Grad-seq guides the discovery of ProQ as a major small RNA-binding protein.
The functional annotation of transcriptomes and identification of noncoding RNA (ncRNA) classes has been greatly facilitated by the advent of next-generation RNA sequencing which, by reading the nucleotide order of transcripts, theoretically allows the rapid profiling of all transcripts in a cell. However, primary sequence per se is a poor predictor of function, as ncRNAs dramatically vary in length and structure and often lack identifiable motifs. Therefore, to visualize an informative RNA landscape of organisms with potentially new RNA biology that are emerging from microbiome and environmental studies requires the use of more functionally relevant criteria. One such criterion is the association of RNAs with functionally important cognate RNA-binding proteins. Here we analyze the full ensemble of cellular RNAs using gradient profiling by sequencing (Grad-seq) in the bacterial pathogen Salmonella enterica, partitioning its coding and noncoding transcripts based on their network of RNA-protein interactions. In addition to capturing established RNA classes based on their biochemical profiles, the Grad-seq approach enabled the discovery of an overlooked large collective of structured small RNAs that form stable complexes with the conserved protein ProQ. We show that ProQ is an abundant RNA-binding protein with a wide range of ligands and a global influence on Salmonella gene expression. Given its generic ability to chart a functional RNA landscape irrespective of transcript length and sequence diversity, Grad-seq promises to define functional RNA classes and major RNA-binding proteins in both model species and genetically intractable organisms.
RNA Biology Group, Institute of Molecular Infection Biology, University of Würzburg, D-97080 Wurzburg, Germany.
Alexandre
0000-0002-9142-7979
A
Smirnov
RNA Biology Group, Institute of Molecular Infection Biology, University of Würzburg, D-97080 Wurzburg, Germany; Core Unit Systems Medicine, University of Würzburg, D-97080 Wurzburg, Germany.
Konrad U
0000-0002-1481-2996
KU
Förstner
RNA Biology Group, Institute of Molecular Infection Biology, University of Würzburg, D-97080 Wurzburg, Germany.
Erik
0000-0001-7834-1487
E
Holmqvist
Institute for Microbiology, University of Greifswald, D-17489 Greifswald, Germany.
Andreas
A
Otto
RNA Biology Group, Institute of Molecular Infection Biology, University of Würzburg, D-97080 Wurzburg, Germany.
Regina
R
Günster
Institute for Microbiology, University of Greifswald, D-17489 Greifswald, Germany.
Dörte
D
Becher
Max Planck Genome Centre Cologne, Max Planck Institute for Plant Breeding Research, D-50829 Cologne, Germany.
Richard
R
Reinhardt
RNA Biology Group, Institute of Molecular Infection Biology, University of Würzburg, D-97080 Wurzburg, Germany; Research Centre for Infectious Diseases (ZINF), University of Würzburg, D-97070 Wurzburg, Germany joerg.vogel@uni-wuerzburg.de.
Jörg
0000-0003-2220-1404
J
Vogel
D001426:Bacterial Proteins / Q000378:metabolism*; D017124:Conserved Sequence / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008024:Ligands; D025341:Principal Component Analysis; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D022661:RNA, Untranslated / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism*; D019779:Salmonella enterica / Q000378:metabolism*
D001426:Bacterial Proteins; D008024:Ligands; D012329:RNA, Bacterial; D022661:RNA, Untranslated; D016601:RNA-Binding Proteins
Hfq; ProQ; RNA–protein interaction; noncoding RNA; small RNA
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