New Peptides Under the s(ORF)ace of the Genome.

Hundreds, perhaps thousands of previously unidentified functional small peptides could exist in most genomes, but these sequences have been generally overlooked. The discovery of genes encoding small peptides with important functions in different organisms has ignited the interest in these sequences and led to an increasing amount of effort towards their identification. Here, we review the advances, both computational and biochemical, that are leading the way in the discovery of putatively functional small open reading frame genes (smORFs), as well as the functional studies that have been carried out as a consequence of these searches. The evidence suggests that smORFs form a substantial part of our genomes, and that their encoded peptides could have a variety of important cellular functions.

Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans.

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.

Evolution of nubbin function in hemimetabolous and holometabolous insect appendages.

Insects display a whole spectrum of morphological diversity, which is especially noticeable in the organization of their appendages. A recent study in a hemipteran, Oncopeltus fasciatus (milkweed bug), showed that nubbin (nub) affects antenna morphogenesis, labial patterning, the length of the femoral segment in legs, and the formation of a limbless abdomen. To further determine the role of this gene in the evolution of insect morphology, we analyzed its functions in two additional hemimetabolous species, Acheta domesticus (house cricket) and Periplaneta americana (cockroach), and re-examined its role in Drosophila melanogaster (fruit fly). While both Acheta and Periplaneta nub-RNAi first nymphs develop crooked antennae, no visible changes are observed in the morphologies of their mouthparts and abdomen. Instead, the main effect is seen in legs. The joint between the tibia and first tarsomere (Ta-1) is lost in Acheta, which in turn, causes a fusion of these two segments and creates a chimeric nub-RNAi tibia-tarsus that retains a tibial identity in its proximal half and acquires a Ta-1 identity in its distal half. Similarly, our re-analysis of nub function in Drosophila reveals that legs lack all true joints and the fly tibia also exhibits a fused tibia and tarsus. Finally, we observe a similar phenotype in Periplaneta except that it encompasses different joints (coxa-trochanter and femur-tibia), and in this species we also show that nub expression in the legs is regulated by Notch signaling, as had previously been reported in flies and spiders. Overall, we propose that nub acts downstream of Notch on the distal part of insect leg segments to promote their development and growth, which in turn is required for joint formation. Our data represent the first functional evidence defining a role for nub in leg segmentation and highlight the varying degrees of its involvement in this process across insects.

RNAi analysis of nubbin embryonic functions in a hemimetabolous insect, Oncopeltus fasciatus.

Although the expression of the POU homeodomain gene nubbin (nub) has been examined in several arthropod species, its function has been studied only in Drosophila. Here, we provide the first insight into functional roles of this gene in a hemimetabolous insect species, Oncopeltus fasciatus. The analysis of its function using RNAi resulted in the altered morphology of antennae and labial tubes in the head, legs in the thorax, and, most notably, the growth of ectopic appendages originating from abdominal segments A2-A6. This change in the morphology of the abdomen can largely be attributed to the altered expression patterns of two hox genes, Ubx and abd-A, in RNAinub embryos. First, abd-A expression is completely abolished in A3-A6. Second, weak Ubx expression expands posteriorly to encompass novel domains in A2 and A3. Concomitant with these changes, limbs on A2 and A3 are small and less developed, whereas limbs on A4-A6 are large thoracic-like legs. These results show that nub function is necessary for normal abd-A expression and thus plays a critical role in suppressing leg formation on the abdomen. The loss of this regulation leads to upregulation of Distal-less, and subsequent development of appendages. In Drosophila, however, abd-A expression is unaffected in a nub-depleted background, indicating that no such regulatory relationship exists between these two genes in the fruit fly. These differences reveal that variation exists in the genetic mechanisms that maintain an ancient insect feature, the limbless abdomen.

Control of Drosophila imaginal disc development by rotund and roughened eye: differentially expressed transcripts of the same gene encoding functionally distinct zinc finger proteins.

The Drosophila rotund gene is required in the wings, antenna, haltere, proboscis and legs. A member of the Rac family of GTPases, denoted the rotund racGAP gene, was previously identified in the rotund region. However, previous studies indicated that rotund racGAP was not responsible for the rotund phenotypes and that the rotund gene had yet to be identified. We have isolated the rotund gene and show that it is a member of the Krüppel family of zinc finger genes. The adjacent roughened eye locus specifically affects the eye and is genetically separable from rotund. However, roughened eye and rotund are tightly linked, and we have therefore also isolated the roughened eye transcript. Intriguingly, we show that roughened eye is part of the rotund gene but is represented by a different transcript. The rotund and roughened eye transcripts result from the utilization of two different promoters that direct expression in non-overlapping domains in the larval imaginal discs. The predicted Rotund and Roughened Eye proteins share the same C-terminal region, including the zinc finger domain, but differ in their N-terminal regions. Each cDNA can rescue only the corresponding mutation and show negative effects when expressed in each others domain of expression. These results indicate that in addition to the differential expression of rotund and roughened eye, their proteins have distinct activities. rotund and roughened eye act downstream of early patterning genes such as dachshund and appear to be involved in Notch signaling by regulating Delta, scabrous and SERRATE: