Periodic Reporting for period 1 - lincPeptEvolDev (LincRNA and encoded small peptides: Functional discovery in development and evolution)
Okres sprawozdawczy: 2015-05-01 do 2017-04-30
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.
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.