A quantitative targeted proteomics approach to validate predicted microRNA targets in C. elegans.
Efficient experimental strategies are needed to validate computationally predicted microRNA (miRNA) target genes. Here we present a large-scale targeted proteomics approach to validate predicted miRNA targets in Caenorhabditis elegans. Using selected reaction monitoring (SRM), we quantified 161 proteins of interest in extracts from wild-type and let-7 mutant worms. We demonstrate by independent experimental downstream analyses such as genetic interaction, as well as polysomal profiling and luciferase assays, that validation by targeted proteomics substantially enriched for biologically relevant let-7 interactors. For example, we found that the zinc finger protein ZTF-7 was a bona fide let-7 miRNA target. We also validated predicted miR-58 targets, demonstrating that this approach is adaptable to other miRNAs. We propose that targeted mass spectrometry can be applied generally to validate candidate lists generated by computational methods or in large-scale experiments, and that the described strategy should be readily adaptable to other organisms.
Jovanovic
Marko
M
Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.
Reiter
Lukas
L
Picotti
Paola
P
Lange
Vinzenz
V
Bogan
Erica
E
Hurschler
Benjamin A
BA
Blenkiron
Cherie
C
Lehrbach
Nicolas J
NJ
Ding
Xavier C
XC
Weiss
Manuel
M
Schrimpf
Sabine P
SP
Miska
Eric A
EA
Grosshans
Helge
H
Aebersold
Ruedi
R
Hengartner
Michael O
MO
D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D017238:Genes, Helminth; D008156:Luciferases / Q000235:genetics; D013058:Mass Spectrometry; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013045:Species Specificity
D029742:Caenorhabditis elegans Proteins; D035683:MicroRNAs; D012333:RNA, Messenger; C474488:let-7 microRNA, C elegans; D008156:Luciferases