Abstract
To accelerate high-density interactome mapping, we developed a yeast two-hybrid interaction screening approach involving short-read second-generation sequencing (Y2H-seq) with improved sensitivity and a quantitative scoring readout allowing rapid interaction validation. We applied Y2H-seq to investigate enzymes involved in protein methylation, a largely unexplored post-translational modification. The reported network of 523 interactions involving 22 methyltransferases or demethylases is comprehensively annotated and validated through coimmunoprecipitation experiments and defines previously undiscovered cellular roles of nonhistone protein methylation.
Highlights
Despite recent conceptual and technical advances[1,2,3], large-scale interactome mapping remains a daunting task, and for most species, only a small fraction of interactions have been mapped
Empirical assessment of data quality has demonstrated that Y2H protein-protein interaction (PPI) data are of high precision but of low coverage, owing to low sensitivity of the methods[1], and require repeated screens to capture most detectable interactions
We applied Y2H-seq for interactome mapping of a largely unexplored set of human proteins: those involved in protein methylation and demethylation[6]
Summary
Mareike Weimann[1,4], Arndt Grossmann[1,4], Jonathan Woodsmith[1,4], Ziya Özkan[1], Petra Birth[1], David Meierhofer[1], Nouhad Benlasfer[1], Taras Valovka[2], Bernd Timmermann[1], Erich E Wanker[3], Sascha Sauer1 & Ulrich Stelzl[1]. To accelerate high-density interactome mapping, we developed a yeast two-hybrid interaction screening approach involving short-read second-generation sequencing (Y2H-seq) with improved sensitivity and a quantitative scoring readout allowing rapid interaction validation. We applied Y2H-seq to investigate enzymes involved in protein methylation, a largely unexplored post-translational modification. We applied Y2H-seq for interactome mapping of a largely unexplored set of human proteins: those involved in protein methylation and demethylation[6]. Top-ranked prey in at least two replicate screens were retested against all baits in a pairwise manner (Supplementary Fig. 4) and yielded 463 protein interactions (Supplementary Table 3). We found 151 interactions (Supplementary Table 3) with 90 prey proteins at a retest success rate of 0.78. Comparison of the Y2H-matrix and the Y2H-seq approaches at the same
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