Pat1 promotes processing body assembly by enhancing the phase separation of the DEAD-box ATPase Dhh1 and RNA

Processing bodies (PBs) are cytoplasmic mRNP granules that assemble via liquid–liquid phase separation and are implicated in the decay or storage of mRNAs. How PB assembly is regulated in cells remains unclear. Previously, we identified the ATPase activity of the DEAD-box protein Dhh1 as a key regulator of PB dynamics and demonstrated that Not1, an activator of the Dhh1 ATPase and member of the CCR4-NOT deadenylase complex inhibits PB assembly in vivo (Mugler et al., 2016). Here, we show that the PB component Pat1 antagonizes Not1 and promotes PB assembly via its direct interaction with Dhh1. Intriguingly, in vivo PB dynamics can be recapitulated in vitro, since Pat1 enhances the phase separation of Dhh1 and RNA into liquid droplets, whereas Not1 reverses Pat1-Dhh1-RNA condensation. Overall, our results uncover a function of Pat1 in promoting the multimerization of Dhh1 on mRNA, thereby aiding the assembly of large multivalent mRNP granules that are PBs.


Introduction 33
Cells are often subjected to severe environmental fluctuations such as nutrient deficiency, 34 temperature changes or osmotic shock. To cope with stresses, a variety of mechanisms have evolved 35 allowing cells to respond acutely and survive. These include changes in the gene expression program, 36 which are often driven by robust transcriptional responses. Yet cells must also inactivate old mRNAs 37 that may interfere with adaptation to the new condition, and therefore, post-transcriptional regulation 38 and RNA turnover are critical to enable rapid changes in gene expression (Ashe et al., 2000;Mager and 39 Ferreira, 1993). 40 The post-transcriptional fate of eukaryotic mRNAs is tightly linked to the complement of proteins that 41 associate with it to form mRNPs (messenger ribonucleoproteins). For example, the presence of the m 7 42 G cap structure at the 5' end is crucial for eIF4E binding, while Pab1 binds the 3' poly-A tail, both of 43 which protect the mRNA against degradation and promote translation initiation. On the other hand, 44 mRNA turnover is thought to initiate via deadenylation by the Ccr4/Pop2/Not1 complex (Muhlrad and 45 mutated to four alanines [Pat1 4A-Dhh1 , see supplemental table S2B for a list of all mutants used in this 146 study] (Figure 2A). 147 We next investigated the effect of the pat1 4A-Dhh1 mutant in the context of the full-length protein on 148 PB formation in vivo. While cells expressing pat1 4A-Dhh1 showed a mild growth defect compared to the 149 wild type PAT1, they grew significantly better than pat1Δ cells, indicating that this protein is at least 150 partially functional ( Figure S2A). To examine whether the interaction between Pat1 and Dhh1 is 151 required for PB formation, we monitored PB assembly upon stress in the absence of glucose in wild-152 type (PAT1) or the pat1 4A-Dhh1 background. As expected, the number of PBs was drastically reduced in 153 the complete absence of Pat1, as visualized by Dhh1-GFP ( Figure S2B). Importantly, pat1 4A-Dhh1 154 expressing cells also demonstrated a drastic reduction in PB number compared to cells expressing PAT1 155 ( Figure 2B, C) and the Pat1 4A-Dhh1 -GFP protein itself showed a significant defect in localizing to PBs 156 ( Figure 2D, E). 157 We were unable to overexpress the Pat1 4A-Dhh1 mutant in yeast. In order to investigate PB formation 158 independently of carbon starvation stress, we therefore treated cells with the drug hippuristanol, 159 which inhibits the eukaryotic translation initiation factor eIF4A (Bordeleau, M.E., et al., 2006). In 160 consequence, hippuristanol prevents translation initiation and robustly induces PB formation within 161 minutes in cells expressing wild-type PAT1 (Chan et al., 2018) [ Figure 2F, Movies 1 and 2]. Cells 162 expressing pat1 4A-Dhh1 however, did not show PB formation until 2hrs after hippuristanol addition. 163 Taken together, our data reveal that direct binding of Pat1 to Dhh1 is required for robust PB assembly. 164

Pat1 phosphorylation status influences RNA binding 165
Pat1 is a target of the cAMP-dependent protein kinase A (PKA) and phosphorylation of two serines 166 (amino acids 456/457) in the C-terminus of Pat1 under glucose-rich conditions was previously shown 167 to negatively regulate PB formation (Ramachandran et al., 2011). To better understand how Pat1 168 phosphorylation controls PB assembly, we sought to characterize the influence of the phosphorylation 169 status of Pat1 on PB formation in the absence of additional stresses. Overexpression of PAT1 WT/SS (WT= 170 wild-type) and pat1 AA (non-phosphorylatable) led to constitutive PB formation as visualized by  GFP and Dcp2-mCherry positive foci. However, upon overexpression of pat1 EE (phospho-mimic), foci 172 number was reduced (Figure 3 A, B). Cells expressing the pat1 EE mutant also showed a defect in PB 173 formation upon stress in agreement with published literature (Figure S3 A, B and Ramachandran et al., 174 2011). 175 Pull-down experiments from cell extracts previously suggested that PKA-dependent phosphorylation 176 of Pat1 diminishes its interaction with Dhh1 (Ramachandran et al., 2011). To test whether this effect 177 is direct, we performed pull-down assays with recombinant Pat1-NC protein and the Pat1-NC EE variant. 178 However, in this assay there was no significant difference in Dhh1 binding between Pat1-NC and Pat1-179 NC EE (Figure 2A). Since the strong DETF binding site in Pat1-N might mask any weaker interaction in 180 Pat1-C, we also tested for direct binding of Pat1-C to Dhh1 but were unable to detect any interaction 181 ( Figure 3C), indicating that the DETF-motif in Pat1-N provides the major interaction surface for Dhh1. 182 Pat1-C was previously shown to bind RNA (Pilkington and Parker, 2008;Chowdhury et al., 2014). We 183 therefore tested whether RNA binding is regulated by PKA-dependent phoshphorylation by performing 184 RNA oligo gel shift assays using recombinant proteins. Whereas wild-type Pat1-C robustly binds the 185 RNA oligo and shifts it to higher molecular weight in a native PAGE gel, Pat1 EE displayed a significantly 186 reduced RNA shift (quantified in Figure 3D). Taken together, our in vivo and in vitro results on the 187 phospho-mimic Pat1 EE mutant suggest that in addition to Pat1-Dhh1 binding, also the interaction 188 between Pat1 and RNA is important for PB formation. 189

Pat1 and Not1 act antagonistically in vivo 190
Our lab previously showed that Not1 promotes PB disassembly by activating the ATPase activity of 191 Dhh1, and cells expressing a Not1 mutant that cannot bind to Dhh1 (not1 9X-Dhh1 ) form constitutive PBs 192 in non-stressed cells. Yet these PBs are less intense and fewer in number than those formed upon 193 glucose starvation (Mugler et al., 2016), indicating that additional mechanisms prevent PB formation 194 in glucose-replete conditions. 195 Since in glucose-rich media PB assembly is also inhibited by the PKA-dependent phosphorylation of 196 Pat1 (Ramachandran et al., 2011), we wanted to test whether expression of the non-phosphorylatable 197 pat1 AA variant enhances PB formation in the not1 9X-Dhh1 strain background. While expression of pat1 AA 198 alone was not sufficient to induce constitutive PBs as observed before (Ramachandran et al., 2011 and 199  constitutive PBs compared to cells expressing not1 9X-Dhh1 alone (Figure 4 A and B). Yet, these PBs are 201 still not as bright and numerous as in stressed wild-type cells. Interestingly, PB intensity strongly 202 increased when these strains were further treated with hippuristanol, a drug that blocks initiation and 203 liberates mRNA molecules from polysomes. The percentage of large PBs was highest in the pat1 AA + 204 not1 9X-Dhh1 strain, followed by the not1 9X-Dhh1 and then pat1 AA . 205 Overall, this suggests that there are at least three determinants that act cooperatively to enhance the 206 formation of PBs: (a) direct interactions between Pat1-Dhh1 and Pat1-RNA; (b) lack of Not1 binding to 207 Dhh1 and in consequence low Dhh1 ATPase activity (Mugler et al., 2016) To test the first hypothesis, we used an in vitro ATPase assay using purified protein components in 217 which Not1 robustly stimulates the ATPase activity of Dhh1 (Mugler et al., 2016). However, we did not 218 observe a significant inhibition of the Not1-stimulated ATPase activity upon addition of Pat1-NC ( Figure  219 S4A), suggesting that Pat1 does not directly interfere with the mechanism of ATPase activation. 220 We had previously shown that PB dynamics are regulated by the ATPase activity of Dhh1 (Carroll et al., 221 2011;Mugler et al., 2016). To test Pat1's effect on the Dhh1 ATPase cycle in vivo, we therefore also 222 examined the turnover of PBs that form upon Pat1 overexpression. The drug cycloheximide (CHX) traps 223 mRNAs on polysomes and thereby stops the supply of new RNA clients to PBs. Since active Dhh1 224 constantly releases mRNA molecules from PBs, a lack of mRNA influx eventually leads to their 225 disassembly and in consequence the catalytic dead mutant of Dhh1 that is locked in the ATP-state 226 (Dhh1 DQAD ) inhibits PB disassembly and was shown to have negligible turnover rates (Mugler et al.,227 2016; Kroschwald et al., 2015). We observed that PBs formed upon Pat1 overexpression were more 228 dynamic than PBs formed in the presence of Dhh1 DQAD and their dis-assembly kinetics was comparable 229 to PBs formed upon stress in a wild-type DHH1 background ( Figure S4B, C and Movies 3,4,5). Thus, 230 overall, our in vitro and in vivo findings are not consistent with the conclusion that Pat1 blocks the 231 ATPase activation of Dhh1. 232 The second hypothesis is that Pat1 promotes higher-order mRNP assembly by directly or indirectly 233 promoting Dhh1 oligomerization, thereby acting as a scaffold and providing additional protein-protein 234 or protein-RNA interactions (Coller and Parker, 2005; Roy and Parker, 2017). We have previously 235 shown that recombinant Dhh1 can undergo liquid-liquid phase separation (LLPS) in the presence of 236 RNA and ATP, and that these Dhh1 droplets can recapitulate aspects of in vivo PB dynamics. (Mugler 237 et al., 2016). We therefore utilized this in vitro system to test Pat1's impact on the phase separation 238 behavior of Dhh1. 239 Dhh1 droplets were assembled from purified components as described previously (Mugler et al., 240 2016). Interestingly, while no LLPS of Pat1 alone was detected ( Figure S5A), addition of increasing 241 concentrations of wild-type Pat1 strongly enhanced the LLPS of Dhh1 in the presence of ATP and RNA 242 as judged by an increase in the area * intensity of Dhh1 droplets ( Figure 5A). Furthermore, Pat1 itself 243 enriched in Dhh1 droplets, suggesting that Pat1 and Dhh1 co-oligomerize with RNA to form a 244 composite phase-separated compartment. 245 To test the specificity of LLPS stimulation, we tested the Pat1 4A-Dhh1 (Dhh1 binding) and Pat1 EE (phospho-246 mimic) mutants both of which both drastically reduce PB formation in vivo (see Figure 2A, 3A and S2B). 247 Consistent with the in vivo results, Dhh1-RNA droplet formation was either not at all or only mildly 248 stimulated upon addition of these Pat1 variants ( Figure 5A). 249 We previously demonstrated that the MIF4G domain of Not1 prevents formation of Dhh1-RNA 250 droplets, presumably by activating the ATPase cycle of Dhh1 (Mugler et al., 2016). We therefore next 251 analyzed whether the Pat1-Dhh1 droplets are still responsive to Not1. Similar to what we observed in 252 vivo (Figure 4), Not1 MIF4G also diminishes formation Dhh1 droplets in the presence of Pat1 in vitro 253 ( Figure 5B). 254 Taken together our results suggest that Pat1 enhances the multivalency of protein-protein and 255 protein-RNA interactions in a Dhh1-Pat1 mRNP and in consequence promotes the formation of higher-256 order, liquid-like mRNP droplets akin to in vivo PBs. 257

In vitro phase separated droplets mimic the stoichiometry of PB components in vivo 258
To examine how well our in vitro droplets resemble in vivo PBs we investigated the stoichiometric ratio 259 of Dhh1:Pat1 in these granules. In order to determine the stoichiometry in vivo, we measured the 260 number of PBs and the fluorescence intensity of GFP-tagged Dhh1 and Pat1 in these foci. Briefly, we 261 glucose starved both Pat1-GFP and Dhh1-GFP expressing cells to induce PBs ( Figure S6A) and used the 262 single particle tracking software Diatrack to count the number and intensity of PBs in an unbiased and 263 automated manner in each strain [Vallotton et al., 2013]. Surprisingly, despite the fact that the cellular 264 concentration of Pat1 is ten-times lower than Dhh1 ( Figure S6B), the ratio of the two PB components 265 was approximately 2:1+/-0.18 (Dhh1:Pat1) after 0.5-4 hours of starvation ( Figure 6A). Extended 266 starvation resulted in enhanced Dhh1 recruitment to PBs until reaching a Dhh1:Pat1 ratio of 2.5:1+/-267 0.23, suggesting that PB composition matures over time ( Figure 6). 268 In order to determine the stoichiometry of Pat1 and Dhh1 droplets in vitro, we imaged Dhh1-mCherry 269 and Pat1-GFP droplets with a confocal microscope ( Figure S6C). The protein concentration of both 270 Dhh1 and Pat1 in the droplet was calculated from a standard curve determined from values measured 271 for different concentrations of the respective soluble fluorophores ( Figure 6B). Remarkably, we found 272 a stoichiometric ratio of 2.7:1 +/-0.13 of Dhh1 to Pat1 in the in vitro phase-separated droplets, closely 273 resembling the Dhh1:Pat1 ratio of mature PBs in vivo ( Figure 6B Overall, our work uncovers a function of Pat1 as an enhancer of PB formation that acts via Dhh1 and 291 counteracts the inhibitory function of Not1. Based on our results, we propose that there are at least 292 three inputs that cooperatively regulate PB dynamics in vivo ( Figure 6C). First, the availability of mRNA 293 clients acting as seeding substrates to initiate PB formation. mRNA availability is inversely proportional 294 to the translation status, which is regulated by different stress responses and the metabolic state of 295 the cell ( Figure 2C). Second, the activity of cAMP-dependent PKA negatively regulates PB assembly in 296 nutrient-rich conditions, at least in part through phosphorylation of Pat1. However, since expression 297 of non-phosphorylatable Pat1 AA at endogenous levels (in contrast to overexpression) is not sufficient 298 to induce PBs, Pat1 availability appears to be limiting ( Figure 6, 3A, S2B and Ramachandran et al., 299 2011). Third, activation of the Dhh1 ATPase cycle by the CCR4-Not1 deadenylation complex stimulates 300 mRNA release and negatively regulates PB assembly. It is of note that Not1 itself might also be subject 301 to post-translational regulation, in response to metabolic state and/or cell cycle stages of the cell 302 (Mugler et al., 2016;Braun et al., 2014). 303 In this model, a tug-of-war between Pat1 and Not1 regulates PB assembly and disassembly: whereas 304 a tight Pat1-Dhh1 interaction in the presence of RNA clients, promotes PB formation, Not1, induces PB 305 turnover via stimulating the ATPase activity of the DEAD box ATPase Dhh1 ( Figure 6C). Remarkably, 306 our in vivo findings are corroborated in vitro wherein, Pat1 enhances the LLPS of Dhh1 and RNA, a step 307 critical for the assembly of large mRNP granules, and by contrast, Not1 reverses the LLPS of Pat1-Dhh1-308 RNA droplets ( Figure 6, 4, 5B). Furthermore, the observed stoichiometry of Dhh1 and Pat1 in PBs is 309 recapitulated in our in vitro phase separation assay. 310 The interaction between Pat1 and Dhh1 is critical for PB assembly 311 Inducing PB formation by Pat1 overexpression or hippuristanol treatment allowed us to dissect distinct 312 mechanisms that govern PB assembly and characterize the crucial role of the Pat1-Dhh1 interaction in 313 this process. The benefit of these modes of PB formation are that they bypass the characteristic 314 stresses associated with PB formation, such as nutrient starvation, that might have more widespread 315 and confounding effects on translation, mRNA degradation, or on the regulation of other PB 316

components. 317
Our experiments reveal that Pat1 functions in PB formation through Dhh1 since PB induction by Pat1 318 overexpression is strictly dependent on the presence of Dhh1 ( Figure 1A). Furthermore, a mutant in 319 the DETF motif in the N-terminus of Pat1 mediating direct binding to Dhh1 (Pat1 4A-Dhh1 variant) fails to 320 promote PB assembly ( Figure 2). These results were further confirmed using the drug hippuristanol 321 that inhibits translation initiation wherein PBs were induced in wild-type cells but not in cells 322 expressing the Pat1 4A-Dhh1 mutant ( Figure 2E, Chan et al., 2018) suggesting that high mRNA load alone 323 is inadequate for PB formation and that a direct physical interaction between Pat1 and Dhh1 is 324 obligatory as well. Since the DETF motif in Pat1 is conserved across evolution and was suggested to 325 regulate PB assembly in human cells as well (Sharif et al., 2013;Ozgur and Stoecklin, 2013), the Pat1-326 Dhh1 interaction likely plays a critical role in PB formation across species. 327 Our results seem to be at odds with a previous publication, which suggests that Pat1 acts 328 independently of Dhh1 to promote PB formation (Coller and Parker 2005). However, in this study Pat1 329 was overexpressed from a plasmid, on top of the endogenous copy. When recapitulating these 330 conditions, we observed that PB components mislocalize to the nucleus [observed by DAPI and a 331 nuclear rim marker, see Figure S7A

Construction of yeast strains and plasmids 390
S. cerevisiae strains used in this study are derivatives of W303 and are described in table S1. ORF 391 deletion strains and C-terminal epitope tagging of ORFs was done by PCR-based homologous 392 recombination, as previously described (Longtine et al., 1998). Plasmids for this study are described 393 in table S2A. Mutations in Pat1 were generated by introducing the mutation in the primer used to 394 amplify the respective Pat1 regions and stitched together with the selection marker from the plasmid 395 (table S2B)

Overexpression of Pat1 wild-type and mutants 398
The samples were grown overnight in synthetic media containing 2% raffinose, diluted to OD600 = 0.05 399 or 0.1 the following day, and grown to mid-log phase (OD600= 0.3-0.4). The culture was split into two 400 and to one-half galactose was added to 2% final concentration and the corresponding protein induced 401 for 2-3 hours. The cells in both raffinose and galactose were imaged using a wide-field fluorescence 402 microscope. 403

PB induction and disassembly kinetics 404
PBs were induced via glucose starvation stress. Samples were grown overnight in synthetic media 405 containing 2% glucose, diluted to OD600 = 0.05 or 0.1 the following day, and grown to mid-log phase 406 were processed using ImageJ software. Brightness and contrast were adjusted to the same values for 419 images belonging to the same experiment and were chosen to cover the whole range of signal 420 intensities. Image processing for PB analysis was performed using Diatrack 3.05 particle tracking 421 software (Vallotton et al., 2013) as described below. 422

Automated image analysis for PB quantification 423
In order to quantify PB formation in live cells, we used an automated image analysis in a manner similar 424 to Mugler et al., 2016. First, PBs were counted using Diatrack 3.05 particle tracking software using local 425 intensity maxima detection, followed by particle selection by intensity thresholding and particle 426 selection by contrast thresholding with a value of 5% (Vallotton et al., 2017). To speed up the analysis, 427 we renamed all our images in a form that can be recognized as a time-lapse sequence by Diatrack, and 428 placed them all in a single directory, such that they all will be analyzed using exactly the same image 429 analysis parameters. Renaming and copying was done by a custom script (supplementary code 1), 430 which also performed cell segmentation using a method adapted from (Hadjidemetriou et al., 2014). 431 Briefly, the method first detects all edges using a Laplacian edge detection step, and then traces 432 normals to those edges in a systematic manner. These normals tend to meet at the cell centre where 433 the high density of normals is detected, serving as seeds to reconstruct genuine cells. Our script thus 434 counts cells and reports their number for each image -information which is output to an excel table. 435 The results from Diatrack PB counting are imported from a text file into that table, and the number of 436 PB is divided by the number of cells for each image.

. (A, B) Overexpression (OE) of p(GAL)-PAT1 WT (wild type) and p(GAL)-pat1 AA (non-606 phosphorylatable) leads to constitutive PB formation but OE of p(GAL)-pat1 EE (phospho-mimic) does 607
not. Cells co-expressing the indicated PB components were grown in SC raffinose media to exponential 608 growth phase after which OE of different Pat1 alleles was induced by galactose addition.  in (SC) raffinose to exponential growth phase, after which galactose was added to 2% final 714 concentration for 2hrs to allow PBs to form. Thereafter, the cells were treated with 50µg/mL 715 cycloheximide (CHX) for 90 mins and disappearance of foci was monitored using fluorescence 716 microscopy. (5 min intervals; movie played at 7 fps). Each frame is a single plane. 717 Movie 5: Cycloheximide treatment of dhh1 DQAD PBs. Dcp2-mCherry expressing cells in the dhh1 DQAD 718 mutant background were grown in (SC) raffinose to exponential growth phase, after which galactose 719 was added to 2% final concentration for 2hrs. The cells were then carbon starved for 30 mins to allow 720 PBs to form after which, 50 µg/mL cycloheximide was added and disappearance of foci was monitored 721 for 90 mins using fluorescence microscopy. (5 min intervals; movie played at 7 fps). Each frame is a 722 single plane. 723 SUPPLEMENTARY