting peptides remains unclear. Here we characterise hemotin, a smORF gene that encodes a transmembrane peptide of 88 amino-acids expressed in Drosophila macrophages, and we show that Hemotin localizes to early endosomes–vesicles involved in the traffic of material between the cell membrane and the cytoplasm. Macrophages found in hemotin mutants have enlarged and abnormal endosomes that delay digestion of phagocytosed bacteria. Accordingly, these mutant flies fight bacterial infections poorly, and die early. In humans we identify Stannin–a peptide previously involved in heavy metal toxicity–as the Hemotin homologue, and show that it shares Hemotin function in macrophages. These results identify the Hemotin-Stannin smORF peptides as regulators of phagocytosis, and suggest that they could have contributed to the ancestral origin of macrophage-like cells. This provides a strong example of a smORF conserved for hundreds of millions of years.

Up to 80% of individuals with myotonic dystrophy type 1 (DM1) will develop cardiac abnormalities at some point during the progression of their disease, the most common of which is heart blockage of varying degrees. Such blockage is characterized by conduction defects and supraventricular and ventricular tachycardia, and carries a high risk of sudden cardiac death. Despite its importance, very few animal model studies have focused on the heart dysfunction in DM1. Here, we describe the characterization of the heart phenotype in a Drosophila model expressing pure expanded CUG repeats under the control of the cardiomyocyte-specific driver GMH5-Gal4. Morphologically, expression of 250 CUG repeats caused abnormalities in the parallel alignment of the spiral myofibrils in dissected fly hearts, as revealed by phalloidin staining. Moreover, combined immunofluorescence and in situ hybridization of Muscleblind and CUG repeats, respectively, confirmed detectable ribonuclear foci and Muscleblind sequestration, characteristic features of DM1, exclusively in flies expressing the expanded CTG repeats. Similarly to what has been reported in humans with DM1, heart-specific expression of toxic RNA resulted in reduced survival, increased arrhythmia, altered diastolic and systolic function, reduced heart tube diameters and reduced contractility in the model flies. As a proof of concept that the fly heart model can be used for in vivo testing of promising therapeutic compounds, we fed flies with pentamidine, a compound previously described to improve DM1 phenotypes. Pentamidine not only released Muscleblind from the CUG RNA repeats and reduced ribonuclear formation in the Drosophila heart, but also rescued heart arrhythmicity and contractility, and improved fly survival in animals expressing 250 CUG repeats.

Small Open Reading Frames (smORFs) of less than 100 codons remain mostly uncharacterised. About a thousand smORFs per genome encode peptides and microproteins about 70–80 aa long, often containing recognisable protein structures and markers of translation, and these are referred to as short Coding Sequences (sCDSs). The characterisation of individual sCDSs has provided examples of smORFs’ function and conservation, but we cannot infer the functionality of all other metazoan smORFs from these. sCDS function has been characterised at a genome-wide scale in yeast and bacteria, showing that hundreds can produce a phenotype, but attempts in metazoans have been less successful. Either most sCDSs are not functional, or classic experimental techniques do not work with smORFs due to their shortness. Here, we combine extensive proteomics with bioinformatics and genetics in order to detect and corroborate sCDS function in Drosophila. Our studies nearly double the number of sCDSs with detected peptides and microproteins and an experimentally corroborated function. Finally, we observe a correlation between proven sCDS protein function and bioinformatic markers such as conservation and GC content. Our results support that sCDSs peptides and microproteins act as membrane-related regulators of canonical proteins, regulators whose functions are best understood at the cellular level, and whose mutants produce little, if any, overt morphological phenotypes.