Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes

ABSTRACT Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the total number of small proteins remains unknown because conventional annotation pipelines usually exclude smal...

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Autores principales:	Jeremy Weaver, Fuad Mohammad, Allen R. Buskirk, Gisela Storz
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2019
Materias:	Ribo-seq small protein alternate ORFs antisense genome annotation leader peptide Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/01f8e0e61cfc4284a906889e9ea5bfc7
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spelling	oai:doaj.org-article:01f8e0e61cfc4284a906889e9ea5bfc72021-11-15T15:55:25ZIdentifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes10.1128/mBio.02819-182150-7511https://doaj.org/article/01f8e0e61cfc4284a906889e9ea5bfc72019-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02819-18https://doaj.org/toc/2150-7511ABSTRACT Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the total number of small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organism Escherichia coli using theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions in E. coli. We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. Not only are the corresponding genes intergenic but they are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context. IMPORTANCE Proteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the functions of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification, and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.Jeremy WeaverFuad MohammadAllen R. BuskirkGisela StorzAmerican Society for MicrobiologyarticleRibo-seqsmall proteinalternate ORFsantisensegenome annotationleader peptideMicrobiologyQR1-502ENmBio, Vol 10, Iss 2 (2019)
institution	DOAJ
collection	DOAJ
language	EN
topic	Ribo-seq small protein alternate ORFs antisense genome annotation leader peptide Microbiology QR1-502
spellingShingle	Ribo-seq small protein alternate ORFs antisense genome annotation leader peptide Microbiology QR1-502 Jeremy Weaver Fuad Mohammad Allen R. Buskirk Gisela Storz Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes
description	ABSTRACT Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the total number of small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organism Escherichia coli using theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions in E. coli. We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. Not only are the corresponding genes intergenic but they are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context. IMPORTANCE Proteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the functions of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification, and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.
format	article
author	Jeremy Weaver Fuad Mohammad Allen R. Buskirk Gisela Storz
author_facet	Jeremy Weaver Fuad Mohammad Allen R. Buskirk Gisela Storz
author_sort	Jeremy Weaver
title	Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes
title_short	Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes
title_full	Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes
title_fullStr	Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes
title_full_unstemmed	Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes
title_sort	identifying small proteins by ribosome profiling with stalled initiation complexes
publisher	American Society for Microbiology
publishDate	2019
url	https://doaj.org/article/01f8e0e61cfc4284a906889e9ea5bfc7
work_keys_str_mv	AT jeremyweaver identifyingsmallproteinsbyribosomeprofilingwithstalledinitiationcomplexes AT fuadmohammad identifyingsmallproteinsbyribosomeprofilingwithstalledinitiationcomplexes AT allenrbuskirk identifyingsmallproteinsbyribosomeprofilingwithstalledinitiationcomplexes AT giselastorz identifyingsmallproteinsbyribosomeprofilingwithstalledinitiationcomplexes
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Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes

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