Discovery and Characterization of Novel smORF-Encoded Polypeptides (SEPs)

Peptides and small proteins have essential physiological roles including metabolism (insulin), sleep (orexin), and stress (corticotropin-releasing hormone). Recent exploration of the human genome and proteome has revealed the existence of hundreds to thousands of short open reading frames (sORFs); however, the extent to which sORFs are translated into polypeptides is unknown. Inline with the current convention, a protein-coding short ORF is defined to be a small ORF or smORF; the protein product as a smORF-encoded polypeptide is called a SEP; and a sORF or smORF upstream from an open reading frame (i.e. in the 5'-UTR) is called an upstream ORF or uORF. The identification of smORFs and SEPs have prompted efforts determine the regulation and biological functions for these molecules. My thesis research focused on improving SEP discovery and the characterization of functional SEPs.;The discovery of novel SEPs contributes to our understanding of composition of the human genome and proteome. My colleagues and I developed and utilized a proteogenomics strategy, which integrates genomics (RNA-Seq) with proteomics, to discover 86 novel human SEPs, the largest number of validated SEPs described at the time. Our findings indicated that SEPs are a large, unappreciated, peptide family. Moreover, our approach was far from optimized and we felt that there were likely many additional SEPs in the human genome. One goal of my thesis work was to improve the SEP discovery methodology to find more human SEPs. My efforts led to the discovery of an over 300 SEPs in cell lines and human tissue.;A second goal of my thesis work was to identify and characterize functional SEPs. To do this I identified the SEPs that are most highly conserved throughout evolution with a program called PhyloCSF. PhyloCSF identifies which SEPs are evolutionary conserved to provide evidence for function. Seven out of the 300 plus SEPs had PhyloCSF scores that indicate that they have been conserved throughout evolution. These seven SEPs included an interesting SEP called SLC35A4-SEP that is generated from a uORF in the SLC35A4 gene. The SLC35A4-SEP had contained a transmembrane domain and analysis of cells revealed the mitochondrial localization of this SEP.;Further characterization of SCL35A4 indicated that this polypeptide interacts with members of the ATP synthase complex. Though this interaction requires further validation the putative interactions suggested a role for SLC35A4-SEP in cellular energetics. Overexpression or knockout of SLC35A4-SEP affected cellular respiration. Ongoing work is testing to see if SLC35A4-SEP also effects mitochondrial membrane potential and structure of ATP-synthase. More generally, this approach highlights how I can begin to identify functional SEPs using a combination of computational and experimental methods. And my work on another functional SEP called NoBody indicates that this strategy is general.