Leishmania PNUTS discriminates between PP1 catalytic subunits through an RVxF–ΦΦ–F motif and polymorphisms in the PP1 C-tail and catalytic domain

Phosphoprotein phosphatase 1 (PP1) associates with specific regulatory subunits to achieve, among other functions, substrate selectivity. Among the eight PP1 isotypes in Leishmania, PP1-8e associates with the regulatory protein PNUTS along with the structural factors JBP3 and Wdr82 in the PJW/PP1 complex that modulates RNA polymerase II (pol II) phosphorylation and transcription termination. Little is known regarding interactions involved in PJW/PP1 complex formation, including how PP1-8e is the selective isotype associated with PNUTS. Here, we show that PNUTS uses an established RVxF–ΦΦ–F motif to bind the PP1 catalytic domain with similar interfacial interactions as mammalian PP1–PNUTS and noncanonical motifs. These atypical interactions involve residues within the PP1-8e catalytic domain and N and C terminus for isoform-specific regulator binding. This work advances our understanding of PP1 isoform selectivity and reveals key roles of PP1 residues in regulator binding. We also explore the role of PNUTS as a scaffold protein for the complex by identifying the C-terminal region involved in binding JBP3 and Wdr82 and impact of PNUTS on the stability of complex components and function in pol II transcription in vivo. Taken together, these studies provide a potential mechanism where multiple motifs within PNUTS are used combinatorially to tune binding affinity to PP1, and the C terminus for JBP3 and Wdr82 association, in the Leishmania PJW/PP1 complex. Overall, our data provide insights in the formation of the PJW/PP1 complex involved in regulating pol II transcription in divergent protozoans where little is understood.

Phosphorylation is a critical regulatory mechanism for over 70% of eukaryotic cellular proteins, and the majority of the phosphorylations occur on serine, threonine, or tyrosine residues (1).More than 420 serine/threonine kinases target specific serine/threonine residues, which account for approximately 98% of all phosphorylation events.On the other hand, fewer than 40 serine/threonine phosphatases are involved in protein dephosphorylation (2,3).Phosphoprotein phosphatase 1 (PP1) is a major serine/threonine phosphatase, estimated to catalyze one-third of all dephosphorylation events in eukaryotic cells and involved in many essential cellular activities (including cardiac muscle contraction, glycogen metabolism, cell cycle transition, and transcription termination) (4)(5)(6).In contrast to protein serine/threonine kinases, although PP1 exhibits some intrinsic preference for pThr versus pSer and motifs surrounding the phosphorylation sites (7)(8)(9), the substrate specificity of PP1 is largely conferred by regulatory interactors of PP1 (RIPPOs) (previously referred to as PP1-interacting proteins) (2,(10)(11)(12)(13).Therefore, to carry out specific functions in a wide variety of cellular activities, PP1 binds over 200 confirmed RIPPOs, forming highly specific holoenzymes in mammalian cells (2,(13)(14)(15).These RIPPOs target PP1 to distinct cellular compartments and/or help direct its activity toward specific substrates (14,16).RIPPOs usually associate with PP1 using a combination of short linear motifs (SLiMs).They bind in a largely extended manner at multiple sites across the top of PP1 (remote from the catalytic site), including the RVxF motif-binding site and the ΦΦ motifbinding site, both of which are used by a large number of RIPPOs.However, many studies have shown that RIPPO binding is usually more complex, with RIPPOs utilizing additional motifs beyond the RVxF and ΦΦ motif for PP1 holoenzyme formation (13).Characterizing these interactions is key to understanding how RIPPOs associate with PP1 and regulate specific biological processes such as transcription and gene expression.
One of the earliest characterized RIPPOs is PNUTS (PP1 nuclear targeting subunit), originally described as a nuclear regulator of PP1 that helps retain PP1 in the nucleus (17)(18)(19).PNUTS has been implicated in PP1-regulated processes, including cell cycle regulation (20), RNA processing (17,21), DNA repair (22), transcription (23), and telomere stability (24).Like most RIPPOs, the PP1-interacting domain in PNUTS is largely unstructured in the unbound state, and PNUTS is included in a group of intrinsically disordered proteins (14,15,25,26).This intrinsic flexibility is important for the formation of extensive interactions with PP1.PNUTS modulation of PP1 is mediated by a central region, employing RVxF-ΦΦ-Phe-Arg motifs (25).The most well-characterized motif is the RVxF motif ([K/R]-X 0-1 -[V/I/L]-X-[F/W], where X can be any amino acid except proline) that is found in 90% of RIPPOs (27)(28)(29). 398TVTW 401 in human PNUTS (hPNUTS) constitutes the canonical RVxF PP1-binding motif, with the second and fourth residues burying deep in two hydrophobic pockets on the PP1 surface, providing an essential stabilizing force (25).As demonstrated for hPNUTS (25,30), mutation of hydrophobic valine and phenylalanine/tryptophan positions in the RVxF-binding motif typically abolishes the ability of RIPPO to bind to PP1.Structure analyses of RIPPO-PP1 holoenzymes (including PNUTS) have identified several additional motifs that make contact with PP1 (25).For example, the ΦΦ motif is a two-hydrophobic residue motif that is usually found five to eight amino acids C terminal to the RVxF motif on RIPPOs (25).hPNUTS-PP1 is found to be associated with two additional structural proteins, Wdr82 and the DNAbinding protein Tox4, in a complex called PTW/PP1 (30).PNUTS is the scaffolding protein in the complex and mediates independent associations of PP1, Wdr82, and Tox4.Tox4 interacts with an N-terminal TFIIS domain in hPNUTS, whereas Wdr82 binds to a C-terminal region in hPNUTS (amino acids 418-619).The PTW/PP1 complex is a negative regulator of RNA polymerase II (pol II) elongation rate and plays a key role in transcription termination.Depletion of individual components in human cells, or ortholog components in yeast, leads to pol II transcription termination defects (23,(31)(32)(33)(34).In the torpedo model of transcription termination, as pol II reaches the poly(A) signal, pre-mRNA is cleaved, providing an entry site for the 5 0 -3 0 exoribonuclease Xrn2 to catch up with pol II and dislodge it from the DNA template, allowing for transcription termination (35)(36)(37).Dephosphorylation of pol II Cterminal domain (CTD) and Spt5, reducing the speed of the polymerase within the so-called termination zone, facilitates this process (38)(39)(40).
The Trypanosomatidae are early divergent protozoan parasites.Several members of the Trypanosomatidae including Trypanosoma brucei and Leishmania major are pathogenic to humans, causing human African trypanosomiasis (African sleeping sickness) and leishmaniasis.In these parasites, hundreds of genes of unrelated functions are arranged into polycistronic transcription units throughout the genome (41,42).Genes in each polycistronic transcription unit are cotranscribed from an initiation site at the 5 0 end to the termination site at the 3 0 end.Pre-mRNAs are processed through transsplicing with the addition of a 39-nucleotide spliced leader sequence to the 5 0 end of mRNAs, which is coupled to the 3 0 polyadenylation of the upstream transcript (43)(44)(45)(46)(47)(48)(49)(50).Very little is understood regarding the pol II transcription cycle (initiation, elongation, and termination) in these important eukaryotic pathogens.Epigenetic markers, such as histone variants (H3V and H4V) and the DNA modification base J, are enriched at pol II transcription termination sites in Leishmania and T. brucei (41,(51)(52)(53).Base J is a glucosylated thymidine (54) and has only been identified in the nuclear DNA of kinetoplastids, Diplonema, and Euglena (55,56).The loss of base J (and H3V) in Leishmania and T. brucei led to readthrough transcription at termination sites, suggesting a critical role of base J in pol II transcription termination (57)(58)(59)(60)(61). Exploring base J function further led to the identification of the PJW/PP1 complex in Leishmania tarentolae composed of PP1-PNUTS-Wdr82 and a base J-binding protein, JBP3 (62,63).LtPNUTS (Leishmania tarentolae PNUTS) is a predictively disordered 29 kDa protein with 23% sequence identity to hPNUTS and contains a putative RVxF PP1-binding motif ( 97 RVCW 99 ) (62).Alanine substitution of the hydrophobic residues in the RVxF motif ( 97 RACA 99 ) has been shown to disrupt LtPNUTS-PP1 association (64).In addition, short synthetic RVxF-containing peptides are sufficient to disrupt the LtPNUTS-PP1 association.Ablation of PNUTS, JBP3, and Wdr82 by RNAi in T. brucei (62), and deletion of PP1-8e and JBP3 in Leishmania (63,64), has been shown to cause pol II termination defects, similar to the defects following the loss of base J-H3V.These in vivo data, along with the recent demonstration that pol II is a direct substrate for PP1-8e as a component of the Leishmania PJW/PP1 complex in vitro (64), support a conserved PNUTS-PP1 regulatory mechanism from trypanosomatids to yeast and mammalian cells.We therefore proposed that similar to the PTW/PP1 complex, LtPNUTS is a scaffolding protein that mediates independent binding of PP1, JBP3, and Wdr82, with JBP3 tethering the complex to the base J-enriched transcription termination sites for PP1-mediated dephosphorylation of pol II.
Eight PP1 isoforms, grouped into five different clades (A-E), are identified in the Leishmania genome (Fig. S1).Among these, only PP1-8e is found associated with the PJW/PP1 complex in vivo and shown to be involved in pol II transcription termination (64).Although the T. brucei genome also harbors eight PP1 isoforms, no obvious PP1 isoform belongs to clade E as a homolog of LtPP1-8e (64).Furthermore, purification of TbPNUTS (Trypanosoma brucei PNUTS) pulls down JBP3 and Wdr82 but not PP1 (62).Presumably, transient/weak association between a TbPP1 isotype and the PNUTS-Wdr-JBP3 complex via the conserved RVxF PP1-binding motif allows a conserved transcription termination mechanism in T. brucei cells (62,64).Unique sequences within PP1-8e may explain isotype selectivity of PNUTS binding in Leishmania (64).However, interactions involved in the selectivity of LtPNUTS for the PP1-8e isoform have not been explored.In fact, while PP1 isoform selectivity is thought to be an important feature of regulatory RIPPOs, limited mechanistic information exists on how this is achieved in any system.The mammalian PP1 isoforms (PP1α, PP1β, and PP1γ) share a sequence identity ranging from 85% to 93%, and sequence variability mainly comes from the divergent N termini and, most notably, C termini, with only a few amino acid residues being different within the catalytic domains (2).Among the regulatory RIPPOs, which display isoform preferences, such as MYPT1 (65,66), spinophilin (67), RepoMan (68), Ki67 (68), ASPP2 (69) and RRP1B (70), specificity is achieved via recognition of the PP1 C terminus or a β/γ specificity pocket within the PP1 catalytic domain.The extreme C terminus of PP1 (PP1α 309-330 ) contains an SH3-binding motif (PPII-xxPxR), which is conserved among all the mammalian PP1 isoforms, and a variable C-tail.The apoptosis stimulation proteins of p53 family (iASPP/ASPP1/ASPP2) utilize an SH3 domain to selectively bind the PP1 C terminus via contacts in the PPII motif and residues in the variable C-tail region to PNUTS-PP1 interactions in Leishmania achieve isoform selectivity (69,71,72).Ankyrin repeats of the myosin phosphatase targeting subunit MYPT1 associate with amino acids in the PP1 C-tail and drive selectivity toward PP1β (2).In the case of RRP1B, RepoMan, and Ki-67, the SLiM (KiR or SLIV) immediately downstream of the RVxF motif determines the preference toward PP1γ through a single amino acid change in the catalytic domain of PP1 (68,70,73).Therefore, isoform specificity is mediated in these RIPPOs by a single amino acid difference in PP1 at position 20, which is an Arg residue in PP1γ/β and a Gln residue in PP1α.
In this study, we employ structural modeling and mutagenesis analysis to help define how LtPNUTS specifically recruits PP1-8e to the PJW/PP1 complex.First, we confirm that LtPNUTS demonstrates substrate specificity for PP1-8e among the identified LtPP1 isoforms in vivo by coimmunoprecipitation (co-IP) analysis.We show that LtPNUTS binds to PP1 via a combination of well-characterized PP1-interacting motifs including the extended RVxF (RVxF-ɸ R -ɸɸ) and Phe motif.We also identified unique termini and motifs within LtPP1-8e catalytic domain, including sites not previously shown to bind any PP1 regulator, which are important for PP1-PNUTS interaction.Finally, we explore the scaffold function of PNUTS by mapping the Wdr82 and JBP3-binding domain at the C terminus of PNUTS and demonstrate that PNUTS protein level is critical for the integrity of the PJW/PP1 complex and function in pol II termination.Together, these data support a model for extensive interactions between LtPNUTS and PP1-8e and provide key insights into the isoform selectivity of LtPNUTS and its scaffold function in overall stability of the PJW/PP1 complex.

LtPNUTS displays PP1 isoform selectivity
Our previous affinity purification-mass spectrometry (MS) data (62) indicated that PNUTS is part of a tightly interlinked protein network comprising the PP1 catalytic subunit PP1-8e, JBP3, and Wdr82 in L. tarentolae cells.While there are eight PP1 isotypes in the Leishmania genome (Fig. S1) (63,64), only PP1-8e was associated with the Leishmania PJW/PP1 complex.In order to understand the specific association of PP1-8e in this complex, we first sought to verify the binary interaction between PNUTS and the L. tarentolae PP1 catalytic subunits by co-IP in vivo.To do this, we hemagglutinin (HA) tagged the endogenous loci of LtPNUTS using Cas9 and overexpressed various Pd-tagged LtPP1 isotypes from a plasmid (Fig. 1A).LtPP1-3 (LtaPh_3411201) is much smaller (167 amino acids) than other PP1 isotypes and is predicted to contain a partial catalytic core.We were unable to overexpress Pd-tagged LtPP1-3, suggesting that it could be a truncated pseudogene.Therefore, we analyzed five of the seven complete PP1 isotypes in L. tarentolae (representing all five clades).Our results show that only PP1-8e can IP a significant fraction of PNUTS, whereas the other PP1 isotypes (PP1-1, PP1-2, PP1-4, and PP1-7) show no detectable interaction with PNUTS by co-IP (Fig. 1B).These data confirm the MS analysis of the purified PNUTS-PP1 complex (62,64) and directly demonstrate for the first time that PNUTS preferentially targets PP1-8e over other isoforms in intact Leishmania cells.

LtPNUTS associates with PP1-8e through an established RVxF-ɸ R -ΦΦ-F motif
To determine the molecular basis of isoform specificity of PNUTS for PP1-8e in L. tarentolae, we used AlphaFold to help define the PNUTS-PP1 interaction interface.We first explored the predicted structure for the LtPP1 isotypes.The PP1 catalytic core is highly conserved across eukaryotes from human to yeast cells, consisting of 10 sets of α-helices (labeled A 0 to I) and 15 sets of β-sheets (numbered 1 0 to 14) (Fig. S2) (74,75).The catalytic core regions of the LtPP1 isotypes are predicted to be of high confidence by AlphaFold, and their structural overlay to the determined human PP1 protein structure (Protein Data Bank ID: 3E7A, Fig. S3A) shows a high structural similarity.An example is the predicted LtPP1-1a structure (Fig. S3B), which shows a high structural identity to hPP1 (Fig. S3C) with an RMSD of 0.580 Å. LtPP1-8e was also predicted with high confidence (Fig. S3D) for the catalytic core region.The predicted LtPP1-8e structure aligns well to the hPP1 structure (Fig. S3E) except three regions within the catalytic core and the N and C terminus that we have identified as unique to PP1-8e (64) (Figs.S2 and S3E).Deletion of these unique regions in LtPP1-8e increases the structural similarity between LtPP1-8e and hPP1 (RMSD: 0.666 Å, Fig. S3G).Thus, as previously predicted based on sequence conservation (64), the structural identity of PP1 catalytic subunits between Figure 1.PNUTS binding is specific for the PP1-8e isoform.A, expression of PNUTS and PP1 isoforms in Leishmania tarentolae.Cell extracts from L. tarentolae cells that endogenously express HA-tagged PNUTS and exogenously express Pd-tagged PP1 isoforms from the pSNSAP1 vector were analyzed by Western blotting with anti-HA and anti-protein A. B, PNUTS-PP1 co-IP analysis.Lysates from the indicated cell lines were purified by anti-protein A affinity resin and analyzed by Western blotting with antiprotein A and anti-HA.Equal cell equivalents of input (In), precipitated immunocomplexes (IP), and flow-through or unbound fraction (FT) were loaded on the gel.EF1A serves as a loading and negative control for the co-IP.co-IP, coimmunoprecipitation; HA, hemagglutinin; PNUTS, PP1 nuclear targeting subunit; PP1, phosphoprotein phosphatase 1.
mammals and Leishmania suggests strong functional conservation during evolution.However, unique sequences in PP1-8e may be important for PP1-8e-specific functions in Leishmania.
We next submitted LtPNUTS and LtPP1-8e sequences together to AlphaFold2 to generate the predicted LtPP1-PNUTS structure (Fig. S4).As expected, the majority of LtPNUTS is unstructured.While only a limited region of PNUTS is confidently predicted to become buried upon complex formation (Fig. S4), this region binds in a largely extended manner at multiple sites across the top of PP1 in a way highly similar to several well-characterized PP1interacting proteins (Fig. 2, B and C), including hPNUTS (Fig. S5) (25), spinophilin (76), and Gm (77).They share multiple well-characterized PP1-binding motifs, including the RVxF-ɸ R -ɸɸ binding motif (Fig. 2, A and C).Furthermore, similar to hPNUTS, LtPNUTS is predicted to bind PP1 remotely away from the PP1 catalytic site, making it fully accessible to substrate.Consistently, we have recently Figure 2. Predicted LtPNUTS-PP1-8e interacting motifs.A, structure-based sequence alignment of the PP1-interacting motifs of LtPNUTS, hPNUTS, spinophilin, and Gm, with PP1-interacting residues indicated.B, predicted structure of the LtPNUTS-PP1-8e complex.LtPNUTS is shown as orange ribbon with key interacting residues shown as sticks and LtPP1-8e is shown as a gray surface.LtPNUTS residues 96 RVCW 99 and L111 are predicted to bind to the RVxF-binding pocket (red), LtPNUTS residues 114 HV 115 are predicted to bind to the PP1 ΦΦ-binding pocket (cyan), and LtPNUTS residue F118 is predicted to bind to the Phe-binding pocket (yellow).The colored regions of PP1-8e correspond to the zoomed in pockets shown in D-F.C, overlay of the RVxF and ΦΦ-F structures of four PP1 regulators, LtPNUTS (orange), hPNUTS (green, 4mp0), spinophilin (blue, 3egg), and Gm (gray, 6dno), with residues binding RVxF-Φ R , ΦΦ, and Phe pockets shown as sticks.The arginine residue (R125) of LtPNUTS that deviates from the hPNUTS and spinophilin structure is indicated.D-F, major binding interactions between LtPNUTS (orange sticks) and PP1 (surface).The well-established SLiM-binding pockets (D, RVxF; E, ΦΦ; F, Phe) are shown.Key interacting residues in LtPP1-8 (black) and LtPNUTS (orange) are labeled.Predicted salt-bridge interactions between PP1 residues E350 and T348 with the PNUTS ΦΦ motif indicated by dashed line in E. Gm, muscle-specific glycogen-targeting subunit of PP1; hPNUTS, human PNUTS; LtPNUTS, Leishmania tarentolae PNUTS; PNUTS, PP1 nuclear targeting subunit; PP1, phosphoprotein phosphatase 1; SLiM, short linear motif.

PNUTS-PP1 interactions in Leishmania
demonstrated that PP1 is catalytically active in the PNUTS-PP1-8e holoenzyme, capable of dephosphorylating model substrates, such as p-nitrophenyl phosphate, as well the LtPol II CTD (64).The first of the key interaction sites in the LtPNUTS-PP1-8e complex is bound by the RVxF-ɸ R motifs (Fig. 2, A-D).Nine residues of PNUTS ( 93 R to D 101 ) adopt an extended conformation and bind to a hydrophobic channel on the PP1 surface formed at the interface of the two β-sheets of the β-sandwich opposite to the catalytic site channel.PNUTS residues 96 RVCW 99 form the RVxF motif, which binds the PP1 RVxF-binding pocket, and V and W are the anchoring hydrophobic residues that bind deeply in this pocket (Fig. 2D).The predicted LtPNUTS-RVxF interaction is highly similar to those observed in other PP1 holoenzyme complexes, including mammalian PNUTS-PP1 (Fig. S5B).Structural and functional studies of the mammalian PP1-PNUTS complex, and modeling of the LtPP1-PNUTS complex here, suggest a dominant role for V97 and W99 in stabilizing the interaction between LtPNUTS and PP1-8e.We have recently shown that the V97A-W99A double mutant is unable to bind PP1-8e (64).To test this hypothesis in more detail, we made single alanine mutations at each of these positions in the LtPNUTS expression plasmid (pSNSAP1) and tested the PNUTS mutants for interaction by co-IP with endogenously HA-tagged PP1-8e.Alanine mutation of W99 completely abolished PP1-PNUTS association, and V97A decreased PP1-PNUTS association by fivefold (Fig. 3, A and B), indicating the importance of the hydrophobic association mediated by the RVxF motif.Inspection of the structure of hPNUTS in complex with PP1 highlighted interfacial PP1 amino acids I169, L243, F257, R261, V264, I266, M283, C291, and F293 that are conserved in LtPP1-8e as I217, L293, F307, R314, L317, L336, V343, C344, and I346 that form the hydrophobic pocket and stabilize V97 and W99 in the PNUTS RVxF motif (Figs.2D  and S5B).To test this, single alanine mutation of I217 was introduced into the LtPP1-8e expression construct and the PP1 mutants tested for interaction by co-IP with endogenously HA-tagged PNUTS.Mutation of I217 PP1 to alanine significantly reduced (50%) the PP1-PNUTS interaction (Figs.3C  and S6B), supporting the importance of the hydrophobic interface with the conserved Val and Trp moieties of the LtPNUTS RVxF motif.We suggest that the VxW motif in LtPNUTS is the putative counterpart of a Vx(F/W) motif that comprises a key part of the PP1 phosphatase-binding site identified in several other PP1 regulatory subunits, including hPNUTS, where the VxW motif binds to a hydrophobic pocket of the phosphatase remote from the phosphatase active site (25) (Fig. S5B).
A short 22-amino acid peptide from PNUTS that contains the RVxF motif is able to disrupt the PP1-PNUTS association, whereas the identical peptide with V97A and W99A substitutions is not (64), further confirming the importance of the RVxF motif in the LtPNUTS-PP1-8e complex.However, WT RVxF peptide did not elute all the PP1s from PNUTS suggesting there may be additional interaction sites that stabilize the PNUTS-PP1 complex (64).PP1 phosphatase-regulatory proteins often have at least one, and often several, basic amino acids preceding the Vx(F/W) motif (78).It has been suggested that this basic region may interact with a negatively charged patch near the RVxF-binding pocket of PP1.In the case of hPNUTS, there is a run of five basic amino acids upstream of VxW (Fig. 2A); two of which engage in salt bridges to acidic side chains of PP1 (Fig. S5G).Similar interactions are predicted for LtPNUTS 94 RKR 96 , which are predicted to have electrostatic interaction with PP1-8e residues D290, E340, and D292, respectively (Fig. S5H).Alanine mutation of R94A PNUTS , or its predicted interacting residue D290 PP1 , leads to a 50% reduction in PP1-PNUTS association (Figs. 3, B and C and S6, A and B).Alanine mutation of K95 PNUTS did not affect PP1-PNUTS interaction (Figs.3B and S6A).However, not all electrostatic interactions mediated by these basic residues contribute equally to the association, and sometimes, simultaneous alanine mutations of all the basic amino acid residues preceding the RVxF motif are required to affect PP1 binding, as observed for the fission yeast PNUTS (79).We were unable to generate the R96A PNUTS mutant, but alanine mutation of its interacting residue D292A PP1 leads to 60% reduction in PP1-PNUTS interaction (Figs.3C and S6B), supporting the importance of R96 PNUTS .Acidic residues C terminal to the RVxF motif are also present in other PP1 regulatory subunits and important for binding PP1.The AlphaFold model shows D101 of LtPNUTS engaging in salt-bridge interactions to R314 PP1 (Fig. S5H).Similar interaction is observed on E405 of hPNUTS (Fig. S5G).Consistent with the prediction, the D101A PNUTS mutant showed roughly 80% decreased interaction with PP1-8e (Figs. 3B and S6A).
The LtPNUTS-PP1-8e model predicts that H114 PNUTS and V115 PNUTS form the PNUTS ΦΦ motif, which binds the PP1 ΦΦ-binding pocket (Fig. 2E).Like the RVxF interaction, the ΦΦ interaction is highly similar to those observed in other PP1 holoenzyme complexes (Fig. 2C).The ɸɸ motif usually consists of two hydrophobic residues of RIPPOs that are buried in a hydrophobic pocket on PP1, but can be degenerate, including sequences, such as VS, VC, VK, IN, and HH (25).The ɸɸ motif of hPNUTS is represented by 410 YF 411 located on a short β strand that hydrogen bonds with β strand β14 of PP1, extending one of its two central β sheets (Fig. S5C).AlphaFold predicts a similar arrangement in the LtPNUTS-PP1 complex (Figs.2E and S5C).The predicted ɸɸ motif of LtPNUTS, 114 HV 115 , is located on a short β strand formed by 112 VKHV 115 that potentially H-bonds with PP1-8e 0 s β strand 14, and the ɸɸ hydrophobic pocket on LtPP1-8e includes residues N100, R104, E350, and T348 (Fig. 2E).To test the significance of the ɸɸ motif, we mutated LtPNUTS 114 VH 115 to alanine and found that the mutation significantly weakens the PP1-PNUTS association (Figs.3B and S6A).While the structure of the hPNUTS-PP1 complex does not indicate any specific interactions between the ɸɸ motif of hPNUTS and the ɸɸ hydrophobic pocket of PP1, we noticed a potential salt-bridge interaction between H114 PNUTS and T348 PP1 in our model (Fig. 2E).Alanine mutation of T348 PP1 , however, had minimal impact on PP1-PNUTS association (Figs.3C and S6B).Presumably, the interaction does not occur, or the alanine mutation of T348 alone is not sufficient to disrupt the stabilizing
An additional potential LtPP1-8e interaction beyond the ΦΦ motif is R125 PNUTS (Fig. 2A).In hPNUTS, R420 is involved in hydrophobic and electrostatic interactions with PP1, representing the so-called Arg motif (Fig. S5E).R420 hPNUTS is buried in a hydrophobic pocket formed by L296, P298, and P270 of hPP1.In addition, E419 hPNUTS and R420 hPNUTS form bidentate salt bridges with R74 PP1 and D71 PP1 , respectively (Fig. S5E).However, this interaction is not predicted by AlphaFold in the LtPNUTS-PP1-8e complex.Rather, an alpha helix (residues 354-362) within the C-terminal tail of PP1-8e occupies the PP1 hydrophobic pocket involved in R420 hPNUTS binding (Fig. S5F).Furthermore, while the Arg motif is presumably conserved on LtPNUTS as R125, the polar S124 PNUTS replaces the negatively charged E419 PNUTS in hPNUTS.Moreover, the interacting charged residue in hPP1, R74 PP1 , is replaced by N100 in LtPP1-8e (Fig. S7).The replacement of charged residues with polar residues may prevent the formation of a bidentate salt bridge important for Arg motif binding.This concept along with the blocking of the Arg pocket by the C-terminal tail of LtPP1-8e could explain the divergence of the LtPNUTS-PP1 binding structure from hPNUTS at this region.R125 on LtPNUTS is therefore not predicted to bind to PP1-8e, and alanine mutation of R125 in LtPNUTS had no effect on PP1-PNUTS association (Figs.3B and S6A).Alanine mutation of R420 hPNUTS , however, does not affect PP1-PNUTS association in human cells (25), although the crystal structure indicates the importance of the Arg motif.Therefore, while the AlphaFold model clearly rules out the interaction, our co-IP results do not completely exclude the possibility that R125 PNUTS mediates interaction with PP1 in L. tarentolae cells.
Taken together, the predicted structure and mutagenesis analyses establishes that LtPNUTS, like a majority of PP1specific regulators, binds LtPP1-8e, in part, using a general RVxF and ɸ R -ΦΦ-F SLiMs.We noticed that a majority of the mutant PNUTS proteins tested here are overexpressed at a lower protein level than WT PNUTS protein (Fig. S8A).This is not observed for PP1 mutants overexpressed from the same plasmid (Fig. S8B), suggesting that PNUTS protein level is sensitive to mutations.However, reduction in the level of overexpressed PNUTS does not necessarily lead to reduction in PP1 binding in the co-IP.For example, R125A PNUTS is one of the lowest expressed PNUTS mutants (Fig. S8A) but showed comparable PP1 association as WT PNUTS (Fig. 3B).Potentially, the reduced expression of the mutant PNUTS protein is sufficient for saturation binding of available PP1, allowing co-IP of PP1 to the same extent as WT PNUTS.Furthermore, confirmation of the PNUTS-PP1 interface based on mutation of PNUTS residues is supported by mutation analysis of the corresponding binding pocket on PP1 where expression levels are not affected by mutagenesis.Therefore, the reduced protein expression level of certain mutant PNUTS protein does not affect our overall conclusions regarding the RVxFɸ R -ΦΦ-F motifs.

PP1-8e isoform-specific residues are involved in PNUTS binding
The mode of PP1-PNUTS interaction described previously, via the established RVxF-ɸ R -ΦΦ-F motif, is typical for a scaffolding function of regulatory proteins but likely does not affect selectivity toward PP1 isoforms.In fact, a majority of the PP1 residues characterized previously as involved in PNUTS-PP1 binding are not restricted to the PP1-8e isotype (Fig. S7), and thus, fail to explain the marked preference of PNUTS for PP1-8e.Therefore, we explored structural features present in LtPP1-8 that would confer specificity to LtPNUTS.As mentioned previously, hPP1 isoforms share a high sequence identity with differences mainly limited to their extremities, and some RIPPOs take advantage of these differences to interact selectively with specific PP1 isoforms.As we recently noted (64), an interesting characteristic of the PP1 isotypes in Leishmania is the diversity of their N-and C-terminal tails and the insertion of several short sequence elements specifically within the catalytic subunit of PP1-8e (Fig. S2).To test the PNUTS-PP1 interactions in Leishmania contribution of each of these unique PP1-8e characteristics to LtPP1-PNUTS association, we performed deletion and alanine mutagenesis (constructs used in this study are illustrated in Figure 4A).As shown in Fig. S8B, all PP1-8e mutants are overexpressed in Leishmania cells to similar levels as WT PP1-8e.Upstream of the highly conserved catalytic domain, LtPP1-8e has a 32 amino N-terminal extension (Fig. S2).Deleting the PP1-8e N-terminal region completely abolished PP1-PNUTS association by co-IP (Figs.4B and S9), suggesting that residues 1 to 32 of LtPP1-8e are essential for this interaction.Sequence differences between mammalian PP1-α and PP1-γ C-terminal 25-amino-acid tails are implicated in isoform-specific binding by ASPP2 (69) and MYPT1 (65).Similarly, LtPP1-8e has a unique extended C-tail of 25 amino acids (Fig. S2) that includes two residues (P352 and I360) that we have demonstrated previously as important for PNUTS-PP1 binding, potentially via stabilization of the Phe motif (Fig. 2F).Deleting the PP1-8e C-terminal tail significantly impacted PNUTS binding in vivo (Fig. 4B).While deletion of the 23 amino acid C-terminal extension of PP1-8e (CΔ23) leads to 80% loss in PNUTS binding, deletion of the final 11 amino acids (CΔ11) resulted in 30% reduction in PNUTS binding (Figs. 4B and   S9).The 12 amino acids of the C-terminal region between these two deletions include a predicted nine-amino acid alphahelix (354-362) rich in charged residues or polar residues (Fig. S2), potentially involved in electrostatic interactions with PNUTS.To test this idea, we did alanine scanning mutagenesis of two regions within this C-terminal helical region.Alanine substitution of four residues within first half of this helix in PP1-8e (354-358A) resulted in 50% reduction in PNUTS binding, and the 359 to 362A mutation of PP1-8e resulted in 80% reduction in PNUTS binding, similar to what we observed in the 23 amino acid deletion (CΔ23) (Fig. 4B).The I360A PP1 mutant led to a similar 80% reduction in LtPNUTS-PP1-8e associations, indicating I360 is a key residue within this C-terminal 359 to 362 helical region.Thus, the unique Cterminal tail of PP1-8e, in particular residues 359 to 362, and the first 32 amino acids at the N terminus are needed for PNUTS binding.
According to the LtPNUTS-PP1-8e model, while the N terminus of PP1-8e is unstructured and thus, difficult to understand how it is involved in isoform selective binding to PNUTS, the C terminus appears to provide additional stabilization to the Phe-binding pocket.PP1-8e, and other LtPP1

PNUTS-PP1 interactions in Leishmania
isotypes, have a Phe-binding pocket similar to the human PP1-PNUTS complex (Figs.2F and S5D and S7).However, the unique C terminus of PP1-8e provides additional residues (including P352 and I360) that may contribute to the Phebinding pocket.To examine this idea further, we determined the AlphaFold model for the LtPNUTS-PP1-1a complex (Fig. 5).LtPP1-1a appears to have a majority of the conserved residues for the RVxF, ɸ R , ɸɸ, and F motif-binding pockets as the hPNUTS-PP1 complex and the predicted LtPNUTS-PP1-8e model (Figs. 5 and S7).However, LtPP1-1a lacks the extended C terminus present in LtPP1-8e (Figs. 5 and S2) and, interestingly, is predicted to associate with LtPNUTS with the RVxF-ɸ R -ɸɸ motifs but not the Phe motif (Fig. 5).
An additional characteristic of PP1-8e is the insertion of three unique sequence motifs within the catalytic domain; a 26 amino acid insertion (residues 109-134) near the N terminus and two smaller ( 260 LPAGVD 265 and 310 DHK 312 ) insertions near the C terminus (Fig. S2).AlphaFold modeling shows the insertions are presented on the surface of PP1-8e at novel sites compared with the human PP1 structure and the LtPP1-1 isoform (Fig. S3, E and F).To test the significance of these regions, we performed deletion and alanine mutagenesis.Deletion of the 26 amino acid insertion in PP1-8e (109-134Δ) results in severely reduced ability (80%) to associate with PNUTS (Fig. 4B).The 26 amino acid region is rich in charged and polar residues that are conserved among Leishmania PP1-8e homologs, potentially involved in electrostatic interactions with PNUTS.To test this idea, we did alanine mutagenesis in three regions of the 26 amino acid insertion: GGVFG (109-114A), DKKR (116-121A), and SDDYS (126-134A) (Fig. 4A).While the 116 to 121A and 126 to 134A mutations had little effect on PNUTS binding, mutation of five residues in 109 to 114A resulted in 80% reduction in PNUTS binding, similar to the effect of deleting the entire 26 amino insert (Fig. 4B).Similar alanine mutagenesis was performed for the two smaller PP1 insertions: 260 LPGVD 265 and 310 DHK 312 (Fig. 4B).The results show that while alanine mutagenesis of 310 DHK 312 leads to a small decrease (20%) in PP1-PNUTS association, alanine mutagenesis of 260 LP 261 abolishes roughly 80% of PP1-PNUTS interaction (Figs.4B and S9).Alanine substitution of the remaining three residues of the 260 LPGVD 265 (263-265A) led to approximately 90% reduction in PNUTS binding, and D265A PP1 mutation only had a moderate effect on PP1-PNUTS interaction (Figs.4B and S9).Thus, unique sequences within the catalytic domain of PP1-8e, in particular residues 109 GGTVFG 114 and 260 LPAGV 264 , are needed for PNUTS interaction.Taken together, these results suggest that LtPNUTS can discriminate between different PP1 isoforms based on the PP1 N and C terminus and unique sequence motifs within the catalytic domain.As such, these regions might underlie the mechanism by which LtPNUTS shows preferential binding to PP1-8e.
PNUTS as a scaffold for the PJW/PP1 complex hPNUTS is a scaffolding protein in the human PTW/PP1 complex, binding Tox4 and Wdr82 with its N and C terminus regions, respectively, and PP1 via the centrally located RVxF motif (30).The hPNUTS is a 114 kDa protein (940 amino acids) with multiple identified protein domains (21).LtPNUTS, which lacks identifiable protein domains or motifs apart from the conserved PP1-interacting RVxF motif discussed previously, is much smaller at 28.6 kDa, consisting of 264 amino acids.To test if LtPNUTS similarly serves as a scaffolding protein and binds to Wdr82 and JBP3 with distinct domains, we overexpressed Pd-tagged PNUTS protein with various N-and C-terminal truncations and studied the interaction between PNUTS truncations and endogenously HAtagged JBP3-Wdr82 using co-IP (Fig. 6A).We find that fulllength PNUTS allows significant co-IP of both JBP3 and Wdr82 (Figs. 6C and S10), consistent with our previous studies of the PJW/PP1 complex in Leishmania and T. brucei (62).Confirming that the RVxF motif and PP1 binding are not required for JBP3 and Wdr82 association with PNUTS, mutation of the PP1 binding RVxF motif (RACA mutant) has little to no effect on Wdr82 or JBP3 binding to PNUTS (Figs. 6C  and S10).Interestingly, PNUTS proteins with three different N-terminal truncations (NΔ27, NΔ47, and NΔ75) are expressed at significantly lower levels than the full-length PNUTS control, and the major species run at lower molecular weights (MWs) than expected on SDS-PAGE gel (Figs.6B  and S8C).As an intrinsically disordered protein, hPNUTS is known to not run to the expected size on the SDS-PAGE gel (17), and we have characterized the altered mobility of TbPNUTS (62).Potentially, the deletion of an N-terminal sequence accentuates the disordered nature and altered mobility of the truncated LtPNUTS polypeptide.In this case, the major species represents the indicated truncated PNUTS protein.Alternatively, N-terminal deletions lead to PNUTS protein instability and further protein cleavage.While it is difficult to obtain accurate measurement of binding with such low protein expression in the parasite, it seems that PNUTS with varying lengths of N-terminal truncations still immunoprecipitated a significant level of Wdr82 or JBP3 compared with the negative control, although not to the same extent as WT PNUTS (as shown in Fig. S10).This is best represented by the NΔ75 PNUTS, with the highest level of expression among the N-terminally truncated PNUTS proteins (Fig. 6B).This would suggest that the N terminus of PNUTS is not essential for JBP3-Wdr82 binding.We noticed that similar N-terminal truncations of the PNUTS homolog in T. brucei does not result in decreased levels of expression (Fig. 6, D and E), allowing further studies of Wdr82-JBP3 association.To do this, we tagged JBP3 and Wdr82 in T. brucei with HA and Myc tags, respectively, and exogenously expressed protein A-tagged PNUTS via a Tet-inducible promoter.Supporting the LtPNUTS analysis, 72 amino acid deletion from the N terminus (NΔ72) tested in TbPNUTS had little to no effect on Wdr82-JBP3 binding (Fig. 6F).In contrast, while all C-terminal truncations of LtPNUTS are expressed at levels similar to full-length in both HA-tagged Wdr82 and JBP3 cell lines (Figs. 6B and S8C), even the smallest 23 amino acid deletion (CΔ23) had a negative effect on both Wdr82 and JBP3 binding to LtPNUTS (Figs. 6C and S10).While the 23 amino acid deletion led to complete loss of JBP3 binding, a small level of Wdr82 association remained that was subsequently lost upon further deletions of the C-terminal end (CΔ37 and CΔ66) (Figs. 6C and S10).Similar to LtPNUTS, C-terminal deletion of TbPNUTS (CΔ82) results in complete loss of JBP3-Wdr82 association (Fig. 6F).CΔ23 and CΔ37 PNUTS had a minor but insignificant effect on LtPNUTS-PP1-8e association (Figs.3B  and S6A), suggesting that PP1 binding into the complex is not dependent on Wdr82 interaction or JBP3 interaction.Taken together, the data suggest that the C terminus of LtPNUTS (and TbPNUTS) is required for binding both Wdr82 and JBP3 and that binding is independent of PP1 binding at the central RVxF motif.
Thus far, we have been unable to produce soluble recombinant protein in Escherichia coli to study the PJW/PP1 complex formation in vitro.Therefore, to further test the scaffold function of PNUTS in the complex and clarify its binding relationship with Wdr82 and JBP3, we utilized the RNAi system in T. brucei.This system would allow us to characterize, for example, the effect of PNUTS knockdown on the interaction between Wdr82 and JBP3 by co-IP.Therefore, we tagged JBP3 and Wdr82 with HA and Myc, respectively, in the PNUTS RNAi cell line.We found that knockdown of TbPNUTS (with mRNA ablated roughly 40% by RT-qPCR analysis) leads to significantly decreased protein levels of both Wdr82 and JBP3 (Fig. 7A).JBP3 is particularly sensitive to PNUTS knockdown, with the majority (>90%) of JBP3 being lost within 24 h of PNUTS RNAi induction.On the other hand, Wdr82 is less affected with 50% reduction in protein level within 24 h, with levels decreasing to 75% reduction upon 72 h postinduction.While this effect does prevent the analysis of JBP3-Wdr82 interactions by co-IP, it is consistent with PNUTS knockdown in human embryonic kidney 293 cells, which leads to loss of both Tox4 and Wdr82 (30), and further supports a scaffold function for LtPNUTS.Interestingly, knockdown of Wdr82 (50% ablation of mRNA) by RNAi similarly leads to a significant reduction in HA-tagged JBP3 protein level but does not affect Myc-tagged PNUTS protein level (Fig. 7B).On the other hand, ablation of JBP3 by RNAi does not lead to any change in PTP-PNUTS or Myc-Wdr82 protein levels (Fig. 7C).PNUTS and Wdr82 association was analyzed by anti-protein A co-IP with or without PNUTS-PP1 interactions in Leishmania JBP3 RNAi induction.The result shows that JBP3 knockdown does not affect PNUTS-Wdr82 co-IP (Fig. 7D), indicating that Wdr82 binds to PNUTS independently of JBP3.The results collectively further suggest that JBP3 associates into the complex via binding to Wdr82, and that complex integrity is essential to Wdr82 and JBP3 protein stability.
In setting up the PP1-PNUTS co-IP analysis and overexpressing LtPNUTS from a plasmid in cells expressing a tagged version of PP1-8e from the endogenous locus, we noticed that transfection with the PNUTS-expressing plasmid led to a 50% decrease in PP1-8e abundance (Fig. 8, A and B).PNUTS overexpression has no effect on PP1-7 protein level, indicating an isotype-specific effect.Interestingly, the effect of PNUTS overexpression on PP1-8e level is not dependent on PP1 binding, since this occurs even upon overexpression of the PNUTS defective for PP1 association, such as RACA PNUTS (Fig. 8A), L111A PNUTS , F118A PNUTS , or R125A PNUTS (Fig. S11A).However, overexpression of C-terminal truncated versions of PNUTS (CΔ23 or CΔ37) did not lead to reduced PP1 protein level to the same extent as other tested PNUTS mutants, indicating that the effect is dependent on the ability of PNUTS to bind Wdr82 and/or JBP3.Furthermore, while PNUTS overexpression had no effect on Wdr82 protein abundance, in a few clones, it led to a shift in mobility of Wdr82 on the SDS-PAGE gel (Figs.8C and S11B).Endogenously, HA-tagged Wdr82 has a predicted MW of 48 kDa, and a majority of the protein runs slightly above the 50 kDa protein ladder marker with a minor lower MW species sometimes visible just below the marker (Fig. 8C).We observed that WT and RACA PNUTS overexpression caused the population of Wdr82 to shift to the lower MW species (Fig. 8C) in two of nine and six clones analyzed, respectively (Fig. S11B).The finding that expression of WT LtPNUTS and the RACA mutants had similar effects on the altered mobility of Wdr82 in vivo indicates that the effect is independent of the ability of PNUTS to bind PP1-8e.Treatment of cell lysates with or without calf intestinal phosphatase and conditions we have demonstrated to dephosphorylate pol II (64) had no effect on Wdr82 gel mobility (data not shown), excluding the possibility that the observed shift in Wdr82 is due to changes in phosphorylation status.The AlphaFold-predicted Wdr82 structure indicates that the N terminus (1-27) of Wdr82 has low prediction confidence, followed by potentially solventexposed 34 FYTGIN 39 sequence susceptible for cleavage by chymotrypsin and thermolysin (Fig. S12A), suggesting a disordered N terminus region prone to proteolytic cleavage.Preliminary MS analyses to confirm the processing of the lower MW form of Wdr82 have been inconclusive.Regardless While it is unclear why LtPNUTS overexpression results in these effects on Wdr82 and PP1-8e, the data further support a scaffold function for PNUTS in the PJW/PP1 complex.Furthermore, as predicted based on previous studies of PJW/ PP1 complex function in vivo in Leishmania and T. brucei, these defects correlate with defects in pol II transcription termination (Fig. 8, E and F).Strand-specific RT-quantitative PCR (qPCR) shows that compared with the parental cells (WT), cells that overexpress WT or RACA PNUTS accumulated nascent transcripts downstream of the analyzed transcription termination site (Fig. 8, E and F).As a positive control, cells treated with DMOG, a drug that inhibits base J synthesis and induces transcription termination defects in Leishmania (58), also accumulated readthrough transcripts.The effect of PNUTS overexpression and corresponding decreased levels of PP1-8e on pol II termination seen here is consistent with the recently characterized role of PP1-8e in pol II phosphorylation and transcription termination in Leishmania (64).To address the impact of Wdr82 cleavage that occurs following overexpression of PNUTS to the termination defects measured here, we repeated the analysis using HAtagged Wdr82 cells and examined the degree of readthrough in cells with or without Wdr82 cleavage (Fig. S11C).Compared with WT cells, C-terminal tagging of Wdr82 leads to increased readthrough transcripts (Fig. S11D), possibly indicating an impaired function for Wdr82-HA, similar to what we observed Cell lysates were analyzed by Western blot with anti-protein, anti-HA, and anti-EF1a.Anti-EF1A serves as a loading control.PP1-tagged control cell lines not transfected with the PNUTS expression plasmid are indicated by the C for control.B, HA-tagged PP1-1 and PP1-8 band intensities were quantified by densitometry.The bar graph represents the mean ± SD of PP1-1 or PP1-8 protein level relative to control cells with no overexpression of PNUTS (WT, black bar; RACA mutant, gray bar).C, Wdr82 was tagged at its endogenous loci with HA tag with or without WT PNUTS overexpression, and cell lines were analyzed by Western blot with anti-protein A and anti-HA.A nonspecific product recognized by the anti-protein A antibody is indicated by an asterisk and serves as a loading control.Shown here are results from two clones (Cl2 and Cl4).See Fig. S11B for results from multiple clones.D, cell extracts from the cell lines in C were purified by anti-protein A affinity resin and analyzed by Western blot with anti-protein A, anti-HA, and anti-EF1a.E and F, effect of PNUTS overexpression on polymerase II transcription termination.E, diagram of the termination site at the end of a polycistronic gene array on chromosome 22 illustrating the strand-specific RT-qPCR analysis of readthrough defects.The dashed line indicates the readthrough transcripts past the transcription termination site (TTS) that accumulate following a defect in polymerase II termination.The location of primers for RT and qPCR (A and B) are indicated by the small arrows.F, RT-PCR analysis for the readthrough transcripts.Strand-specific cDNAs were synthesized from RNAs extracted from cells treated with the indicated concentrations of DMOG or from cells with either WT or RACA mutant PNUTS overexpression using primer RT.Fold change of the readthrough transcripts relative to the WT ± SD is based on qPCR analysis with primer A and B, normalized to tubulin RNA.cDNA, complementary DNA; HA, hemagglutinin; LtPNUTS, Leishmania tarentolae PNUTS; PP1, phosphoprotein phosphatase 1; qPCR, quantitative PCR.
for C-terminally tagged PP1-8e in L. major (64).However, we see no difference in the degree of readthrough transcription stimulated by PNUTS overexpression in cells that resulted in Wdr82 cleavage or not (Fig. S11C).Therefore, altered processing of Wdr82 in the PNUTS-expressing cell lines had no additional negative effect on pol II transcription termination.Taken together, similar to the termination defects measured in the Leishmania PP1-8e KO (64), alterations in PJW/PP1 complex formation and levels of PP1-8e following PNUTS overexpression lead to defects in pol II transcription termination.

Discussion
RIPPOs are essential regulators of PP1 substrate specificity and cellular localization.RIPPOs share little sequence or overall structural identity but use short SLiMS (5-8 amino acids long) that are combined within an unstructured domain to render RIPPOs' high affinity to PP1 (13,16,76).According to this PP1 binding code (13), the unique combination of PP1binding motifs (SLiMS) allows RIPPOs to interact with PP1 in a highly specific manner.PNUTS-PP1 complex involved in regulating transcription termination is conserved from mammalian to yeast cells (30,80,81), and recent studies indicate that the binary interaction and function also exists in trypanosomatids (62,63).Purification of the PNUTS complex from L. tarentolae identified a specific interaction with the PP1-8e isoform among the eight encoded in the Leishmania genome (62,63) suggesting that PNUTS selectively targets PP1-8e to the complex.However, the isoform selectivity of PP1 targeting in intact parasites had not been established.Here, we show that PNUTS selectively targets PP1-8e to the complex, and targeting requires both the nonisoform selective canonical PP1-binding motif and additional domains located throughout the PP1-8e sequence.Previous studies have shown that LtPNUTS is a highly disordered protein, and mutation of its putative RVxF motif disrupts its interaction with LtPP1-8e, indicating its importance in PP1-PNUTS interaction (62).In the current study, we used AlphaFold to predict the LtPNUTS-PP1-8e holoenzyme complex and identified additional SLiMs beyond the canonical RVxF motif that are typically difficult to recognize based on sequence analysis alone, because they are short and highly degenerate.Our predicted LtPNUTS-PP1-8e holoenzyme complex and biochemical studies reveal that LtPNUTS binds PP1-8e using an extended RVxF-ɸ R -ɸɸ-Phe motif used by several other RIPPOs including the hPNUTS-PP1 complex.Furthermore, our studies suggest that additional interactions are involved that are atypical compared with any previously studied regulator.These include unique sequences at the ends and within the catalytic domain of PP1-8e that modulate isoform-specific recruitment as well as increasing overall stability of the holoenzyme complex.
Compared with the other seven LtPP1 isoforms, LtPP1-8e has a long and unique C-tail with residues 354 to 362 predicted to form an α-helix secondary structure and the remaining 12 residues (363-374) unstructured.The model indicates that two residues within the C-terminal α-helix (P352 and I360) accommodate the Phe motif in LtPNUTS (F118 PNUTS ).Supporting this model, C-terminal deletion and alanine scanning mutagenesis of PP1-8e indicate the importance of residues 359 to 363 of this alpha-helical region in LtPNUTS-PP1-8e interactions.The strong negative effects of the F118A PNUTS and I360A PP1-8e mutants on complex formation further support this idea.Therefore, although the residues that constitute the conserved RVxF, ɸ R , ɸɸ, and F motif-binding sites are present in all LtPP1 homologs, the PP1-8e C-tail may provide a stabilizing force to the PNUTS Phe SliM and represent a significant component of isoform selectivity.While human PP1 isoforms have a short divergent N terminus (6 amino acids), a role of the N terminus in RIPPO binding or isoform selection has not been described in other systems.Deletion of the LtPP1-8e N terminus (1-32) leads to a dramatic decrease in LtPP1-PNUTS association, indicating its significance, but how it mediates association with LtPNUTS is unclear.The low confidence of the AlphaFold structure for this region makes it difficult to understand how the N terminus is involved in isoform selectivity.BLASTP search against the National Center for Biotechnolgy Information nonredundant protein sequence database using as query either the N-terminal (residues 1-32) or C-terminal (residues 241-264) sequences of L. tarentolae PP1-8, gave no significant similarity to any nontrypanosomatid protein.We also now identify unique inserts within the PP1-8e catalytic region ( 260 LPAGVD 265 and 310 DHK 312 and the 26 amino acid 109-134 motif) where deletion or alanine mutagenesis completely abolishes or significantly decreases PP1-PNUTS association.Mutagenesis analysis has indicated residues 260 LPGV 264 and 109 GGTVFG 114 as key residues within these inserted motifs, essential for PNUTS-PP1-8e complex formation.Overall, the results suggest that polymorphisms within the PP1-8e catalytic domain and N and C terminus are essential for PNUTS binding.As such, these regions might underlie the mechanism by which LtPNUTS shows preferential binding to PP1-8e.However, the position/orientation of the LtPP1-8 polymorphisms were, in some cases, predicted with low confidence by AlphaFold.Therefore, how they contribute to PNUTS association cannot be easily inferred.Interestingly, the LtPNUTS-PP1-8e model predicts two of these unique regions of PP1-8e (C-terminal and the 26 amino acid internal motif) to be in close proximity to regions 116 to 121 of PNUTS that includes the Phe SLiM (F118) (Fig. 5).The predicted role of the C-terminal region forming an essential part of the Phe-binding pocket is discussed previously.Within the 26-amino acid insertion polymorphism in the PP1-8e catalytic domain, 113 FG 114 is predicted to be in close proximity of Y117 PNUTS (Fig. 5).The importance of this region is supported by our co-IP studies where alanine mutagenesis of residues 109 to 114 of PP1-8e, in contrast to mutagenesis of the remaining part of this 26-amino acid insert, significantly affected PP1-PNUTS association (Fig. 4).The absence of both these regions in LtPP1-1 could therefore explain the altered Phe motif interactions in the PNUTS-PP1-1 holoenzyme model (Fig. 5).Taken together, the data support two unique PNUTS-PP1 interactions in Leishmania regions of the PP1-8e isotype making critical interactions with PNUTS Phe motif that may help explain the isotype-specific stable association of the LtPNUTS-PP1-8e complex.
As mentioned previously, our model predicts that LtPNUTS binds PP1-8e via RVxF-ɸ R -ɸɸ-Phe motifs, similar to the hPNUTS-PP1 complex.hPNUTS, like most RIPPOs, is able to bind all PP1 isoforms.Presumably, the additional contacts with PP1-8e-specific sequences we describe here allow isoformspecific binding of LtPNUTS.However, the conservation of residues involved in interactions with the extended RVxF motif in all eight LtPP1 isoforms (Fig. S7) suggests, as described for mammalian isoform-specific RIPPOs, some low level of PNUTS binding in vivo by the remaining PP1 isoforms.This characteristic would explain the ability of other PP1 isoforms to functionally compensate for the loss of PP1-8e in Leishmania (64).The PNUTS-PP1-8e complex has been shown to regulate transcription termination in Leishmania potentially through PP1-8e-mediated dephosphorylation of pol II CTD (64).KO of PP1-8e in L. major causes transcription termination defects, which can be rescued, albeit to a limited degree, by overexpression of PP1-1 or PP1-7 (64).Both proteins have conserved residues constituting the RVxF motif-binding pocket and are predicted to interact with PNUTS through a majority of the extended RVxF motif.However, they do lack the PP1-8e unique motifs we demonstrate as critical for the PNUTS-PP1-8e co-IP, including the C-tail and the 113 FG 114 residues we predict essential for stable Phe SLiM binding and thus increase overall stability of the holoenzyme complex.Therefore, it is conceivable that while the enhanced affinity for PNUTS allows LtPP1-8e to outcompete other PP1 isotypes for PNUTS binding in the WT cell, in its absence, the remaining PP1 isotypes can form unstable or transient interaction with PNUTS to partially compensate for the loss of LtPP1-8e.Similarly, the lack of these LtPP1-8e-specific polymorphisms essential for the PNUTS-PP1 co-IP in all eight TbPP1 isoforms may explain the failure to identify a PP1 isoform associated with PNUTS in T. brucei.While TbPNUTS has a conserved RVxF motif, purification of PNUTS from T. brucei cells identified the Wdr82 and JBP3 homologs but no catalytic PP1 component (62).Knockdown of TbPNUTS, TbJBP3, or TbWdr82 led to defects in pol II transcription termination (62).Thus, we predict that a similar mechanism of pol II termination involving PP1-mediated pol II dephosphorylation via the PJW/PP1 complex exists in T. brucei as we characterized in Leishmania.The inability to demonstrate TbPNUTS-PP1 binding using co-IP suggests that the two proteins do not interact directly or interact in such a transient manner or a weak manner that PP1 dissociates from the complex during affinity purification process.Although we cannot exclude the possibility that the T. brucei PNUTS complex functions without the association of the catalytic PP1 component, the presence of an RVxF motif in TbPNUTS and lack of the polymorphisms we demonstrate as critical for stable LtPNUTS-PP1-8e interactions in the co-IP in all T. brucei PP1 isoforms support our model.
The PNUTS-PP1 complex in mammalian cells is found associated with structural factors Wdr82 and Tox4, forming the PTW/PP1 complex (30).hPNUTS is a large protein with multiple domains; including the RVxF motif (KSVTW) for PP1 binding and the N-terminal TFIIS-like domain required for Tox4 binding (21).hPNUTS serves as a scaffolding protein in the PTW/PP1 complex, and its ablation in human embryonic kidney 293 cells causes a complete loss of Tox4 and a significant reduction in Wdr82 protein level (30).LtPNUTS, on the other hand, is much smaller with no recognizable domains other than the central RVxF motif and extended SLiMs identified here involved in PP1 binding.For the first time, we now describe that PNUTS performs similar scaffolding function in the PJW/PP1 complex in kinetoplastids, representing a key regulator of complex formation/stability.We show that ablation of TbPNUTS leads to a complete loss of JBP3, the counterpart of Tox4, and a 50% reduction in Wdr82 protein level.Moreover, overexpression of LtPNUTS leads to reduction in PP1-8e level and processing of Wdr82 (discussed later).We demonstrate that JBP3 and Wdr82 bind the C terminus of LtPNUTS and TbPNUTS, independent of PP1 binding.The LtPNUTS defective for PP1 binding (RACA) has no detectable loss of binding to Wdr82 or JBP3, and C-terminal mutants, unable to bind Wdr82-JBP3, bind PP1 with WT level of efficiency.While there is no apparent interaction between Wdr82-JBP3 and PP1, C-terminal deletions had a significant negative effect on both Wdr82 and JBP3 association, suggesting interdependence of PNUTS binding by Wdr82 and JBP3.Alternatively, structural alteration in PNUTS caused by C-terminal deletion could explain the negative effects on the binding of both factors.While we are not able to rule this out, the effect would have to be localized to the C terminus as the deletions have no measurable effect on PP1 binding.Furthermore, the use of RNAi in T. brucei supports the interdependence of PNUTS binding by Wdr82 and JBP3, where primary interactions between PNUTS and Wdr82 regulate JBP3 binding.While the ablation of JBP3 has no effect on Wdr82 levels or interactions with TbPNUTS, Wdr82 ablation leads to specific decrease in JBP3.Presumably, in T. brucei, the stability of JBP3 depends on interactions with Wdr82 (and PNUTS).Additional work is needed to fully elucidate specific interactions involved in PJW/PP1 complex formation.However, taken together, the results from the in vivo studies suggest that PNUTS is a scaffolding protein in the PJW/PP1 complex that mediates the independent binding of PP1 and Wdr82, and JBP3 association with the complex depends, at least partially, on interactions with Wdr82.
The effects of overexpression of LtPNUTS, and ablation of TbPNUTS, on the PJW/PP1 complex, support its key role as a scaffolding factor for the complex and indicate the concentration of PNUTS is finely tuned in vivo in kinetoplastids.Presumably, overexpression of PNUTS in Leishmania leads to stoichiometric imbalance that affects PJW/PP1 complex formation and stability of associated factors, including PP1-8e.LtPNUTS overexpression had no detectable effect on levels of PP1-7 isotype that is not associated with the PJW/PP1 complex.Interestingly, the specific decrease of LtPP1-8e protein level is not dependent on the ability of LtPNUTS to bind PP1 but on its ability to bind Wdr82-JBP3.
Overexpression of C-terminally truncated LtPNUTS (CΔ23 and CΔ37) with significantly lower affinity to Wdr82-JBP3 did not lead to a loss of LtPP1-8 as seen following overexpression of WT PNUTS or PP1-8e binding mutants.These results suggest that the integrity of the PJW-PP1 complex is important for PP1-8e protein level.Excess LtPNUTS (regardless of its ability to bind PP1) could lead to decreased levels of Wdr82-JBP3 (or other unidentified cofactors) available to PNUTS-PP1 to form a stable functional complex.The shift in MW of Wdr82 in a percentage of clones overexpressing PNUTS is currently unclear.We have addressed the possibility of a shift because of phosphorylation and proposed that it represents proteolytic cleavage at the unstructured N terminus.Further work is needed to understand the effect of LtPNUTS overexpression on Wdr82 processing.However, this effect is not linked to the ability of PNUTS to bind PP1.While it is unclear if this altered Wdr82 processing affects cellular function, it has no apparent consequence on the ability of Wdr82 to bind PNUTS.
Overall, the current study identified PP1-binding motifs on LtPNUTS and discovered novel sequences on PP1-8e that could confer isoform selectivity, thereby enhancing our understanding of the PP1 binding code modulating the interaction between PP1 and PP1-interacting proteins.Moreover, our results indicate the conserved role of PNUTS as a scaffolding protein and that its protein level is critical for PJW/PP1 complex stability.The finding that PJW/PP1 complex defects associated with PNUTS overexpression led to readthrough transcription at pol II termination sites provides additional support for the involvement of the complex in the mechanism of pol II transcription termination in kinetoplastids.Additional studies regarding the PJW/PP1 complex formation will help dissect the novel pol II transcription cycle in these divergent eukaryotes.

Protein structure modeling with AlphaFold2
The predicted models were generated using the AlphaFold2 algorithm (82) via the ColabFold platform (83).In the open source Google CoLabFold platform, sequences were pasted in the query sequence box and the complex prediction was run with the default settings.The AlphaFold model was represented by five top-scored conformations along with estimates of prediction reliability (pLDDT), as described elsewhere (82).The protein models were analyzed and displayed with UCSF ChimeraX, version 1.5 (84).

DNA constructs and cell line generation
Endogenous HA tagging in L. tarentolae.A background L. tarentolae cell line was established to express Cas9 and T7 polymerase following transfection with PacI-digested pTB007 plasmid (59) as previously described (64).To tag the endogenous PP1-8e, PP1-7d, PNUTS, JBP3, or Wdr82 locus with 6xHA tag, the Cas9/T7-expressing cell line was transfected with guide RNAs and donor fragments, as previously described (64).Single guide RNAs (sgRNAs) were designed with Leish-GEdit.Appropriate DNA fragments were generated via PCR using sgRNA primers and product transfected to cells to generate guide RNAs in vivo.sgRNA primer sequences are provided: GAAATTAATACGACTCACTATAGGGCTTT GGAGAAGTCTGGCAAGGTTTTAGAGCTAGAAATAGC (PNUTS sgRNA); GAAATTAATACGACTCACTATAGGG ACGCGTAAGGATCTAAAGAGTTTTAGAGCTAGAAAT AGC (PP1 sgRNA); GAAATTAATACGACTCACTA-TAGGGACGTCCTCGTAGACAAGTGGTTTTAGAGCTAG AAATAGC (Wdr82 sgRNA); GAAATTAATACGACTCACT ATAGGGGAACGAAAGCACACAGCAGGTTTTAGAGCTA GAAATAGC (JBP3 sgRNA); AAAAGCACCGACTCGGTGC-CACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAA CTTGCTATTTCTAGCTCTAAAAC (universal reverse).The donor fragments were amplified via PCR from the pGL2314 plasmid with 30-nucleotide homology flanks specific to the target loci.For overexpressing C-terminal-tagged proteins in L. tarentolae, the open reading frame of LtPNUTS or LtPP1 was PCR amplified without a stop codon and inserted into the pSNSAP1 vector at the BamH1 and Xba1 sites as previously described (64).The obtained constructs were referred to as PNUTS-Pd or PP1-Pd.The desired PP1 mutants or PNUTS mutants were generated by oligonucleotide-mediated sitedirected mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit; Agilent Technologies) following the manufacturer's instructions.All final constructs were sequenced prior to electroporation.PNUTS-Pd plasmid was transfected into the PP1-PNUTS-PP1 interactions in Leishmania 8e-HA cell line and WT L. tarentolae and the PP1-Pd plasmid transfected into the PNUTS-HA and WT cell line.
Endogenous tagging in T. brucei.For tagging the 3 0 end of the TbPNUTS, Wdr82, and JBP3 with 3xHA or Myc tag in T. brucei cells, a PCR-based approach was used with the pMOTag4H or pMOTag3M vectors as described (62).For tagging PNUTS with the PTP tag in T. brucei, the 3 0 end of TbPNUTS was cloned in the ApaI and Not1 sites of the Pc-PTP-Neo vector (86) where the neomycin resistance drug marker was replaced with a blasticidin resistance drug marker.The vector was then linearized by restriction enzyme digestion within the 3 0 end of the TbPNUTS gene and used in transfection.For tetracycline-regulated expression of PNUTS in T. brucei, the ORF with a C-terminal PTP tag was amplified by PCR and cloned into the HindIII and BamH1 sites of the pLew100V5 plasmid.The final construct (PNUTS-PTP-pLew100) was linearized with NotI prior to transfection.To induce PNUTS expression, tetracycline was added at 2 μg/ml.All final constructs were sequenced prior to electroporation.Primer sequences used are available upon request.

RNAi analysis
Conditional silencing of PNUTS, JBP3, and Wdr82 in T. brucei BF SMC was performed as previously described (62).Briefly, a fragment of the representative ORF was integrated into the BamHI site of the p2T7-177 vector.I-SceI-linearized p2T7-177 constructs were transfected into BF SMC for targeted integration into the 177 bp repeat locus.All final constructs were sequenced prior to transfection.RNAi was induced with 2 μg/ml tetracycline, and growth was monitored daily in triplicate.

Co-IP
About 5 × 10 8 of L. tarentolae cells were lysed in lysis buffer, and Pd-tagged protein was affinity purified using 50 μl immunoglobulin G Sepharose beads as previously described (37).After incubation with cell extract for 4 h at 4 C, the immunoglobulin G beads were washed three times in 10 ml PA-150 buffer.After the final wash, the beads were boiled for 5 min in 1× SDS-PAGE sample buffer.Samples were run on 10% SDS-PAGE and transferred to nitrocellulose membrane for Western blotting with anti-protein A and anti-HA antibodies.About 1.2 × 10 8 of T. brucei cells expressing PTPtagged protein was used for co-IP as described previously.Samples were run on 10% SDS-PAGE and transferred to nitrocellulose membrane for Western blotting with antiprotein A, anti-HA, and anti-Myc antibodies.

Strand-specific RT-qPCR
Total RNA was extracted using the Tripure Isolation Reagent (Roche).To synthesize complementary DNA (cDNA), 1 μg of Turbo DNase-treated total RNA (ThermoFisher) was reverse-transcribed with strand-specific oligonucleotides using Superscript III kit (ThermoFisher), following the manufacturer's instructions.Quantification of the resulting cDNA was conducted using an iCycler with an iQ5 multicolor real-time PCR detection system (Bio-Rad).Triplicate cDNA samples were assessed and normalized against tubulin cDNA.For the qPCR, a 15 μl mixture containing 5 μl of cDNA, 4.5 pmol each of sense and antisense primers, and 7.5 μl of 2× iQ SYBR Green super mix (Bio-Rad Laboratories) was used.Standard curves were generated for each gene using fivefold dilutions of a known quantity (100 ng/l) of WT genomic DNA.The quantities were determined using the iQ5 optical detection system software.

Figure 3 .
Figure3.LtPNUTS binds LtPP1-8e using an extended RVxF-Φ R -ɸ ɸɸ ɸ-Phe motif.A, co-IP assay of PP1-8e binding to PNUTS and their derivatives.PNUTS IP; PP1-8e was endogenously tagged with HA tag, and WT or indicated PNUTS mutants with Pd tag were overexpressed from the pSNSAP1 vector.Cell extracts from the indicated cell lines were purified by anti-protein A affinity resin and analyzed by Western blot with anti-protein A and anti-HA.EF1α provides a loading control and negative control for the IP.PP1 IP; PNUTS was endogenously tagged with HA tag, and WT or indicated PP1-8e mutants with Pd tag were overexpressed from the pSNSAP1 vector.The levels of PNUTS pulled down in the PP1 IP were assessed by Western blot as described previously.Additional PNUTS and PP1-8e mutations analyzed by co-IP are shown in Fig.S6.B and C, the relative binding (%IP) between PNUTS and PP1-8e (WT and variants) determined by the ratio of the band intensity of IP to that of In.B, PNUTS-Pd IP.The bar graph represents the mean ± SD from three independent experiments, with the percent IP of PP1 using WT PNUTS set to 1.The PNUTS binding motif that corresponds to the residue tested, according to the model in Figure2, is indicated at the bottom of the graph.CΔ23 and CΔ37 refer to C-terminal truncations of PNUTS described in Figure4.C, PP1-8e-Pd IP.The percent IP of PNUTS from the PP1 pull-down (WT and mutants) was determined as in B. The PNUTS-binding pocket represented by each residue of PP1 is indicated below.F307, I346, and R314 are predicted to be key residues of both the RVxF and Φ R -binding pocket of PP1-8e.Co-IP, coimmunoprecipitation; FT, flow through or nonbound supernatant; HA, hemagglutinin; In, input (equivalent to the amount of protein added to the IP reaction mixture); IP, 100% of the precipitated immunocomplexes; LtPNUTS, Leishmania tarentolae PNUTS.

Figure 4 .
Figure 4. Noncanonical sites on PP1-8e are essential for PNUTS binding.A, PP1-8e constructs.The conserved PP1 catalytic domain is shown as gray boxes.Isoform differences between LtPP1-8e and LtPP1a and hPP1 are indicated by the lines within the catalytic domain and at the N and C terminus of LtPP1-8e.Sequence is provided for all these regions in LtPP1-8e, except the N terminus, and residues subjected to alanine mutagenesis (red) or deletion are indicated.Residues in the predicted α-helix at the C terminus are indicated by the line above the sequence.B, the percent IP of PNUTS from the PP1 pulldown (WT and indicated variants) was determined as described in C. The bar graph represents the mean ± SD from three independent experiments, with the %IP from WT PP1 set to 1. See Fig. S9.PNUTS, PP1 nuclear targeting subunit; Pol II, RNA polymerase II; PP1, phosphoprotein phosphatase 1.

Figure 5 .
Figure 5.The predicted LtPNUTS-PP1-1a structure.A, the predicted holoenzyme structure of LtPNUTS (pink ribbon) and LtPP1-1a (white surface).The RVxF-binding pocket (red) and ΦΦ-binding pocket (cyan) are shaded on PP1 surface.B, structural comparison of the PP1-binding domains of LtPNUTS in complex with PP1-8e (green) or PP1-1a (pink).Structure of hPNUTS (blue) bound to hPP1 is also shown.C, close-up view of F118 PNUTS (pink) in complex with LtPP1-1a (white surface, left) or (green) with LtPP1-8e (white surface, right).F118 and Y119 of PNUTS are shown as sticks and labeled.Conserved Phe-binding pocket residues (according to the hPNUTS-PP1 structure) are shown in blue sticks.LtPNUTS-PP1-8e complex (right).The C terminus of LtPP1-8e is shown in red carton, and key residues (P352 and I360) shown to be important for LtPNUTS binding are shown in sticks and labeled.Residues 109 GGTVFG 114 within the PP1 catalytic motif and important for PNUTS binding are also shown in red, and residue 113 F is shown as sticks and labeled.hPNUTS, human PNUTS; LtPNUTS, Leishmania tarentolae PNUTS; PNUTS, PP1 nuclear targeting subunit; PP1, phosphoprotein phosphatase 1.

Figure 6 .
Figure 6.JBP3 and Wdr82 bind to the C terminus of PNUTS.A-C, coimmunoprecipitation analysis of LtPNUTS and Wdr82 and JBP3.A, schematic diagram of PNUTS depicting PP1-specific RVxF SLiM (RVCW).Constructs used in this study are illustrated.B, Western blot showing the protein expression of Pd-tagged PNUTS (WT and truncation mutants) in JBP3-HA tagged Leishmania tarentolae cells.Dots indicate the proposed products representing the indicated N-terminal truncations.Anti-EF1A Western blot is shown as a loading control.C, analysis of JBP3-Wdr82 binding to PNUTS by co-IP.PNUTS truncations (C) or mutants (D) were tested for interaction with HA-tagged Wdr82 or JBP3 by co-IP analysis.%IP of WT Wdr82 and JBP3 by PNUTS were set to 1, and relative %IP of the indicated mutants was determined as described in Figure 3.The bar graph represents the mean ± SD from three independent experiments.See Fig. S10.D-F, co-IP analysis of TbPNUTS and Wdr82 and JBP3.D, schematic representation of the TbPNUTS truncations.The putative RVxF motif is indicated by a gray box.E, JBP3 and Wdr82 were endogenously tagged with HA and Myc tags, respectively.The protein expression of the indicated TbPNUTS was induced by addition of tetracycline (Tet) for 24 h, and lysates were analyzed by Western blot with anti-protein A, anti-HA, anti-Myc, or anti-EF1a.EF1a serves as a loading control.F, lysates of the indicated cell lines with or without tetracycline induction were purified by anti-protein A affinity resin and analyzed by Western blot with anti-protein A, anti-HA, anti-Myc, and anti-EF1a antibodies.Asterisk indicates the IgG cross-reactive signal in the IP fraction from anti-Myc.Protein A purification results in low background JBP3-HA signal in the absence of protein A-tagged PNUTS.%IP is quantified from two replicates and shown below for the corresponding cell lines.co-IP, coimmunoprecipitation; FL, full-length (WT) TbPNUTS; HA, hemagglutinin; IgG, immunoglobulin G; LtPNUTS, Leishmania tarentolae PNUTS; PNUTS, PP1 nuclear targeting subunit; PP1, phosphoprotein phosphatase 1; SLiM, short linear motif; TbPNUTS, Trypanosoma brucei PNUTS.

Figure 7 .
Figure 7. TbPNUTS functions as a scaffold factor.RNAi knockdown of the Trypanosoma brucei PJW complex components.Endogenous loci of the indicated genes were tagged with HA, PTP, or Myc tags.Cells were then transfected with the indicated RNAi construct, and knockdown of PNUTS (A), Wdr82 (B), or JBP3 (C) was induced by tetracycline (Tet) addition.Cell lysates were collected at the indicated time points and analyzed by Western blot with antiprotein A, anti-HA, or anti-Myc.Anti-EF1a serves as a loading control.Bands were quantified by densitometry.The bar graphs represent the mean ± SD from three independent experiments for the indicated protein level relative to protein level prior to the induction of RNAi.The bar graphs on the right, in A and B, show depletion of the RNAi targeted transcript upon Tet induction by qRT-PCR analysis and represent the mean ± SD from three independent experiments, with levels at uninduced (Tet-) set to 1. D, effect of JBP3 knockdown on PNUTS-Wdr82 binding.JBP3 RNAi was induced for 48 h, and PNUTS-PTP was purified from cell extracts by anti-protein A affinity resin and analyzed by Western blot.The %IP of Wdr82 by PNUTS IP with or without JBP3 RNAi induction was determined as described in Figure 3.The bar graph on the right represents the mean ± SD from three independent experiments, with the %IP from the uninduced cells set to 1. HA, hemagglutinin; qRT-PCR, quantitative RT-PCR; TbPNUTS, Trypanosoma brucei PNUTS.

Figure 8 .
Figure 8. LtPNUTS overexpression alters PP1 and Wdr82 stability and transcription termination.A-D, effect of PNUTS overexpression on PP1 and Wdr82.A, PP1-1 or PP1-8 was tagged with HA tag at its endogenous loci, and either WT or RACA mutant PNUTS protein was exogenously overexpressed.Cell lysates were analyzed by Western blot with anti-protein, anti-HA, and anti-EF1a.Anti-EF1A serves as a loading control.PP1-tagged control cell lines not transfected with the PNUTS expression plasmid are indicated by the C for control.B, HA-tagged PP1-1 and PP1-8 band intensities were quantified by densitometry.The bar graph represents the mean ± SD of PP1-1 or PP1-8 protein level relative to control cells with no overexpression of PNUTS (WT, black bar; RACA mutant, gray bar).C, Wdr82 was tagged at its endogenous loci with HA tag with or without WT PNUTS overexpression, and cell lines were analyzed by Western blot with anti-protein A and anti-HA.A nonspecific product recognized by the anti-protein A antibody is indicated by an asterisk and serves as a loading control.Shown here are results from two clones (Cl2 and Cl4).See Fig. S11B for results from multiple clones.D, cell extracts from the cell lines in C were purified by anti-protein A affinity resin and analyzed by Western blot with anti-protein A, anti-HA, and anti-EF1a.E and F, effect of PNUTS overexpression on polymerase II transcription termination.E, diagram of the termination site at the end of a polycistronic gene array on chromosome 22 illustrating the strand-specific RT-qPCR analysis of readthrough defects.The dashed line indicates the readthrough transcripts past the transcription termination site (TTS) that accumulate following a defect in polymerase II termination.The location of primers for RT and qPCR (A and B) are indicated by the small arrows.F, RT-PCR analysis for the readthrough transcripts.Strand-specific cDNAs were synthesized from RNAs extracted from cells treated with the indicated concentrations of DMOG or from cells with either WT or RACA mutant PNUTS overexpression using primer RT.Fold change of the readthrough transcripts relative to the WT ± SD is based on qPCR analysis with primer A and B, normalized to tubulin RNA.cDNA, complementary DNA; HA, hemagglutinin; LtPNUTS, Leishmania tarentolae PNUTS; PP1, phosphoprotein phosphatase 1; qPCR, quantitative PCR.