To start with, we created a new bioinformatics pipeline to process genomic data to look for conserved micropeptides. Specifically, the pipeline takes expressed genomic sequences (the transcriptome) from insect embryos of various developmental stages and various species spanning the breadth of the insect phylum, and compares analogous sequences. We looked for small proteins expressed in polyadenylated transcripts that do not overlap known protein coding genes, do not possess any known functional domains (which may indicate a mis-annotated known protein coding transcript), of 10-40 amino acids in length, and with at least 60% sequence similarity across species. Using a series of pairwise comparisons among the species in our pipelines, we recovered over 10,000 candidate micropeptides that are conserved between at least two of our study insect species. I began work on 4 of the top candidates that were detectably conserved in at least 5 of our insect species in this study. I designed small molecular beacons (probes) that allow me to detect each expressed gene ("transcript") in insect embryos, using an in situ detection method called in situ hybridization. I looked at the expression of each of these genes in the different species to look at where each transcript is expressed in the embryo, which can sometimes give clues about its function. During this time, some genome sequence work I was doing led me to notice that one of the conserved micropeptides I was studying is conserved not only among all the insects examined in our study (spanning around 300 million years of evolutionary time), but also all the way up through humans. Such a degree of conservation is a strong hallmark of functional essentialness, since over such a long evolutionary time, anything non essential would have accumulated so many mutations randomly as to be unrecognizable. I began to focus my work on this candidate micropeptide, which I called Peptide X.
I used a recently discovered genome editing system called CRISPR/Cas9 to mutate the genomes of fruit flies and of zebrafish, as a representative vertebrate species, to change the sequence of Peptide X so it would no longer encode a functional small protein. It took many generations to establish stable mutant lines of each animal. In the mean time, we also generated an antibody that would specifically recognize our Peptide X. This reagent would allow us to "see" the peptide in tissue, to understand where it is made, if that pattern is the same as the transcript that encodes it, and to gain some further insights into its potential function. The antibody was used on embryos and adults of both fruit flies and zebrafish. Peptide X seems to be expressed almost everywhere. But in both animals, we discovered that its expression is enriched in the gut. When we eventually were able to recover mutant animals completely lacking Peptide X, I found that in zebrafish, these animals also exhibit some defects in the gut, supporting a role Peptide X in the tissue where we observed its strong expression.
My current work in completing this project is establishing how Peptide X functions in the gut, with whom it interacts, and what its significance is for healthy gut function and possibly in disease. Many small peptides play important roles in the gut, from signaling in appetite and nutritional sensing, to modulating metabolism, fending off bacterial infection, and many more functions. We still don't know what Peptide X does in the gut, but we will determine its function in each species, and determine whether its network and function are as conserved between distant species as its sequence is. We hope that this discovery will lead to deeper understanding of human gut physiology and disease pathology.
