- Research article
- Open Access
Cotranslational protein-RNA associations predict protein-protein interactions
© Duncan and Mata; licensee BioMed Central Ltd. 2014
- Received: 5 November 2013
- Accepted: 13 February 2014
- Published: 22 April 2014
Most cellular proteins function as part of stable protein complexes. We recently showed that around 38% of proteins associate with mRNAs that encode interacting proteins, reflecting the cotranslational formation of the complex between the bait protein and the nascent peptides encoded by the interacting mRNAs. Here we hypothesise that these cotranslational protein-mRNA associations can be used to predict protein-protein interactions.
We found that the fission yeast Exo2 protein, which encodes an exonuclease of the XRN1 family, coimmunoprecipitates with the eti1 mRNA, which codes for a protein of unknown function and uninformative sequence. Based on this protein-mRNA association, we predicted that the Exo2 and Eti1 protein are part of the same complex, and confirmed this hypothesis by coimmunoprecipitation and colocalization of the proteins. Similarly, we show that the cotranslational interaction between the Sty1 MAP kinase and the cip2 mRNA, which encodes an RNA-binding protein, predicts a complex between Sty1 and Cip2.
Our results demonstrate that cotranslational protein-mRNA associations can be used to identify new components of protein complexes.
- Fission Yeast
- Stress Granule
- Glucose Starvation
- Bait Protein
- Nascent Peptide
The Sty1 protein is a MAP kinase that mediates stress responses in fission yeast. RIP-chip experiments revealed that Sty1 interacts with two mRNAs : pyp2, which encodes a protein phosphatase known to physically interact with Sty1 , and cip2, which codes for an RNA-binding protein that regulates the response to oxidative stress . We have shown that the interaction between the Sty1 protein and the cip2 mRNA requires active translation of cip2, as it is disrupted when translation is inhibited using puromycin and when the cip2 initiation codon is mutated . This strongly suggested that Sty1 binds to Cip2 as the Cip2 protein is being synthesised on the polysome. We therefore predicted that the Sty1 and Cip2 proteins interact with each other. To test this hypothesis we epitope-tagged Sty1 and Cip2, and used strains carrying the tagged proteins for coimmunoprecipitation experiments. As predicted, Sty1-myc copurified specifically with Cip2-TAP (Figure 1B). To confirm the specificity of the interactions we repeated the experiment using Sty1-myc and Cip2-GFP. Consistent with the previous result, Cip2-GFP copurified with immunoprecipitated Sty1-myc (Figure 1C). Moreover, the interaction Cip2-TAP and Sty1-myc was not disrupted by treatment with RNase, indicating that the associations are not mediated by the cip2 mRNA, and thus that both proteins are part of the same complex (Additional file 1: Figure S1A). These results show that RIP-chip data can be used to predict protein-protein interactions.
To prove that the association also takes place in vivo, we tagged Exo2 and Eti1 with different fluorescent proteins. Exo2 and Eti1 could be detected by fluorescence microscopy in very weak cytoplasmic foci reminiscent of Processing bodies (PBs) in vegetatively growing cells (Additional file 1: Figure S2A). PBs are large cytoplasmic RNA – protein complexes that include multiple proteins involved in mRNA degradation , such as components of the decapping complex (Dcp1 and Dcp2) and the 5′- > 3′ exonuclease Exo2 . However, PBs do not contain the poly(A) binding protein Pabp . To investigate the identity of these granules we coexpressed Exo2 and Eti1 with the PB marker Dcp2. As previously reported , we could observe Dcp2 in discrete but weak cytoplasmic foci (Additional file 1: Figure S2A). Although the weakness of the fluorescence signals of the tagged proteins precluded us from performing extensive the colocalization studies, we detected structures containing both Dcp2 and Eti1 (Additional file 1: Figure S2B), suggesting that Eti1 may be a component of PBs.
Finally, we tested whether Exo2 and Eti1 required each other for their localization. In eti1Δ cells, as in a wild type background, Exo2-GFP was visualized as very weak foci in vegetative cells, but moved to stress granules upon glucose starvation (Additional file 1: Figure S4A). Pabp-GFP also relocalized normally, indicating that Eti1 is not required for stress granule assembly (Additional file 1: Figure S4B). By contrast, Eti1 was present in bright granules in exo2Δ cells even in unstressed cells (Additional file 1: Figure S4C). Inhibition of RNA decay by inactivation of XRN1/Exo2 in yeast causes an increase in the number and size of PBs in budding yeast cells, presumably due to the accumulation of decay intermediates . Eti1-containing granules in exo2Δ mutants did not contain Pabp (Additional file 1: Figure S4D), consistent with the idea that these foci represent PBs.
Altogether, biochemical and cytological data confirm that Exo2 and Eti1 are part of the same complex, and suggest that Eti1 is a novel component of stress granules.
Our results demonstrate that cotranslational mRNA-protein interactions detected using RIP-chip can be used to predict protein-protein associations. The nature and behaviour of the proteins we identified suggest that the interactions may have biological importance. For example, there is evidence that Sty1 regulates the phosphorylation of Cip2: Cip2 is phosphorylated in response to oxidative stress, and this phosphorylation does not take place in sty1 mutants . Our results demonstrate that Sty1 and Cip2 are associated, and suggest that Sty1 may directly phosphorylate Cip2. In the case of Eti1, the interactions between Exo2 and eti1 allowed us to identify a novel component of stress granules, which might be involved in RNA decay. Although only a small fraction of protein-protein interactions appear to form cotranslationally , RIP-chip experiments often identify key partners of high biological significance and have a very low false positive rate. For example, RIP-chip with the Cdc2 protein, the fission yeast CDK1, identified mRNAs encoding key regulators of the G1/S transition (rum1) and of S phase initiation (cdc18) . It is important to note that the associations revealed by RIp-chip may not be direct. For example, a preformed protein complex might interact with a nascent peptide. We propose that RIP-chip can serve as a complementary method to mass spectrometry-based techniques to identify interacting partners of a given protein.
Standard methods were used for growth and manipulation of fission yeast cells . All strains used in this work are listed in Additional file 1: Table S1. All experiments were performed with cells grown in Edinburgh Minimal Medium (EMM) with the appropriate supplements at 32°C. For glucose starvation experiments, cells were incubated in EMM that did not contain glucose for 1 hour. For coimmunoprecipitation experiments cell extracts were prepared as described in . Proteins were TAP-, GFP-, and myc- tagged using one-step PCR methods [18, 19]. A full list of oligonucleotides is presented in Additional file 1: Table S2. TAP-tagged proteins were immunoprecipitated using monoclonal antibodies against protein A (Sigma), and detected by Western Blot using peroxidase-anti-peroxidase soluble complexes (Sigma). Myc-tagged proteins were immunoprecipitated and detected by Western blot using the 9E11 monoclonal antibody (Abcam). GFP-tagged proteins were detected with the B2 monoclonal antibody (Santa Cruz). The requirement of the interactions for intact RNA was tested by treating 250 μl of cell extract with 200 units of RNase I (Life Technologies) for 15 minutes at room temperature. For microscopy experiments, living cells expressing the indicated fusion proteins were visualized using an AxioImager M1 by Carl Zeiss MicroImaging. RIp-chip experiments, puromycin and EDTA treatments were carried out as previously described . Custom-designed oligonucleotide microarrays were manufactured by Agilent, and were processed and analysed as previously described .
We thank Gerry Smith for providing reagents and Rafael Carazo-Salas and James Dodgson for help with microscopy. This work was funded by the following Biotechnology and Biological Sciences Research Council (BBSRC) research grants to Juan Mata: BB/G011869/1 and BB/J007153/1.
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