Summary Genome-wide studies have identified abundant small, non-coding RNAs including snRNAs, snoRNAs, cryptic unstable transcripts (CUTs), and upstream regulatory RNAs (uRNAs) that are transcribed by RNA polymerase II (pol II) and terminated by a Nrd1-dependent pathway. Here, we show that the prolyl isomerase, Ess1, is required for Nrd1-dependent termination of ncRNAs. Ess1 binds the carboxy terminal domain (CTD) of pol II and is thought to regulate transcription by conformational isomerization of Ser-Pro bonds within the CTD. In ess1 mutants, expression of ∼10% of the genome was altered, due primarily to defects in termination of snoRNAs, CUTs, SUTs and uRNAs. Ess1 promoted dephosphorylation of Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase, and we provide evidence for a competition between Nrd1 and Pcf11 for CTD-binding that is regulated by Ess1-dependent isomerization. This is the first example of a prolyl isomerase required for interpreting the “CTD code.”

23High-throughput screening (HTS) using new approach methods is revolutionizing 24 toxicology. Asexual freshwater planarians are a promising invertebrate model for neurotoxicity 25 HTS because their diverse behaviors can be used as quantitative readouts of neuronal function. 26Currently, three planarian species are commonly used in toxicology research: Dugesia japonica, 27Schmidtea mediterranea, and Girardia tigrina. However, only D. japonica has been demonstrated 28 to be suitable for HTS. Here, we assess the two other species for HTS suitability by direct 29 comparison with D. japonica. Through quantitative assessments of morphology and multiple 30 behaviors, we assayed the effects of 4 common solvents (DMSO, ethanol, methanol, ethyl acetate) 31 and a negative control (sorbitol) on neurodevelopment. Each chemical was screened blind at 5 32 concentrations at two time points over a twelve-day period. We obtained two main results: First, 33 G. tigrina and S. mediterranea planarians showed significantly reduced movement compared to 34 D. japonica under HTS conditions, due to decreased health over time and lack of movement under 35 red lighting, respectively. This made it difficult to obtain meaningful readouts from these species. 36Second, we observed species differences in sensitivity to the solvents, suggesting that care must 37 be taken when extrapolating chemical effects across planarian species. Overall, our data show that 38 D. japonica is best suited for behavioral HTS given the limitations of the other species. 39Standardizing which planarian species is used in neurotoxicity screening will facilitate data 40 comparisons across research groups and accelerate the application of this promising invertebrate 41 system for first-tier chemical HTS, helping streamline toxicology testing. 42 43 Keywords 44 Planarian, high-throughput screening, invertebrate, developmental neurotoxicity, solvents 45 . CC-BY-NC 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a 65 We have developed the asexual freshwater planarian Dugesia japonica as a promising new 66 invertebrate model for high-throughput neurotoxicity and DNT screening (Hagstrom et al., 2016(Hagstrom et al., , 67 2015Zhang et al., 2019aZhang et al., , 2019b. We have shown that it possesses comparable sensitivity to more 68 . CC-BY-NC 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a established new approach methods and is predictive of mammalian DNT (Hagstrom et al., 2019, 69 2015Zhang et al., 2019aZhang et al., , 2019b. The key advantage of the planarian system is its sufficiently 70 complex behavioral repertoire which enables distinct behaviors to be used as a multifaceted 71 quantitative readout of neuronal function (Hagstrom et al., 2019;Zhang et al., 2019aZhang et al., , 2019b. The 72 planarian nervous system is of medium size (~10,000 neurons), possessing >95% gene homology 73 and sharing most of the same neurotransmitte...

In response to noxious stimuli, planarians cease their typical ciliary gliding and exhibit an oscillatory type of locomotion called scrunching. We have previously characterized the biomechanics of scrunching and shown that it is induced by specific stimuli, such as amputation, noxious heat, and extreme pH. Because these specific inducers are known to activate Transient Receptor Potential (TRP) channels in other systems, we hypothesized that TRP channels control scrunching. We found that chemicals known to activate TRPA1 (allyl isothiocyanate (AITC) and hydrogen peroxide) and TRPV (capsaicin and anandamide) in other systems induce scrunching in the planarian species Dugesia japonica and, except for anandamide, in Schmidtea mediterranea. To confirm that these responses were specific to either TRPA1 or TRPV, respectively, we tried to block scrunching using selective TRPA1 or TRPV antagonists and RNA interference (RNAi) mediated knockdown. Unexpectedly, co-treatment with a mammalian TRPA1 antagonist, HC-030031, enhanced AITC-induced scrunching by decreasing the latency time, suggesting an agonistic relationship in planarians. We further confirmed that TRPA1 in both planarian species is necessary for AITC-induced scrunching using RNAi. Conversely, while co-treatment of a mammalian TRPV antagonist, SB-366791, also enhanced capsaicin-induced reactions in D. japonica, combined knockdown of two previously identified D. japonica TRPV genes (DjTRPVa and DjTRPVb) did not inhibit capsaicin-induced scrunching. RNAi of DjTRPVa/DjTRPVb attenuated scrunching induced by the endocannabinoid and TRPV agonist, anandamide. Overall, our results show that although scrunching induction can involve different initial pathways for sensing stimuli, this behavior’s signature dynamical features are independent of the inducer, implying that scrunching is a stereotypical planarian escape behavior in response to various noxious stimuli that converge on a single downstream pathway. Understanding which aspects of nociception are conserved or not across different organisms can provide insight into the underlying regulatory mechanisms to better understand pain sensation.

AbstractHigh-throughput screening (HTS) using new approach methods is revolutionizing toxicology. Asexual freshwater planarians are a promising invertebrate model for neurotoxicity HTS because their diverse behaviors can be used as quantitative readouts of neuronal function. Currently, three planarian species are commonly used in toxicology research: Dugesia japonica, Schmidtea mediterranea, and Girardia tigrina. However, only D. japonica has been demonstrated to be suitable for HTS. Here, we assess the two other species for HTS suitability by direct comparison with D. japonica. Through quantitative assessments of morphology and multiple behaviors, we assayed the effects of 4 common solvents (DMSO, ethanol, methanol, ethyl acetate) and a negative control (sorbitol) on neurodevelopment. Each chemical was screened blind at 5 concentrations at two time points over a twelve-day period. We obtained two main results: First, G. tigrina and S. mediterranea planarians showed significantly reduced movement compared to D. japonica under HTS conditions, due to decreased health over time and lack of movement under red lighting, respectively. This made it difficult to obtain meaningful readouts from these species. Second, we observed species differences in sensitivity to the solvents, suggesting that care must be taken when extrapolating chemical effects across planarian species. Overall, our data show that D. japonica is best suited for behavioral HTS given the limitations of the other species. Standardizing which planarian species is used in neurotoxicity screening will facilitate data comparisons across research groups and accelerate the application of this promising invertebrate system for first-tier chemical HTS, helping streamline toxicology testing.

Organophosphorus pesticides (OPs) are a chemically diverse class of commonly used insecticides. Epidemiological studies suggest that low dose chronic prenatal and infant exposures can lead to life-long neurological damage and behavioral disorders. While inhibition of acetylcholinesterase (AChE) is the shared mechanism of acute OP neurotoxicity, OP-induced developmental neurotoxicity (DNT) can occur independently and/or in the absence of significant AChE inhibition, implying that OPs affect alternative targets. Moreover, different OPs can cause different adverse outcomes, suggesting that different OPs act through different mechanisms. These findings emphasize the importance of comparative studies of OP toxicity. Freshwater planarians are an invertebrate system that uniquely allows for automated, rapid and inexpensive testing of adult and developing organisms in parallel to differentiate neurotoxicity from DNT. Effects found only in regenerating planarians would be indicative of DNT, whereas shared effects may represent neurotoxicity. We leverage this unique feature of planarians to investigate potential differential effects of OPs on the adult and developing brain by performing a comparative screen to test 7 OPs (acephate, chlorpyrifos, dichlorvos, diazinon, malathion, parathion and profenofos) across 10 concentrations in quarter-log steps. Neurotoxicity was evaluated using a wide range of quantitative morphological and behavioral readouts. AChE activity was measured using an Ellman assay. The toxicological profiles of the 7 OPs differed across the OPs and between adult and regenerating planarians. Toxicological profiles were not correlated with levels of AChE inhibition. Twenty-two “mechanistic control compounds” known to target pathways suggested in the literature to be affected by OPs (cholinergic neurotransmission, serotonin neurotransmission, endocannabinoid system, cytoskeleton, adenyl cyclase and oxidative stress) and 2 negative controls were also screened. When compared with the mechanistic control compounds, the phenotypic profiles of the different OPs separated into distinct clusters. The phenotypic profiles of adult vs. regenerating planarians exposed to the OPs clustered differently, suggesting some developmental-specific mechanisms. These results further support findings in other systems that OPs cause different adverse outcomes in the (developing) brain and build the foundation for future comparative studies focused on delineating the mechanisms of OP neurotoxicity in planarians.