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LincRNA and encoded small peptides: Functional discovery in development and evolution

Periodic Reporting for period 1 - lincPeptEvolDev (LincRNA and encoded small peptides: Functional discovery in development and evolution)

Reporting period: 2015-05-01 to 2017-04-30

Recent technological advances in DNA sequencing have led to a rapidly growing body of genomic information, wherein the genetic material from hundreds of plant and animal species, thousands of unique individuals, and many cancer types are now being catalogued. With this glut of genomic information, we are also making important discoveries about the complexity of our genomes, discovering new classes of functional elements that were previously unknown. These include functional RNA molecules from regions of the genome previously thought to be junk (including "long non-coding RNAs") and even new small protein units encoded in regions of coding and non-coding genes that were previously thought to be only regulatory.

This project sought to identify novel functional protein molecules, called "micropeptides", a recently discovered class of tiny proteins that exist in all living things. We used a variety of insect model systems to identify potential functional micropeptides, by looking at their genomic sequences. We used evolutionary conservation as a beacon for candidates of interest, since evolutionary pressure causes a signature of protein sequence conservation, in a background where random mutation renders non-essential genome elements largely unrecognizable over time. Since insect development has been very well studied, and since genetic perturbations of these programs result in strong and distinctive defects, we focused on looking for conserved micropeptides that are expressed during development, and hoped that their mutation would lead to defects that would point us toward the part of development that is specifically affected. Identifying the functions of micropeptides is still one of the most challenging aspects of their study, since even by introducing mistakes into their sequence, it can be challenging to identify what consequence this has for a whole organism unless it is extremely significant. Still, this area of study is of great current interest, since small proteins like this class of naturally existing micropeptides, are able to integrate into a variety of cellular settings, and may represent a fruitful area of development for novel therapeutics that are able to affect a variety of cellular processes in a specific manner.

Our overall objectives were to identify conserved insect micropeptides and to identify and study their functions, by introducing mutations that eliminate the function of these molecules, in order to identify the consequences of this loss of function. Our hope was that discovering and characterization of these tiny proteins would deepen our understanding of the functional capabilities of this class of molecules in general, and also specifically identify new functional elements that are critical components of developmental gene networks.
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.
If indeed Peptide X has a function in any process similar to those known for gut peptides to date- nutrition sensing and metabolism, microbiome modulation, protection of gut epithelium integrity, hormonal signaling, immunity- it could have major implications for drug targeting or mimetics, depending on the function. It may improve our understanding of normal function of the gut, and may eventually lead to better, actionable understanding of gut architecture, function, and maintenance.