An Integrated Approach for Microprotein Identification and Sequence Analysis

Summary

The protocol described here provides detailed instructions on how to analyze genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser. Additionally, several tools and resources are recommended to further investigate sequence characteristics of identified microproteins to gain insight into their putative functions.

Abstract

Next-generation sequencing (NGS) has propelled the field of genomics forward and produced whole genome sequences for numerous animal species and model organisms. However, despite this wealth of sequence information, comprehensive gene annotation efforts have proven challenging, especially for small proteins. Notably, conventional protein annotation methods were designed to intentionally exclude putative proteins encoded by short open reading frames (sORFs) less than 300 nucleotides in length to filter out the exponentially higher number of spurious noncoding sORFs throughout the genome. As a result, hundreds of functional small proteins called microproteins (<100 amino acids in length) have been incorrectly classified as noncoding RNAs or overlooked entirely.

Here we provide a detailed protocol to leverage free, publicly available bioinformatic tools to query genomic regions for microprotein-coding potential based on evolutionary conservation. Specifically, we provide step-by-step instructions on how to examine sequence conservation and coding potential using Phylogenetic Codon Substitution Frequencies (PhyloCSF) on the user-friendly University of California Santa Cruz (UCSC) Genome Browser. Additionally, we detail steps to efficiently generate multiple species alignments of identified microprotein sequences to visualize amino acid sequence conservation and recommend resources to analyze microprotein characteristics, including predicted domain structures. These powerful tools can be used to help identify putative microprotein-coding sequences in noncanonical genomic regions or to rule out the presence of a conserved coding sequence with translational potential in a noncoding transcript of interest.

Introduction

The identification of the complete set of coding elements in the genome has been a major goal since the initiation of the Human Genome Project, and remains a central objective toward the understanding of biological systems and the etiology of genetic-based diseases^1,2,3,4. Advances in NGS techniques have led to the production of whole genome sequences for an extensive number of organisms, including vertebrates, invertebrates, yeast, and plants⁵. Additionally, high-throughput transcriptional sequencing methods have fur....

Protocol

The protocol outlined below details steps to load and navigate the PhyloCSF browser tracks on the UCSC Genome Browser (generated by Mudge et al.⁴⁹). For general questions regarding the UCSC Genome Browser, an extensive Genome Browser User's Guide can be found here: https://genome.ucsc.edu/goldenPath/help/hgTracksHelp.html.

1. Loading the PhyloCSF Track Hub to the UCSC Genome Browser

Discussion

The protocol presented here provides detailed instructions on how to interrogate genomic regions of interest for microprotein-coding potential using PhyloCSF on the user-friendly UCSC Genome Browser^48,49,50,51. As detailed above, PhyloCSF is a powerful comparative genomics algorithm that integrates phylogenetic models and codon substitution frequencies to identify evolutionary signatures that a.......

Materials

Name	Company	Catalog Number	Comments
Website	Website Address	Requirements
Clustal Omega Multiple Sequence Alignment Tool	https://www.ebi.ac.uk/Tools/msa/clustalo/	Web browser	Multiple sequence alignment program for the efficient alignment of FASTA sequences (i.e. for cross-species comparison of identified microproteins)
COXPRESSdb	https://coxpresdb.jp	Web browser	Provides co-regulated gene relationships to estimate gene functions
EMBL-EBI Bioinformatics Tools FAQs	https://www.ebi.ac.uk/seqdb/confluence/display/JDSAT/Bioinformatics+Tools+FAQ	Web browser	Frequently Asked Questions (FAQs) for EMBL-EBI tools. Includes the color coding key for protein sequence alignments
European Bioinformatics Institute (EMBL-EBI), Tools and Data Resources	https://www.ebi.ac.uk/services/all	Web browser	Comprehensive list of freely available websites, tools and data resources
Expasy - Swiss Bioinformatics Resource Portal	https://www.expasy.org	Web browser	Suite of bioinformatic tools and resources for protein sequence analysis that is maintained by the Swiss Institute of Bioinformatics (SIB)
National Center for Biotechnology Information (NCBI) Conserved Domain Search	https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi	Web browser	Search tool to identify conserved domains within protein or coding nucleotide sequences
Pfam 35	http://pfam.xfam.org	Web browser	Protein family (Pfam) database, provides alignments and classification of protein families and domains
PhyloCSF Track Hub Description	https://genome.ucsc.edu/cgi-bin/hgTrackUi?hgsid=1267045267_TEc99h2oW5Q edaCd4ir8aZ65ryaD&db=mm10 &c=chr2&g=hub_109801_ PhyloCSF_smooth	Web browser	Detailed description of the Smoothed PhyloCSF tracks and PhyloCSF Track Hub
SignalP 6.0	https://services.healthtech.dtu.dk/service.php?SignalP-6.0	Web browser	Predicts the presence of signal peptides and the location of their cleavage sites
TMHMM - 2.0	https://services.healthtech.dtu.dk/service.php?TMHMM-2.0	Web browser	Prediction of transmembrane helices in proteins
UCSC Genome Browser BLAT Search	https://genome.ucsc.edu/cgi-bin/hgBlat	Web browser	Tool used to find genomic regions using DNA or protein sequence information
UCSC Genome Browser Gateway	https://genome.ucsc.edu/cgi-bin/hgGateway	Web browser	Direct link to the UCSC Genome Browser Gateway
UCSC Genome Browser Home	https://genome.ucsc.edu/	Web browser	Home website for the UCSC Genome Browser
UCSC Genome Browser Track Data Hubs	https://genome.ucsc.edu/cgi-bin/hgHubConnect#publicHubs	Web browser	Direct link to Track Data Hubs/Public Hubs database to search for and load the PhyloCSF Tracks
UCSC Genome Browser User Guide	https://genome.ucsc.edu/goldenPath/help/hgTracksHelp.html	Web browser	Comprehensive user guide detailing how to navigate the UCSC Genome Browser
WoLF PSORT	https://wolfpsort.hgc.jp	Web browser	Protein subcellular localization prediction tool