Actin Patch mRNA Localization and Dynamics patches and the relative timing of arrival of proteins involved in the regulation and formation of the endosome. Proteins encoded by mRNAs associated with Myo3p are colored in red as in parts (B) and (C). doi:10.1371/journal.pone.0031912.g001 the need for a chemical crosslinker (Figure 1A). In both the case of latrunculin B and formaldehyde treatment, we also performed IPs in which the untagged parent yeast strains were processed for IP and microarray analysis in a manner identical to that of the tagged strains. We used the significance analysis of microarrays (SAM) algorithm [48] to identify mRNAs that were specifically enriched in association with a tagged motor protein and not in the mock IPs, as well as to estimate a corresponding false discovery rate (FDR). Using these methods for Myo4, which served as our positive control, we identified most of the RNAs previously reported as Myo4 mRNA targets with both the formaldehyde crosslinking and latrunculin B procedures at an FDR of less than 5% ([18]; Dataset S1). With the same procedures, we identified mRNAs specifically associated with Myo1, a type II myosin motor that localizes to the actomyosin ring at the bud neck and is required for proper cytokinesis and cell separation [49,50,51]. Using both the formaldehyde crosslinking and latrunculin B procedures, Myo1 associated with no mRNAs under a local FDR cutoff of 5%. However, Gene Ontology (GO) analysis [52,53,54] of the top 465 Myo1-associated mRNAs (global FDR,7%) upon treatment with latrunculin B revealed that the ‘‘protein kinase activity,’’ ‘‘cellular bud neck,’’ and ‘‘site of polarized growth’’ categories are specifically enriched, including the mRNAs BUD4, IQG1, BOI2, NIS1, and SWE1, among others, whose protein products localize to the bud neck (Dataset S1; [55,56,57,58,59]). Finding Myo1 associated with mRNAs encoding regulators of cytokinesis and budding suggests that perhaps these mRNAs co-localize with Myo1 at the bud neck for localized protein production and regulation of cell separation. We identified several mRNAs specifically and reproducibly associated with Myo2 (Dataset S1), a type V actomyosin motor that has been previously reported to travel on actin cables and associate with a variety of organelles such as mitochondria [60,61,62], vacuoles [63,64], peroxisomes [65,66], and vesicles [67] for the establishment of polarity and the delivery of factors into the emerging bud. In contrast to previous reports which identified more than 2,300 Myo2-associated mRNAs [68], we found far fewer mRNAs significantly associated (FDR,5%) with Myo2; in IPs from Myo2:9Myc tagged cells treated with latrunculin B, we found 429 mRNAs, while IPs of Myo2:9Myc and Myo2:GFP from crosslinked cells yielded only 58 and 12 mRNAs, respectively. Intriguingly, among the Myo2-associated mRNAs were several previously found to associate with Myo4, including ASH1, IST2, TCB3, and CLB2 [13,18]. In addition, we found mRNAs enriched in the GO categories of ‘‘actin cytoskeleton organization’’ and ‘‘maintenance of cellular polarity,’’ including WSC2, BNI1, SLA1, CAP2, SPA2, BUD8, and BUD9. Furthermore, Myo2 was selectively associated with mRNAs encoding components of organelle membranes, including Mmr1, Vac8, and Vac17, which have been previously reported to facilitate Myo2 localization of bud-localized mitochondria and the vacuole, respectively [61,69,70]. The kinesin-like proteins Kip1, Kip2, and Smy1 [71], and the dynein protein, Dyn1, did not reproducibly associate with any mRNAs in our assays (Dataset S2). The kinesin-8 type Kip3 protein, however, associated with 327 mRNAs at ,5% FDR, among which mRNAs with roles in mitochondria were enriched. While Kip3 has not been linked to mitochondria in S. cerevisiae, a kinesin-8 type motor has been found associated with mitochondria in Drosophila [72], suggesting the possibility for further Kip3 roles in budding yeast. The Kar3 kinesin-like motor, which is required for nuclear fusion during mating [73], was selectively associated with mRNAs encoding factors that play roles in conjugation and cellular fusion including Cdc12, a septin ([74]; Dataset S2). Myo3 Associates with mRNAs Encoding Actin Patch Nucleating Proteins Myo3, a type I actomyosin motor that, like Myo5, localizes to actin patches at the cortex and plays a role in actin nucleation and the regulation of endocytosis [75,76,77,78,79], reproducibly associated with a specific set of mRNAs encoding key regulators of actin branching and the endocytic process. The mRNAs BBC1, VRP1, and LAS17 were specifically enriched with Myo3 after formaldehyde crosslinking (Figure 1B, inset; Dataset S1). LAS17 encodes the yeast homolog of WASP, which functions as a nucleation promoting factor (NPF) that interacts with type I myosins [14,77] and activates the Arp2/3 complex to facilitate actin branching at the site of endocytosis [80]. Vrp1, otherwise known as WIP, also serves to stimulate the NPF activity of the type I myosin, Myo5 [79]. Bbc1 is recruited to actin patches with Myo5 and has been suggested to regulate the activity of both Myo5 and Las17 [79,81]. Immunopurification of Myo3 from non-crosslinked cells pre- incubated with latrunculin B also selectively enriched VRP1 and LAS17 mRNAs (Figure 1C, inset; Dataset S1) along with three other mRNAs encoding factors that regulate actin patch nucleation: UBP7, SLA1, and PAN1. Sla1 binds to the Arp2/3 regulator, Pan1, and Ubp7 is a ubiquitin-specific protease that has previously been shown to interact with Myo3 [82,83,84]. Additional mRNAs that consistently co-purified with Myo3 in the presence of latrunculin B include RNAs encoding Csf1, a protein localized to mitochondria [85]; Sgd1, a nuclear protein involved in the osmoregulatory response to glycerol [86]; Hal5, a putative protein kinase involved in halide tolerance [87]; Gcn1, involved in translational control [88,89]; and Fmt1, which catalyzes initiator-Met-tRNA formylation in mitochondria [90]. We focused our subsequent investigations on the Myo3- associated mRNAs for a number of reasons. First, actin patches are known to be dynamic structures [91,92] whose regulation is tightly controlled through the sequential recruitment of a series of proteins (Figure 1D; [79,81,93]). Thus, dynamic localization of mRNAs to these structures could play a role in the localized production of proteins to aid in the regulation of endocytosis and budding. Second, Myo3-associated mRNAs encode the key regulators of the actin branching process crucial for successful invagination and release of endosomes from the cell cortex [79,81,93,94,95]. However, Myo3 does not appear to associate with every mRNA that encodes a protein known to interact at the actin patch (Figure 1D; Dataset S1 and Table S2). The other type I myosin present at actin patches, Myo5, associated with the actin patch (AP)-regulating mRNAs SLA1 and VRP1 with a FDR of 23% upon treatment with latrunculin B (Dataset S1), reflecting a less significant and reproducible interaction with the AP mRNAs. Additional IPs using the same procedure with genetically tagged Las17 failed to enrich for the AP RNAs that associate with Myo3 (Dataset S1); Vrp1 was associated with its own transcript upon crosslinking, but did not significantly associate with other AP- PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31912