Discovery and annotation of small proteins using genomics, proteomics, and computational approaches

Abstract

Small proteins (10-200 amino acids [aa] in length) encoded by short open reading frames (sORF) play important regulatory roles in various biological processes, including tumor progression, stress response, flowering, and hormone signaling. However, ab initio discovery of small proteins has been relatively overlooked. Recent advances in deep transcriptome sequencing make it possible to efficiently identify sORFs at the genome level. In this study, we obtained~2.6 million expressed sequence tag (EST) reads from Populus deltoides leaf transcriptome and reconstructed full-length transcripts from the EST sequences. We identified an initial set of 12,852 sORFs encoding proteins of 10-200 aa in length. Three computational approaches were then used to enrich for bona fide protein-coding sORFs from the initial sORF set: (1) codingpotential prediction, (2) evolutionary conservation between P. deltoides and other plant species, and (3) gene family clustering within P. deltoides. As a result, a high-confidence sORF candidate set containing 1469 genes was obtained. Analysis of the protein domains, non-protein-coding RNA motifs, sequence length distribution, and protein mass spectrometry data supported this high-confidence sORF set. In the high-confidence sORF candidate set, known protein domains were identified in 1282 genes (higher-confidence sORF candidate set), out of which 611 genes, designated as highest-confidence candidate sORF set, were supported by proteomics data. Of the 611 highest-confidence candidate sORF genes, 56 were new to the current Populus genome annotation. This study not only demonstrates that there are potential sORF candidates to be annotated in sequenced genomes, but also presents an efficient strategy for discovery of sORFs in species with no genome annotation yet available.

SearchDOGS Bacteria, Software That Provides Automated Identification of Potentially Missed Genes in Annotated Bacterial Genomes

“…Identification of short bacterial proteins using SearchDOGS. Very small genes are notoriously difficult to accurately identify and annotate by experimental, ab initio, and homology-based approaches (8,(48)(49)(50)(51). Using the February 2013 release of the E. coli K-12 MG1655 genome (GenBank accession number U00096.2) as a gold standard and the same set of test genomes (Table 1), we tested the ability of SearchDOGS to identify unannotated homologs of short genes showing both conserved sequence similarity and synteny with their annotated counterparts.…”

Autographa californicaNucleopolyhedrovirus Ac76: a Dimeric Type II Integral Membrane Protein That Contains an Inner Nuclear Membrane-Sorting Motif

“…However, SM fusion proteins were detected at the predicted molecular masses rather than the dimeric sizes by SDS-PAGE. This may be owing to the large size of these chimeric proteins, as all of these proteins are much larger than the small proteins, which are usually less than 200 amino acids in length (36). Thus, their self-association may be unstable and can be broken by SDS.…”

Discovering Elusive Small Genes

“…Integration of information from sequence features, conservation, and transcriptomic, translatomic, and proteomic analyses, will most likely provide the best strategy for obtaining the most complete picture of the coding potential of prokaryotic and eukaryotic organisms [23]. Table 1: Total number and conserved genes identified among annotated or newlypredicted genes in 1000 bacterial genomes [12].…”