A protein domain-based interactome network for C. elegans early embryogenesis

Abstract

Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or "interactome" networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early-embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms.

Figure 1. Strategy for generating the AD-Fragment library and effect on Y2H sensitivity and specificity

(A) Primer placement. Primers are designed to start within a 55 bp window surrounding the ideal start positions (lines above ORF). (B) Fragments generated by combining primers. (C) Distances in between primers and fragment sizes produced for ORFs of the indicated lengths. (D,E) Literature derived interactions and random protein pairs tested as full-length fusions (results from Venkatesan et al. personal communication) and using an AD-Fragment library. Green boxes indicate detection of an interaction. Protein names correspond to Entrez names.

Figure 2. Properties of the Y2H protein-protein interaction network

(A) Network graph of the protein-protein interactions between early embryogenesis proteins, compiled from data in the most recent release of the worm interactome (CCSB-WI8), and from the AD-cDNA and AD-Fragment screens described here. (B) Retest rate of interactions in MAPPIT. Green bar: interactions derived from literature (results from Simonis et. al. personal communication). Random protein pairs did not interact. Blue bars: retest of 355 interactions described here, split into: (1) all 355 interactions, (2) those found as full-length fusions (124 interactions), and (3) those found as truncated fusions only (225 interactions). Error bars correspond to binomial standard error. (C) Overlap between AD-cDNA and AD-Fragment library derived interactions within the early embryogenesis protein space. (D) Fraction of interactions found as full-length fusions in AD-cDNA and AD-Fragment library screens. (E) Comparison of connectivity of bait and prey proteins. (F) Comparison of connectivity of prey proteins that were found as full-length at least once, with those that were never found as full-length.

Figure 3. Enrichment in similar phenotypes, GO terms, and mRNA expression profiles for interacting protein pairs

(A) Examples of interactions between proteins assigned to the same functional class based on their RNAi phenotypes. Red lines: new Y2H interactions. Blue lines: known Y2H interactions re-identified. Blue dotted lines: known Y2H interactions not found. (B) Enrichment in phenotypic correlation for interacting protein pairs relative to average value of all possible protein pairs in the interaction network. (C) Enrichment in shared GO terms at different levels of specificity. (D) Pearson correlation coefficients (PCCs) for the mRNAs corresponding to each pair of proteins in the interaction data sets (red lines), the protein space searched (blue lines), and the entire worm genome (dotted grey lines). Early embryogenesis genes already have highly similar expression profiles compared to the entire worm genome, hence no further enrichment can be observed for interactions derived from the AD-Fragment library (left panel).

Figure 4. Y2H results of nuclear pore complex (NPC) and centrosome screens

(A) Schematic drawing of NPC. Shown are nuclear membrane (grey) with membrane rings (green), inner and outer scaffold rings (orange), FG nucleoporins (green), cytoplasmic tendrils (yellow), and nuclear basket (blue). Left: approximate localization of mammalian proteins within the NPC. C. elegans homologs of proteins in black were used as baits in our screens. Right: Interactions found between C. elegans NPC and import/export machinery proteins. (B) Diagram of centrosome assembly pathway. Green arrows represent localization dependencies, dotted blue lines previously described binary interactions, red lines Y2H interactions discovered here, and dotted boxes co-IP complexes.

Figure 5. Identification and validation of minimal regions required for interaction (MRIs)

(A) Example of identification of an MRI. The AD-Fragment library was screened with full length DB∷RAN- 1 and DB::IMB-4. Grey lines indicate protein fragments of NPP-9 that interacted with RAN-1 or IMB-4. (B) Sizes of MRIs identified in the AD-Fragment library screens expressed as percentage of corresponding full-length protein and absolute amino acids. (C) MRIs identified in proteins involved in centrosome assembly. Green bars represent full-length proteins. Yellow bars represent regions of the full-length protein required for interaction with the indicated binding partner (e.g. the N-terminal region of TPXL-1 is required for binding to AIR-1). Pfam-A domain signatures are drawn as red boxes. CC = coiled-coil prediction. The region of RSA-2 that mediates binding to SPD-5 was further refined manually (not shown).

Figure 6. Comparison of MRIs with computational domain predictions

(A) Three cases where interacting regions differ between C. elegans and the orthologous proteins in human. (B) Localization of GFP fusions of full-length RSA-2 and SAS-5 and their MRIs required for binding to SPD-5 and SAS-6, respectively. (C) Fraction of amino acids of MRIs and the corresponding full proteins that are covered by computationally predicted domains of the indicated types. (D) Fraction of MRIs classified as ‘known folding region,’ ‘predicted folding region,’ ‘unstructured,’ or ‘novel folding region,’ based on overlap with computational predictions.