Article hnRNPDL Phase Separation Is Regulated by Alternative Splicing and Disease-Causing Mutations Accelerate Its Aggregation Graphical Abstract Authors Cristina Batlle, Peiguo Yang, Maura Coughlin, ..., Hong Joo Kim, J. Paul Taylor, Salvador Ventura Correspondence jpaul.taylor@stjude.org (J.P.T.), salvador.ventura@uab.cat (S.V.) In Brief Batlle et al. show that alternative splicing controls heterogeneous ribonucleoprotein D-like (hnRNPDL) phase separation, aggregation, and solubility. Mutations that cause LGMD1G accelerate hnRNPDL aggregation and promote insolubility in Drosophila. Highlights d hnRNPDL requires both N- and C-terminal IDRs to phase separate d The absence of N-terminal IDR facilitates aggregation d The unique combination of IDRs in each isoform determines its cellular behavior d D378N/H mutations accelerate hnRNPDL aggregation and compromise its solubility Batlle et al., 2020, Cell Reports 30, 1117–1128 January 28, 2020 ª 2019 The Author(s). https://doi.org/10.1016/j.celrep.2019.12.080 Cell Reports Article hnRNPDL Phase Separation Is Regulated by Alternative Splicing and Disease-Causing Mutations Accelerate Its Aggregation Cristina Batlle,1 Peiguo Yang,2 Maura Coughlin,2 James Messing,2,3 Mireia Pesarrodona,4,5 Elzbieta Szulc,4,5 Xavier Salvatella,4,5,6 Hong Joo Kim,2 J. Paul Taylor,2,3,* and Salvador Ventura1,7,* 1Institut de Biotecnologia i Biomedicina and Departament de Bioquı́mica i Biologia Molecular, Universitat Autónoma de Barcelona, Bellaterra 08193, Spain 2Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA 3Howard Hughes Medical Institute, Chevy Chase, MD 201815, USA 4Institute for Research in Biomedicine (IRB Barcelona), The Barcelona Institute of Science and Technology, Baldiri Reixac 10, 08028 Barcelona, Spain 5Joint BSC-IRB Research Programme in Computational Biology, Baldiri Reixac 10, 08028 Barcelona, Spain 6ICREA, Passeig Lluı́s Companys 23, 08010 Barcelona, Spain 7Lead Contact *Correspondence: jpaul.taylor@stjude.org (J.P.T.), salvador.ventura@uab.cat (S.V.) https://doi.org/10.1016/j.celrep.2019.12.080 SUMMARY complexity. These IDRs are enriched in specific amino acids, such as glutamine, asparagine, serine, glycine, and tyrosine, be- Prion-like proteins form multivalent assemblies and ing similar in composition to the disordered domains of yeast phase separate into membraneless organelles. Het- prions, and thus referred to as prion-like domains (PrLDs) (King erogeneous ribonucleoprotein D-like (hnRNPDL) is et al., 2012). Interestingly, prion-like proteins often have the abil- a RNA-processing prion-like protein with three alter- ity to phase separate into liquid droplets and this may contribute native splicing (AS) isoforms, which lack none, one, to the formation of MLOs in the nucleus or cytoplasm (Boey- naems et al., 2018). MLOs are dynamic structures and their for- or both of its two disordered domains. It has been mation is usually reversible, but these assemblies may become suggested that AS might regulate the assembly irreversible when proteins aggregate within MLOs due to muta- properties of RNA-processing proteins by controlling tions, prolonged stress, or changes in protein concentration. the incorporation of multivalent disordered regions Protein aggregation is linked to the onset of a growing list of hu- in the isoforms. This, in turn, would modulate their man disorders (Harrison and Shorter, 2017). Not surprisingly, activity in the downstream splicing program. Here, increasing evidences indicate a connection between patholog- we demonstrate that AS controls the phase separa- ical states and MLOs proteins malfunction (Ito et al., 2017). tion of hnRNPDL, as well as the size and dynamics ATX2 (Kato et al., 2019), EWSR1 (Maharana et al., 2018), FUS of its nuclear complexes, its nucleus-cytoplasm (Patel et al., 2015), hnRNPA1 (Kim et al., 2013; Molliex et al., shuttling, and amyloidogenicity. Mutation of the 2015), hnRNPA2 (Kim et al., 2013; Ryan et al., 2018), HTT (Pe- highly conserved D378 in the disordered C-terminal skett et al., 2018), TAF15 (Maharana et al., 2018), Tau (Wegmann et al., 2018), TDP43 (Babinchak et al., 2019), and TIA1 (Macken- prion-like domain of hnRNPDL causes limb-girdle zie et al., 2017) are well-characterized proteins involved in the muscular dystrophy 1G. We show that D378H/N dis- formation of MLOs; their mutation being associated with age- ease mutations impact hnRNPDL assembly proper- related disorders such as amyotrophic lateral sclerosis (ALS), ties, accelerating aggregation and dramatically frontotemporal dementia (FTD), or inclusion-body myopathy reducing the protein solubility in the muscle of (IBM) (Harrison and Shorter, 2017; Ito et al., 2017; Nedelsky Drosophila, suggesting a genetic loss-of-function and Taylor, 2019). Despite the increasing interest in MLOs, the mechanism for this muscular disorder. molecular mechanisms that govern the transition between their functional and pathologic states are still not well understood. INTRODUCTION Alternative splicing (AS) is an important mechanism underlying evolution complexity (Baralle and Giudice, 2017). Many MLOs Eukaryotic cells contain a variety of compartments or organelles proteins have AS isoforms with unknown functions (Gueroussov with specialized functions. There are membrane-bound organ- et al., 2017). Indeed, AS events are frequent in prion-like elles like the nucleus or mitochondria, and membraneless organ- proteins, especially at their PrLDs, affecting their ability to elles (MLOs) such as stress granules or P-bodies (Boeynaems establish multivalent interactions and to form higher-order et al., 2018). MLOs are enriched in a peculiar type of polypep- complexes (Gueroussov et al., 2017). AS also alters the phase tides known as prion-like proteins (March et al., 2016). These separation properties of prion-like proteins’ isoforms as polypeptides consist of one or more globular domains with adja- observed for FUS protein with or without exon 8 inclusion (Guer- cent long intrinsically disordered regions (IDRs) of low oussov et al., 2017). Cell Reports 30, 1117–1128, January 28, 2020 ª 2019 The Author(s). 1117 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Figure 1. LLPS Propensity of hnRNDPL Iso- forms (A) Schematic diagram of hnRNPDL isoforms as SUMO fusion constructs. RNA recognition motifs (RRMs; blue) according to Pfam (El-Gebali et al., 2019), Arg-rich (orange), and Tyr-rich (green) IDR spliced regions according to Uniprot (Bateman et al., 2015) and their respective amino acid splicing positions are shown. hnRNPDL nuclear localization signal (NLS) sequence is as described in Kawamura et al. (2002). (B) SUMO-hnRNPDL isoform 1 (DL1) LLPS at different protein concentrations in 50 mM HEPES pH 7.5 and 150 mM NaCl. (C) 50-mM DL1 LLPS at different salt concentra- tions. (D) 50-mM SUMO-DL2 and SUMO-DL3 LLPS in 150 mM salt with or without the presence of 10% Ficoll, and 50 mM salt. (E) LLPS diagram of hnRNPDL isoforms in the absence of crowding agent. Green circles indicate positive and red diamonds indicate negative for the appearance of droplets at the indicated NaCl/ protein concentration combinations. (Tsuchiya et al., 1998) and it is the pre- dominant isoform in all mouse and human tissues (Akagi et al., 2000). It is 301 amino acids long, constituted by two contiguous canonical RNA recognition motifs (RRMs) and one predicted PrLD at the C terminus, enriched in Gly and Tyr residues. DL1 is a longer isoform of 420 amino acids comprising an additional predicted to be disordered and Arg-enriched domain at the N terminus (Kamei et al., 1999). DL1 expression levels are 4-fold lower than those of DL2 and the transcript is mainly present in brain and testis (Akagi et al., 2000). DL3 is the shorter and minor iso- form, with only 244 amino acids missing both N and C terminus disordered regions (Kawamura et al., 2002). hnRNPDL bears a 25-residue C-termi- nal PY nuclear localization signal (NLS) and its transport is mediated by the M9-transportin-1 (TNPO1) pathway We focus this study on the heterogeneous ribonucleoprotein (Kawamura et al., 2002). Interestingly, only DL2 and DL3 D-like (hnRNPDL), an RNA-binding protein displaying AS iso- are able to shuttle between the cytoplasm and the nucleus, forms, linked to disease and with motifs similar to those associ- whereas DL1 remains strictly nuclear. hnRNPDL isoforms share ated with liquid-liquid phase separation (LLPS) in well-character- the same shuttling sequence, except for DL3, which misses ized human prion-like proteins. four residues that are not required for TNPO1 interaction (Fig- hnRNPDL is a highly conserved nuclear RNA binding protein ure 1A; Kawamura et al., 2002). Therefore, the basis of this involved in mRNA biogenesis located in the genomic position differential translocation is unknown, but it may indicate 4q21 (Kamei et al., 1999). The HNRNPDL gene contains nine hnRNPDL isoforms playing different roles in cells. exons and eight introns, and three isoforms are produced by HNRNPDL constitutes one of the four genes present in the AS, named here as hnRNPDL isoform 1 (DL1), hnRNPDL isoform smallest deletion of the 4q21 microdeletion syndrome, being 2 (DL2), and hnRNPDL isoform 3 (DL3) (Figure 1A). DL2 was the associated with growth retardation and hypotonia (Hu et al., first isoform discovered as a JKT41 binding protein 1 (JKTBP1) 2017). Moreover, hnRNPDL expression is upregulated in 1118 Cell Reports 30, 1117–1128, January 28, 2020 different types of cancers, such as prostate cancer, chronic aqueous phase to form liquid-like protein droplets under physio- myeloid leukemia, colon cancer, and hepatocellular carcinoma logical ionic strength (Figure 1B). DL1 undergoes LLPS in a pro- (Zhou et al., 2014; Liu et al., 2007; Wu et al., 2008; Zhang tein concentration-dependent manner in the absence of any et al., 2018). Finally, genome sequencing of Brazilian, Chinese, crowding agent even at low protein concentrations (2 mM). For Uruguayan, and Argentinian families affected by limb-girdle a given protein concentration, phase separation is enhanced muscular dystrophy 1G (LGMD1G, or LGMDD3 in the new with decreasing ionic strength (Figure 1C). DL2, missing the nomenclature; Straub et al., 2018) detected D378N and D378H Arg-enriched disordered domain at its N terminus, also can un- point substitutions in HNRNPDL, indicating a mutation hotspot dergo phase separation, but its propensity is much lower than for this disease (Berardo et al., 2019; Sun et al., 2019; Vieira that of DL1 (Figure 1E). At physiological ionic strength, DL2 et al., 2014). LGMD1G is an autosomal dominant inherited requires the presence of a crowding agent (10% Ficoll) to subtype of LGMD, the fourth most common muscular dystrophy, phase separate (Figure 1D). When transferred to low ionic characterized by progressive weakness of hip- or shoulder-gir- strength (50 mM NaCl), DL2 rapidly coalesces into micron-sized dle muscles (Liewluck and Milone, 2018; Nigro and Savarese, spherical structures without any crowding agent, but this pro- 2014). cess requires higher protein concentrations than for DL1 (Figures Interestingly, hnRNPDL D378N/H point mutations reside in the 1D and 1E). The fact that, in the absence of a crowding agent, PrLD of hnRNPDL (Navarro et al., 2015). Disease-causing muta- DL2 only forms droplets at low ionic strength suggests that tions in PrLDs have been discovered in other prion-like proteins, its LLPS depends on electrostatic interactions. Indeed, the for example in hnRNPA1 and hnRNPA2 proteins (Kim et al., C-terminal IDR is predicted to behave as a weak polyampholyte, 2013), in both cases involving the replacement of a single Asp a kind of molecule that displays reduced LLPS as the salt con- residue, as in hnRNPDL. These mutations have been reported centration increases (Das and Pappu, 2013). DL3, the isoform to increase protein aggregation and result in the formation of devoid of any IDRs, does not phase separate in any tested con- cytoplasmic inclusions in patients (Harrison and Shorter, 2017; dition, neither at 50 mM NaCl nor after crowding agent addition Kim et al., 2013; Mackenzie et al., 2017). Immunohistochemical (Figures 1D and 1E). analysis of LGMD1G patients identified nuclear condensates The radii of hydration of hnRNPDL isoforms were analyzed of hnRNPDL co-localizing with TNPO1 (Vieira et al., 2014). Con- by dynamic light scattering (DLS) at physiological salt concentra- gophilic deposits have however not been detected in LGMD1G tion without any crowding agent (Figure S1). The results corre- patients and it remains unknown whether hnRNPDL mutations lated with the propensity to undergo LLPS (Figure 1E), with impact the protein aggregation propensity. DL1 forming assemblies with a radius of hydration > 1,000 nm, In this study, we show how AS results in hnRNPDL isoforms which could not be observed in DL2 and DL3 proteins, that dis- with dramatically different self-assembling properties in vitro played average radii of hydration of 4.5 and 3.5 nm respectively, and in vivo. We also demonstrate how Arg and Tyr residues, compatible with a monomeric state. segregated in two distant IDRs in hnRNPDL, act as crucial deter- These data indicate that the absence of the N-terminus Arg- minants for both LLPS and aggregation. This spatial segregation enriched IDR of hnRNPDL reduces LLPS propensity and the of multivalent interacting residues explains how AS controls the absence of both the N- and C-terminus IDRs completely abol- propensity to form high-order intranuclear assemblies in ishes LLPS, suggesting that these domains are required for mammalian cells, likely accounting for the different shuttling hnRNPDL self-assembly. The results provide experimental properties of the hnRNPDL isoforms. Finally, we confirm that, support for the hypothesis that AS controls LLPS in hnRNPDL as in other prion-like proteins, hnRNPDL disease-causing muta- (Feng et al., 2019). tions accelerate protein aggregation, resulting in completely insoluble variants when expressed in Drosophila muscle. Interactions between Arg and Tyr Residues of DL1 IDRs Promote Its Phase Separation RESULTS Recent studies have reported the importance of Arg-rich do- mains in providing intermolecular interactions that contribute to hnRNPDL Alternative Splicing Isoforms Display LLPS (Boeynaems et al., 2017). Moreover, Arg residues have Different Phase Separation Behavior been shown to establish interactions with Tyr residues in FUS hnRNPDL has been shown to undergo LLPS in vitro (Wang et al., (Bogaert et al., 2018; Vernon et al., 2018; Wang et al., 2018). In 2018). To evaluate the molecular determinants that govern this the previous section, we have shown how the absence of the process, we took advantage of the different domain architec- Arg- and Tyr-rich IDRs in hnRNPDL protein affects LLPS tures of hnRNPDL isoforms and we tested their propensity behavior. Therefore, we hypothesized that these residues to undergo phase transition. We expressed and purified might be interacting and promoting hnRNPDL phase separation. hnRNPDL isoforms as fusions with solubility-enhancing His- To confirm this hypothesis, we generated three hnRNPDL SUMO tags (Figure 1A), hereinafter referred to as DL1, DL2, variants: (1) all N-terminus Arg mutated to Lys (R/K), (2) all C-ter- and DL3. As expected, the three hnRNPDL isoforms bear distinct minus Tyr mutated to Phe (Y/F), and (3) both Arg to Lys and Tyr to LLPS propensity (Figures 1B–1E). Phe mutations (R/K+Y/F) (Figure 2A). These mutations were The DL1 isoform, containing Arg- and Tyr-enriched IDRs at designed to maintain the aromatic and basic character of Tyr the N- and C terminus, respectively, displays the strongest and Arg, respectively, while preserving the ability to establish tendency to phase separate (Figure 1E). Upon salt dilution, the p-cation contacts. The identity of the basic and positive residues solution becomes turbid spontaneously, demixing from an determines the interaction strength, with Lys-Tyr, Arg-Phe, and Cell Reports 30, 1117–1128, January 28, 2020 1119 Figure 2. DL1 Mutants LLPS Behavior (A) Schematic diagram of DL1 mutants as SUMO fusion constructs: (1) all N-terminus Arg mutated to Lys (R/K); (2) all C-terminus Tyr mutated to Phe (Y/ F); (3) both Arg to Lys and Tyr to Phe mutations (R/ K+Y/F); and (4) N-terminus (Nt) IDR alone. (B) LLPS of DL1 and mutants at 50 mM in 50 mM HEPES pH 7.5 and 150 mM NaCl. (C) LLPS of DL2 green labeled mixed with hnRNPDL-Nt red labeled, in a 1:1 ratio, at different protein concentrations and 150 mM salt. with DL2 at a 1:1 ratio, we completely recover LLPS under physiological condi- tions and DL2 phase separated at concentrations as low as 6.25 mM in the absence of any crowding agent (Figure 2C). The above results indicate that DL1 LLPS is likely governed by intermolecular interactions between the Arg residues at the N-terminal domain and Tyr residues at the C terminus. DL1 Forms High-Order Complexes in the Nucleus of Mammalian Cells In the previous sections, we observed how hnRNPDL isoforms bear different self-assembly propensities in vitro. hnRNPDL is located in the cell nucleus; consequently, we addressed their prop- erties in the nuclear context. To this aim, we examined the isoforms localization in Lys-Phe contacts all being weaker than Arg-Tyr (Wang et al., a HeLa hnRNPDL knockout (KO) cell line (HeLaDL-KO) (Fig- 2018). Therefore, these protein variants would allow us to ure S3A). Cells were transfected with EGFP-tagged fusion evaluate the role of Arg and Tyr interaction strength in hnRNPDL constructs or EGFP alone, as control, and immunostained LLPS. with anti-G3BP antibody as a cytoplasmic marker (Figure 3A). The three hnRNPDL mutants showed a clear reduction of All hnRNPDL isoforms are nuclear (Figure 3A). Both DL1 and LLPS compared to DL1, being unable to phase separate at DL2 are distributed throughout the nucleoplasm, but excluded physiological conditions without crowding agents (Figures 2B from the nucleolus (Figure S4A). In contrast, DL3 was completely and S2A). This supports the idea that hnRNPDL phase separa- diffuse in the nucleus, suggesting that the hnRNPDL C-terminus tion relies on the complementarity of Tyr and Arg contacts (Brady IDR, the only region present in DL1 and DL2 and absent in DL3, et al., 2017) and not on generic p-cation interactions, as might determine the intranuclear compartmentalization of the observed also for FUS protein (Bogaert et al., 2018; Wang isoforms. DL1 and DL2 undergo LLPS in vitro, but they do not et al., 2018). show nuclear puncta indicative of MLO formation. This behavior The reduced LLPS propensity of the R/K mutant (Figure S2A) might be caused by the high RNA concentration in the nucleus, is similar to that of the DL2 isoform (Figure 1E). The C terminus since high RNA/protein ratios have been shown to diminish in DL2 is sufficient to promote LLPS, likely through Tyr-Tyr inter- LLPS in other prion-like RNA binding proteins, such as TDP43 actions (Burke et al., 2015; Murthy et al., 2019; Wang et al., and FUS (Maharana et al., 2018). In order to verify this idea, we 2018), but its ability to undergo LLPS under physiological ionic added increasing concentrations of total RNA to DL1 in vitro strength is weaker than that of DL1 (Figure 1E). To further confirm and we observed a clear decrease in its LLPS propensity (Fig- that the C-terminus Tyr-rich domain and the N-terminal ures S2B and S2C). RNA completely dissolved DL1 droplets at Arg-rich domain can indeed form interactions responsible for 100 ng/mL (Figure S2B). This suggests that the nucleic acid ti- the high LLPS propensity of DL1, we designed a construct trates DL1 from the droplets. Accordingly, the droplets reap- consisting only of the Arg-rich domain of DL1 (hnRNPDL-Nt) peared after RNase addition (Figure S2B). (Figure 2A). hnRNPDL-Nt was unable to phase separate by itself We conducted fluorescent recovery after photobleaching (Figures 2B and S2A). However, when hnRNPDL-Nt was mixed (FRAP) analysis of a small region of the nucleus of HeLaDL-KO 1120 Cell Reports 30, 1117–1128, January 28, 2020 Figure 3. Cellular Localization and Mobility of hnRNPDL Isoforms in Mammalian Cells (A) Cellular localization by immunofluorescence of EGFP-DL1, EGFP-DL2, EGFP-DL3, and unfused EGFP after expression in HeLaDL-KO. Cells were stained with G3BP antibody (red) as cytoplasmic marker and DAPI (blue) as nuclear marker. (B and C) Graph of normalized fluorescence in- tensity (B) and rate of recovery average (C) after FRAP in HeLaDL-KO cells expressing EGFP-DL1, EGFP-DL2, and EGFP-DL3 and unfused EGFP (***p value < 0.0001). (D) Example of a size exclusion chromatography elution pattern after individual EGFP-hnRNPDL isoforms expression in HeLaDL-KO cells. Elution volumes of EGFP-DL1 (74 kDa), EGFP-DL2 (61 kDa), and EGFP-DL3 (55 kDa) are indicated by arrows. Letters along the x axis indicate the elution volumes upon column calibration: A, col- umn void volume; B, ferritin (440 kDa); C, aldolase (158 kDa); D, conalbumin (75 kDa); and E, oval- bumin (44 kDa). both hnRNPDL IDRs confers an in- crease in multivalency, enhancing protein-protein or protein-nucleic acids interactions and, consequently, the formation of larger or more stable complexes in the nucleus as reported cells for all EGFP-tagged hnRNPDL isoforms. FRAP analysis also for other proteins (Gueroussov et al., 2017; Ying et al., showed that the three isoforms associate and dissociate within 2017). the nucleus on different timescales of seconds (Figure 3B). The fluorescence recovery half-times after photobleaching were Transcription Inhibition Affects hnRNPDL Nuclear 0.25, 0.84, and >7.0 s for DL3, DL2, and DL1, respectively (Fig- Localization ure 3C). The low mobility of DL1, relative to DL2 and DL3, sug- hnRNPs are predominantly located in the cell nucleus. Some gests that it might be involved in the formation of larger or hnRNPs, for example hnRNPA1, are known to shuttle between more stable complexes within the nucleoplasm. The DL2 and the nucleus and cytoplasm in a transcription-dependent manner, DL3 mobilities are also significantly different, indicating that but others do not, for example hnRNPC (Dreyfuss et al., 2002; Pi- these isoforms are also associated with nuclear complexes ñol-Roma and Dreyfuss, 1991, 1992). Actinomycin D (ActD) that differ in identity or stability, although the assemblies that is an anti-tumor chemical that inhibits transcription by interca- they form are likely smaller and more dynamic than the ones lating into transcriptionally active regions, resulting in a signifi- formed by DL1. cant reduction in RNA synthesis in the nucleus (Su et al., To confirm that DL1, DL2, and DL3 could be involved in the for- 2013). This treatment is usually performed to determine whether mation of different complexes, we performed size exclusion protein localization is dependent on active transcription, its inhi- chromatography (SEC) of individual HeLaDL-KO cellular extracts bition resulting in protein translocation to the cytoplasm (Bou- after transfection with each of the different constructs (Figures nedjah et al., 2014; Piñol-Roma and Dreyfuss, 1991, 1992; 3D and S5). The DL1 isoform elution pattern differed significantly Zhang et al., 2005). Interestingly, in contrast to untreated cells from that of DL2 and DL3, the protein being eluted in the void vol- (Figure 3A), after 5 mg/ml ActD treatment for 3 h, hnRNPDL ume of the column corresponding to molecular size complexes shows different transport depending on the considered isoform. larger than 5 million Da. Instead, DL2 and DL3 were eluted in vol- Only the DL2 and DL3 isoforms were translocated to the cyto- umes consistent with them being in their monomeric form. In or- plasm indicating nuclear import that is dependent upon active der to prove that the EGFP-tag does not alter the elution pattern transcription; meanwhile, the DL1 isoform remained nuclear of the isoforms, the retention of endogenous hnRNPDL in a HeLa (Figure 4), consistent with previous observations (Kamei and Ya- wild-type (WT) cell line was analyzed (Figure S5). The elution pro- mada, 2002; Kawamura et al., 2002). The three isoforms bear file of endogenous DL2, the predominant isoform, is similar to the same shuttling sequence (Figure 1A) and, thus, in principle, that of EGFP-DL2. The levels of endogenous DL1 and DL3 are they could be transported in the same manner. In the previous too low to be detected in this experiment. section, we showed that DL1 forms higher- or more-stable-order These results correlate well with the observed hnRNPDL complexes (Figures 3B–3D), larger than 5 million Da in the nu- isoforms in vitro self-assembly behavior. The presence of cleus, suggesting extensive protein-protein or protein-nucleic Cell Reports 30, 1117–1128, January 28, 2020 1121 binding after 4 days (Figure 5A). We also used the alternative amyloid-specific dye Congo Red (CR) to confirm the presence of amyloid-like assemblies (Wu et al., 2012). The absorbance spectra of CR shifts in the presence of amyloid aggregates, and in agreement with the ThT results, only the DL2 isoform promoted this red shift in CR spectrum after 4 days (Figure 5B). These results suggest that, from the three hnRNPDL isoforms, only DL2 displays significant amyloid aggregation propensity. In fact, the DL2 aggregates also bind the amyloid dyes Thiofla- vin-S (ThS) and Proteostat, and exhibit CR birefringence under polarized light (Figure S6A). Finally, we observed by transmission electron microscopy (TEM) the morphological features of the three hnRNPDL isoforms after 4 days. Negative staining indi- cated that DL2 formed typical amyloid fibrillary structures without any significant accumulation of amorphous material, DL1 forms amorphous aggregates, and DL3 formed only small aggregates (Figure 5C). Figure 4. Transcription Inhibition Effects on hnRNPDL Localization These results suggest that the Tyr-rich IDR, including a pre- EGFP-DL1, EGFP-DL2, and EGFP-DL3 and unfused EGFP localization after dicted PrLD (Figure 6A), is responsible for hnRNPDL self-assem- their expression in HeLaDL-KO cells and 5 mg/ml Actinomycin D (ActD) treat- bly into ordered amyloid-like structures, but also that this reac- ment for 3 h. Cells were stained with G3BP antibody (red) as cytoplasmic tion only occurs in the absence of the positively charged marker and DAPI (blue) as nuclear marker. N-terminal IDR, which would act as a kind of intramolecular chaperone. The data correlate with the relative solubility of acid interaction networks. This observation could provide an the endogenous or transfected isoforms in HeLa WT or explanation for DL1 nuclear retention, which ultimately would hnRNPDL KO cells, respectively, DL2 being the more insoluble depend on the presence of both IDRs. of the three variants (Figure S3). ActD treatment disrupts the nucleolus (Figure S4B) and relo- cates DL1 and DL2 isoforms within the nucleus, now exhibiting Disease-Causing Mutations Accelerate hnRNPDL a speckled pattern (Figure 4). Interestingly, DNA staining with Aggregation DAPI also showed nuclear puncta, but they do not colocalize Disease-causing mutations in PrLDs are common in prion-like with DL1 and DL2 foci. Indeed, the DL1 and DL2 foci tend to proteins and they have been linked with an acceleration of the coincide with nuclear areas exhibiting poor DAPI staining, aggregation kinetics (Harrison and Shorter, 2017). Mutation of suggesting that high local DNA concentrations might prevent a specific Asp residue in the PrLDs of hnRNPA1 and hnRNPA2 DL1 and DL2 foci formation. In contrast to DL1 and DL2, DL3 re- proteins mapping to evolutionarily conserved regions of these mains diffusely distributed in the nucleus. Therefore, we can IDRs are linked to ALS or MSP (Kim et al., 2013). The conserved ascribe the observed DL1 and DL2 localization patterns in the Asp is involved in destabilizing electrostatic interactions and absence of ActD to the presence of the C-terminal Tyr-rich the removal of repulsion by mutation seems to be responsible IDR. Similar observations have been reported for FUS and for the increased propensity of the mutated PrLDs to self-asso- TAF15 proteins (Marko et al., 2012; Zinszner et al., 1997). ciate and aggregate. Mutation of Asp378 of hnRNPDL to either The data show that only DL2 and DL3 isoforms can shuttle Asn or His has been associated with LGMD1G (Berardo et al., between nucleus and cytoplasm after transcription inhibition, 2019; Sun et al., 2019; Vieira et al., 2014). As in the case of whereas only DL1 and DL2 form nuclear foci. This indicates hnRNPA1 and hnRNPA2, this Asp maps at the PrLD of hnRNPDL that the unique combination of IDRs in each particular variant (Figure 6A; Navarro et al., 2015) and it is strictly conserved in is an important determinant of its localization. vertebrates (Figure S7). Therefore, we examined how these mutations affect LLPS and the aggregation of hnRNPDL protein. hnRNPDL Alternative Splicing Isoforms Have Different DL2 is reported to be the predominant isoform in tissues Aggregation Propensity (Akagi et al., 2000), as observed by western blot of HeLa WT cells Prion-like proteins are well known for their aggregation propen- (Figure S3). Moreover, in the previous section we showed that sity, usually mediated by their PrLDs (March et al., 2016). DL2 is also the isoform with higher amyloid potential (Figure 5). hnRNPDL isoforms were highly insoluble in bacteria, which is Consequently, we focused the study on Asn and His mutants why we studied them as His-SUMO fusions. Once purified, we of the DL2 isoform (DL2N and DL2H) located at position D259; proceeded to analyze their in vitro aggregation properties this position corresponds to D378 in DL1 because DL2 lacks (Figure 5). We used the amyloid-specific dye Thioflavin-T (ThT) the first 119 amino acids (Figure 6A). (Biancalana and Koide, 2010) to follow the kinetics of hnRNPDL We first checked how DL2 mutations affect LLPS behavior un- isoform aggregation at 50-mM protein concentration, 150 mM der physiological salt conditions (Figure 6B). In the absence of NaCl, and 37 C with agitation. Interestingly, after 2 days DL2 Ficoll, DL2H did not show detectable structures by light micro- already exhibited a ThT signal in the plateau phase, DL1 started scopy, whereas in DL2N small irregularly shaped and clustered to bind ThT only after 3 days, and DL3 did not show any ThT structures were observed. In the presence of Ficoll, DL2H 1122 Cell Reports 30, 1117–1128, January 28, 2020 Figure 5. hnRNPDL Isoform Aggregation Propensity Evaluation of Thioflavin T (ThT) binding over time (A), Congo Red (CR) binding (B), and transmission electron microscopy (TEM) (C) at final ag- gregation time point of 50-mM SUMO-hnRNPDL isoforms in 50 mM HEPES pH 7.5 and 150 mM NaCl. Aggregation was conducted at 37 C and 600 rpm. Scale bars of TEM images in (C) represent 400 nm. absorbance spectra of DL2 WT, DL2H, and DL2N samples indicated the pres- ence of a major band at 1,628 cm 1, which can be assigned to the presence of an intermolecular b sheet (Figure S6B). This component accounts for 25%–26% of the spectral area in the three cases (Table S2), which sharply coincides with the proportion of residues mapping at the PrLD in the respective DL2 constructs (25.8%). No major secondary structure differences were observed between the showed aggregated particles, whereas DL2N shows liquid drop- three amyloid assemblies, indicating that, in vitro, the impact of lets morphology similar to DL2 WT. These results indicate that the mutations is mostly kinetic. the mutations in DL2 PrLD affect its LLPS propensity. Overall, these results, provide evidences that mutation of Comparison of DL2 mutants’ in vitro aggregation propensity Asp378 in hnRNPDL PrLD (position 259 in DL2) increases its over time with that of the WT isoform confirmed the impact aggregation propensity, as previously described for hnRNPA1 of the Asp mutation on aggregation. Both DL2 mutants exhibited and hnRNPA2. faster aggregation kinetics, as monitored by ThT, displaying a shorter lag phase and reaching the plateau phase signifi- Disease-Causing Mutations Are Located in the Insoluble cantly before than the WT form (Figure 6C). Accordingly, TEM Fraction of Drosophila Muscle analysis indicates that DL2H and DL2N proteins have already To evaluate the effect of disease-causing mutations in hnRNPDL assembled into amyloid fibrils after 24 h, whereas the DL2 WT in vivo, we generated transgenic Drosophila expressing WT or remains protofibrillar (Figure 6D). This is in agreement with mutant forms of human DL2 by using the UAS/GAL4 system. the predictions of ZipperDB, a structure-based threading Multiple transgenic lines expressing a single copy of DL2 WT algorithm that scores six-amino acid-sequence stretches or mutant (DL2N or DL2H) were generated by site-specific according to their propensity to form ‘‘steric zippers’’ in the 4C31integrase-mediated transgenesis into Drosophila chromo- spine of amyloid fibrils (Goldschmidt et al., 2010). ZipperDB some 3. This approach permits equal levels of expression be- predicted higher steric zipper propensity for DL2H and DL2N, tween independent lines, as demonstrated empirically for the relative to DL2 WT (Table S1). The AMYCO algorithm, which five independent lines expressing WT DL2 (Figure 7A). However, evaluates the impact of mutations on the aggregation of prion- we observed that fly lines expressing mutant forms of DL2 (DL2N like proteins (Iglesias et al., 2019), also predicts increases in or DL2H) consistently exhibited lower levels of DL2 protein aggregation propensities of 7% and 10% for the DL2H and compared to flies expressing WT DL2 (Figure 7A). DL2N variants, respectively. Expression of either WT or mutant (DL2N or DL2H) forms The acceleration of the aggregation reaction in the mutant of DL2 in Drosophila indirect flight muscle led to mild degenera- variants resulted in smaller and less-ordered amyloid fibrils tion, showing disorganized muscle fibers as demonstrated at the end of the reaction (4 days), as observed by TEM (Fig- by phalloidin staining (Figure 7B). Immunohistochemical analysis ure 6D), which displayed a lower, but still significant, binding showed that both WT and mutant forms of DL2 localized pre- to CR than DL2 WT fibrils (Figure 6E). The amyloid-like nature dominantly to myonuclei (Figure 7B). Although we did not of the DL2H and DL2N aggregates was further confirmed by observe clear differences between WT and mutant proteins in staining with ThS, Proteostat, and by CR birefringence their localization patterns, we did observe that the solubility of (Figure S6A). DL2 protein was strongly impacted by disease-causing muta- The secondary structure content of the aggregates was tions. Specifically, mutant DL2 (D259H or D259N) proteins assessed using attenuated total reflection Fourier transform were largely recovered from the detergent-insoluble fraction, infrared spectroscopy (ATR-FTIR) in the amide I region of whereas the WT DL2 protein is found both in the detergent-sol- the spectrum (1,700–1,600 cm 1). Deconvolution of the FTIR uble and detergent-insoluble fractions (Figure 7C). This result is Cell Reports 30, 1117–1128, January 28, 2020 1123 Figure 6. DL2 Disease-Causing Mutation Aggregation Propensity (A) Schematic diagram of DL2, as a SUMO fusion construct. RRMs (blue) according to Pfam (El- Gebali et al., 2019) and PLAAC (Lancaster et al., 2014) predicted PrLD (light green) are indicated. The Tyr-rich IDR (dark green) and the disease- causing mutations (red) within the PrLD are shown with their respective amino acid positions. (B–E) LLPS behavior (B), Thioflavin T (ThT) binding over time (C), transmission electron microscopy (TEM) at 24 h and 4 days (D), and Congo Red (CR) binding (E) at final aggregation time point of 50-mM DL2 and the disease-causing mutations D259N and D259H (DL2N and DL2H) in 50 mM HEPES pH 7.5 and 150 mM NaCl. Aggregation was conduct- ed at 37 C and 600 rpm. Scale bars of TEM images in (D) represent 400 nm. actions (Boeynaems et al., 2018). Recently, it was described that FUS LLPS was governed by interactions be- tween Tyr residues of the PrLD and Arg residues of the RNA binding domain (Wang et al., 2018). We hypothesized that hnRNPDL LLPS would follow the same mechanism than FUS, where Tyr residues from the C terminus and Arg residues from the N terminus would mediate interactions leading to phase separation. We experimentally validated this hypothesis generating hnRNPDL var- iants that maintain the aromatic and cationic residue content but are predicted to establish weaker interactions. These variants could not undergo LLPS under physiological conditions, indicating that, in hnRNPDL, Arg and Tyr residue interac- tions are required for phase separation. The absence of the N-terminus Arg-rich consistent with in vitro data that indicate enhanced aggregation IDR significantly reduces the DL2 LLPS propensity. However, propensity by disease-causing mutations (Figure 6C). The lower DL2 can still undergo LLPS, most probably by Tyr-Tyr interac- levels of DL2 mutant proteins relative to the WT protein in tions of the C-terminal PrLD, as it occurs with purified PrLDs of fly muscle might respond to an attempt of the proteostatic proteins such as FUS (Wang et al., 2018). machinery to degrade misassembled insoluble species. Different self-assembly properties of the hnRNPDL isoforms suggest that they might participate in distinct biological func- DISCUSSION tions. We experimentally validated that hnRNPDL isoforms exhibit a significantly different behavior in mammalian cells, the In this work, we first characterized the molecular properties that data indicating that they might be involved in the formation of govern the self-assembly of hnRNPDL isoforms in vitro. The distinct nuclear complexes. DL1 and DL2 are excluded from three AS isoforms exhibit different LLPS propensities according the nucleolus, while DL3 is diffusely distributed. This exclusion to their IDRs composition: DL1, with both Arg-rich and Tyr-rich from the nucleolus has been also reported for other prion-like IDRs, displays a strong LLPS behavior; DL2, with only the Tyr- proteins like FUS (Yang et al., 2014), TAF15 (Marko et al., rich IDR, has a mild LLPS propensity, requiring a crowding agent 2012), or EWSR1 (Tannukit et al., 2008). The only common region to phase separate at physiological ionic strength; and DL3, in DL1 and DL2 and absent in DL3 is the C-terminus Tyr-rich with none of the IDRs, does not phase separate. Protein self-as- PrLD. Therefore, hnRNPDL PrLD seems to determine the intra- sembly driving forces can be mediated by p-cation, p-p, hydro- nuclear compartmentalization of the isoforms. This is consistent phobic, electrostatic, dipole-dipole, or hydrogen bonding inter- with the observation that the deletion of the N-terminal PrLD of 1124 Cell Reports 30, 1117–1128, January 28, 2020 Figure 7. hnRNPDL Isoform 2 Disease- Causing Mutation Effects in Drosophila (A) Expression levels of DL2 and disease- causing mutations (DL2N and DL2H) in trans- genic flies. Thoraxes of adult flies were pro- cessed for western blot analysis with an anti- body against hnRNPDL. Actin was blotted as a loading control. (B) Adult flies were dissected to expose the dorsal longitudinal indirect fly muscle (DLM) and stained with Alexa Fluor 647-phalloidin (purple), hnRNPDL (red), and DAPI (blue). (C) Thoraxes of adult flies were dissected. Sequential extractions were performed to examine the solubility profile of hnRNPDL. FUS resulted in a protein variant evenly distributed in the entire DL1 and DL2 form bright nuclear foci after RNA synthesis inhi- nucleus (Yang et al., 2014), as it occurs with the natural DL3 iso- bition, while DL3 does not. This necessarily involves the Tyr-rich form. hnRNPDLs have LLPS propensity, but in the nucleus it is PrLD in the process. Interestingly, similar observations were diffusely distributed, without the detection of nuclear puncta described for hnRNPD, TAF15 and FUS after ActD treatment corresponding to MLOs. It has been shown that high RNA/pro- (Arao et al., 2000; Jobert et al., 2009; Marko et al., 2012; Zinszner tein ratios, as occur in the nucleus, prevent prion-like proteins et al., 1997). For the three proteins the formation of nuclear foci from LLPS (Maharana et al., 2018). This could be the underlying was depended on their PrLD (Marko et al., 2012; Zinszner reason of the diffuse distribution of the DL1 and DL2 variants in et al., 1997). Nuclear foci after ActD may represent the retention the nucleus. Accordingly, we experimentally validated that DL1 of the protein in transient subnuclear compartments by interac- LLPS is strongly dependent on RNA levels. We were also inter- tion with proteins recruited to this site (Zinszner et al., 1997) ested in assessing whether hnRNPDL isoforms exhibit different thanks to the characteristic compositional bias of PrLDs. mobilities in cells due to their differential self-assembly proper- Prion-like proteins are well known for their aggregation ties. In excellent agreement with in vitro behavior, DL2 and DL3 behavior and involvement in disease. Therefore, we evaluated form dynamic and small complexes in the nucleus, whereas the aggregation propensity of hnRNPDL isoforms. Not surpris- DL1 forms more rigid and bigger complexes. This last behavior ing, DL2, the predominant isoform in humans, hence the most can be univocally ascribed to the DL1 Arg-rich N terminus IDR, probable isoform being involved in disease, is the isoform with which increases DL1 multivalency thus favoring protein-protein higher amyloidogenic propensity. The data indicate that the and protein-nucleic acids interactions and, consequently, the amyloidogenic potential concentrates in the PrLD, since DL3, formation of the observed large complexes. containing the two RRM domains, remains soluble. A reasonable We further validated that DL2 and DL3 are the only two iso- explanation for the amyloidogenicity of DL2 is that it lacks the forms able to shuttle between the nucleus and cytoplasm, their N-terminus IDR Arg-rich domain, which may act as a mechanism nuclear import being dependent on ongoing transcription, as of protection in DL1, precluding rapid aggregation through previously reported (Kamei and Yamada, 2002; Kawamura electrostatic repulsion between positively charged regions. et al., 2002). Similar differences in isoform shuttling have been Therefore, this domain plays a different role in LLPS and amyloid reported for hnRNPD, a member of the same heterogeneous formation, promoting the first reaction and inhibiting the second ribonucleoprotein (hnRNP) family, where p37 and p40 isoforms one. Importantly, this suggests that, for hnRNPDL, the process shuttle between nucleus and cytoplasm but p45 and p42 iso- of phase separation plays a protective role against fibril forms remain in the nucleus (Arao et al., 2000). hnRNPD p37 formation. and p40 result from AS and lack exon 7, which encodes for a As observed for other prion-like proteins, disease-causing region of 50 residues corresponding to the only Tyr-rich IDR mutations in the PrLD enhance the aggregation propensity of region (28% Tyr) in its PrLD. The p45 and p42 isoforms’ nuclear hnRNPDL. We experimentally demonstrated a significant accel- retention is suggested to be mediated through interaction with eration of DL2 aggregation after introduction of the D378N and the nuclear SAF-B protein, p37 and p40 not interacting with it. D378H LGMD1G-associated mutations (DL1 nomenclature). In SAF-B contains a large Arg-rich IDR region (26% Arg) and there- Drosophila muscle, hnRNPDL mutants locate in the nucleus fore it is plausible that p-cation contacts could contribute to the but show a clear reduction in solubility compared to the WT interactions with the p45 and p42 isoforms. DL1 possesses both protein. Most pathogenic mutations in prion-like proteins are types of IDRs and therefore can establish a potentially larger reported to promote the formation of cytoplasmic inclusions in number of interactions. The fact that the protein is retained in patient tissues, a phenotype that can be recapitulated in cell- the nucleus when RNA levels are decreased by ActD treatment based assays and in animal models of the disease (Kim et al., suggests that these interactions are of proteic nature. 2013). This is not the case for hnRNPDL. Analysis of muscle Cell Reports 30, 1117–1128, January 28, 2020 1125 tissues from LGMD1G patients bearing the hnRNPDL genetic B Phase separation mutations we studied here did not revealed the existence of con- B Dynamic light scattering (DLS) gophilic deposits or any type of inclusions (Sun et al., 2019; Vieira B Protein aggregation et al., 2014), nor did we observe them in Drosophila. The mech- B Transmission Electron Microscopy (TEM) anism by which hnRNPDL mutations elicits the LGMD1G pheno- B Congo Red (CR) binding type is still unknown. Here, we uncover that DL2 is inherently B Thioflavin-T (Th-T) aggregation kinetics assay aggregation prone and that the disease-causing mutations B Thioflavin-S (ThS) and proteostat staining exacerbate this propensity, resulting in a protein that despite B Congo Red (CR) birefringence being diffusely distributed in the nucleus of Drosophila myocytes B Fourier transform infrared (FT-IR) spectroscopy is totally insoluble. This evidence makes us think that LGMD1G B Mammalian molecular cloning is caused by a loss-of-function mechanism, in which DL2 B Mammalian cell culture and transfection function is lost because the protein aggregates or it is degraded B Immunofluorescence by the cellular protein quality control machinery, in line with the B Fluorescence recovery after photobleaching (FRAP) lower levels of mutant proteins detected in Drosophila. Consis- B Size exclusion chromatography (SEC) tent with this view, blocking the translation of hnRNPDL in B Drosophila stocks and culture zebrafish to reduce the endogenous protein levels results in d QUANTIFICATION AND STATISTICAL ANALYSIS restricted and uncoordinated animal movements and disorga- d DATA AND CODE AVAILABILITY nized myofibers (Vieira et al., 2014). Thus, it is feasible that LGMD1G patients would exhibit lower levels of soluble DL2 iso- SUPPLEMENTAL INFORMATION form in their muscular tissue, although this extent should be further confirmed. Importantly, like in the case of hnRNPA2 Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2019.12.080. and hnRNPA1, aggregation in hnRNPDL is triggered by the mutation of a well-conserved Asp residue in the PrLD. This recur- ACKNOWLEDGMENTS rent mutational change within three different hnRNP proteins indicates that these residues play a protective role against ag- We are grateful to Prof. T. Mittag, Dr. C. Mathieu, and Dr. M. Purice from St. gregation, likely by promoting electrostatic repulsion among Jude Children’s Research Hospital, USA, and UAB microscopy service of Bar- side chains of neighboring monomers (Murray et al., 2018). celona for their technical advice. We acknowledge Leticia Nogueira, Antonio hnRNPDL protein is an RNA-binding protein involved in tran- Fernando Ribeiro Jr., Mariz Vainzof, and Mayana Zatz from the Human scription and AS regulation (Li et al., 2019). The presence of Genome and Stem Cell Center, Biosciences Institute, University of Sa ~o Paulo, Brazil, for their help with muscle samples. S.V. acknowledges funding from the exon corresponding to its PrLD is responsible for the incor- MINECO (BIO2016-78310-R) and ICREA (ICREA-Academia 2016). J.P.T. ac- poration of hnRNPDL into multivalent hnRNP assemblies that, knowledges the Howard Hughes Medical Institute, National Institutes of Health in turn, control the AS of other genes (Gueroussov et al., 2017). (R35NS097974), and the St. Jude Research Collaborative on Membrane-less A recent study indicates that SRSF1 protein regulates AS of Organelles. X.S. acknowledges funding from Obra Social ‘‘la Caixa,’’ AGAUR hnRNPDL, generating the isoforms containing the PrLD, and (2017 SGR 324), MINECO (BIO2012-31043 and BIO2015-70092-R), and the therefore the execution of the downstream splicing program European Research Council (CONCERT, contract number 648201). IRB Bar- (Feng et al., 2019). It was suggested that SRSF1 may serve celona is the recipient of a Severo Ochoa Award of Excellence from MINECO (government of Spain). C.B. acknowledges funding from ‘‘Ministerio de Educa- as an upstream regulator of phase separation. We demonstrate ción y Formación Profesional.’’ here that this is the case. Indeed, we speculate that by regulating hnRNPDL AS, SRSF1 might also determine the size and dy- AUTHOR CONTRIBUTIONS namics of the complexes this protein forms in the nucleus, its nucleus-cytoplasm shuttling ability, and its amyloidogenic C.B. designed and performed the experiments, analyzed the data, and wrote potential. the manuscript. P.Y., M.C., J.M., M.P., and E.S. contributed to specific exper- iments. P.Y. supervised the experiments. H.J.K. designed the Drosophila ex- periments. M.C. and H.J.K. wrote the Drosophila section and composed Fig- STAR+METHODS ure 7. P.Y., M.P., E.S., X.S., H.J.K., and J.P.T. edited the manuscript. J.P.T. and S.V. designed the project. S.V. wrote the manuscript. Detailed methods are provided in the online version of this paper and include the following: DECLARATION OF INTERESTS d KEY RESOURCES TABLE The authors declare no competing interests. d LEAD CONTACT AND MATERIALS AVAILABILITY d EXPERIMENTAL MODEL AND SUBJECT DETAILS Received: August 5, 2019 B Bacterial Cell Culture Revised: November 12, 2019 Accepted: December 19, 2019 B Mammalian cell Culture Published: January 28, 2020 B Drosophila Culture d METHOD DETAILS REFERENCES B Isoforms sequence B Bacterial molecular cloning Akagi, T., Kamei, D., Tsuchiya, N., Nishina, Y., Horiguchi, H., Matsui, M., B Protein expression and purification Kamma, H., and Yamada, M. (2000). Molecular characterization of a mouse 1126 Cell Reports 30, 1117–1128, January 28, 2020 heterogeneous nuclear ribonucleoprotein D-like protein JKTBP and its tissue- Harrison, A.F., and Shorter, J. (2017). RNA-binding proteins with prion-like do- specific expression. Gene 245, 267–273. mains in health and disease. Biochem. J. 474, 1417–1438. Arao, Y., Kuriyama, R., Kayama, F., and Kato, S. (2000). A nuclear matrix-asso- Hu, X., Chen, X., Wu, B., Soler, I.M., Chen, S., and Shen, Y. (2017). Further ciated factor, SAF-B, interacts with specific isoforms of AUF1/hnRNP D. Arch. defining the critical genes for the 4q21 microdeletion disorder. Am. J. Med. Biochem. Biophys. 380, 228–236. Genet. A. 173, 120–125. Babinchak, W.M., Haider, R., Dumm, B.K., Sarkar, P., Surewicz, K., Choi, J.K., Iglesias, V., Conchillo-Sole, O., Batlle, C., and Ventura, S. (2019). AMYCO: and Surewicz, W.K. (2019). The role of liquid-liquid phase separation in aggre- evaluation of mutational impact on prion-like proteins aggregation propensity. gation of the TDP-43 low-complexity domain. J. Biol. Chem. 294, 6306–6317. BMC Bioinformatics 20, 24. Baralle, F.E., and Giudice, J. (2017). Alternative splicing as a regulator of devel- Ito, D., Hatano, M., and Suzuki, N. (2017). RNA binding proteins and the path- opment and tissue identity. Nat. Rev. Mol. Cell Biol. 18, 437–451. ological cascade in ALS / FTD neurodegeneration. Sci. Transl. Med. 9, Bateman, A., Martin, M.J., O’Donovan, C., Magrane, M., Apweiler, R., Alpi, E., eaah5436. Antunes, R., Arganiska, J., Bely, B., Bingley, M., et al.; UniProt Consortium Jobert, L., Pinzón, N., Van Herreweghe, E., Jády, B.E., Guialis, A., Kiss, T., and (2015). UniProt: a hub for protein information. Nucleic Acids Res. 43, D204– Tora, L. (2009). Human U1 snRNA forms a new chromatin-associated snRNP D212. with TAF15. EMBO Rep. 10, 494–500. Berardo, A., Lornage, X., Johari, M., Evangelista, T., Cejas, C., Barroso, F., Du- Kamei, D., and Yamada, M. (2002). Interactions of heterogeneous nuclear ribo- brovsky, A., Bui, M.T., Brochier, G., Saccoliti, M., et al. (2019). HNRNPDL- nucleoprotein D-like protein JKTBP and its domains with high-affinity binding related muscular dystrophy: expanding the clinical, morphological and MRI sites. Gene 298, 49–57. phenotypes. J. Neurol. 266, 2524–2534. Kamei, D., Tsuchiya, N., Yamazaki, M., Meguro, H., and Yamada, M. (1999). Biancalana, M., and Koide, S. (2010). Molecular mechanism of Thioflavin-T Two forms of expression and genomic structure of the human heterogeneous binding to amyloid fibrils. Biochim. Biophys. Acta 1804, 1405–1412. nuclear ribonucleoprotein D-like JKTBP gene (HNRPDL). Gene 228, 13–22. Boeynaems, S., Bogaert, E., Kovacs, D., Konijnenberg, A., Timmerman, E., Kato, M., Yang, Y.-S., Sutter, B.M., Wang, Y., McKnight, S.L., and Tu, B.P. Volkov, A., Guharoy, M., De Decker, M., Jaspers, T., Ryan, V.H., et al. (2019). Redox State Controls Phase Separation of the Yeast Ataxin-2 Protein (2017). Phase Separation of C9orf72 Dipeptide Repeats Perturbs Stress via Reversible Oxidation of Its Methionine-Rich Low-Complexity Domain. Granule Dynamics. Mol. Cell 65, 1044–1055.e5. Cell 177, 711–721.e8. Boeynaems, S., Alberti, S., Fawzi, N.L., Mittag, T., Polymenidou, M., Rous- Kawamura, H., Tomozoe, Y., Akagi, T., Kamei, D., Ochiai, M., and Yamada, M. seau, F., Schymkowitz, J., Shorter, J., Wolozin, B., Van Den Bosch, L., et al. (2002). Identification of the nucleocytoplasmic shuttling sequence of heteroge- (2018). Protein Phase Separation: A New Phase in Cell Biology. Trends Cell neous nuclear ribonucleoprotein D-like protein JKTBP and its interaction with Biol. 28, 420–435. mRNA. J. Biol. Chem. 277, 2732–2739. Bogaert, E., Boeynaems, S., Kato, M., Guo, L., Caulfield, T.R., Steyaert, J., Kim, H.J., Kim, N.C., Wang, Y.-D., Scarborough, E.A., Moore, J., Diaz, Z., Ma- Scheveneels, W., Wilmans, N., Haeck, W., Hersmus, N., et al. (2018). Molecu- cLea, K.S., Freibaum, B., Li, S., Molliex, A., et al. (2013). Mutations in prion-like lar Dissection of FUS Points at Synergistic Effect of Low-Complexity Domains domains in hnRNPA2B1 and hnRNPA1 cause multisystem proteinopathy and in Toxicity. Cell Rep. 24, 529–537.e4. ALS. Nature 495, 467–473. Bounedjah, O., Desforges, B., Wu, T.D., Pioche-Durieu, C., Marco, S., Hamon, King, O.D., Gitler, A.D., and Shorter, J. (2012). The tip of the iceberg: RNA- L., Curmi, P.A., Guerquin-Kern, J.L., Piétrement, O., and Pastré, D. (2014). binding proteins with prion-like domains in neurodegenerative disease. Brain Free mRNA in excess upon polysome dissociation is a scaffold for protein mul- Res. 1462, 61–80. timerization to form stress granules. Nucleic Acids Res. 42, 8678–8691. Lancaster, A.K., Nutter-Upham, A., Lindquist, S., and King, O.D. (2014). Brady, J.P., Farber, P.J., Sekhar, A., Lin, Y.H., Huang, R., Bah, A., Nott, T.J., PLAAC: a web and command-line application to identify proteins with prion- Chan, H.S., Baldwin, A.J., Forman-Kay, J.D., and Kay, L.E. (2017). Structural like amino acid composition. Bioinformatics 30, 2501–2502. and hydrodynamic properties of an intrinsically disordered region of a germ Li, R.Z., Hou, J., Wei, Y., Luo, X., Ye, Y., and Zhang, Y. (2019). hnRNPDL exten- cell-specific protein on phase separation. Proc. Natl. Acad. Sci. USA 114, sively regulates transcription and alternative splicing. Gene 687, 125–134. E8194–E8203. Liewluck, T., and Milone, M. (2018). Untangling the complexity of limb-girdle Burke, K.A., Janke, A.M., Rhine, C.L., and Fawzi, N.L. (2015). Residue-by-Res- muscular dystrophies. Muscle Nerve 58, 167–177. idue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Liu, Y., Zhu, X., Zhu, J., Liao, S., Tang, Q., Liu, K., Guan, X., Zhang, J., and Polymerase II. Mol. Cell 60, 231–241. Feng, Z. (2007). Identification of differential expression of genes in hepatocel- Das, R.K., and Pappu, R.V. (2013). Conformations of intrinsically disordered lular carcinoma by suppression subtractive hybridization combined cDNA mi- proteins are influenced by linear sequence distributions of oppositely charged croarray. Oncol. Rep. 18, 943–951. residues. Proc. Natl. Acad. Sci. USA 110, 13392–13397. Mackenzie, I.R., Nicholson, A.M., Sarkar, M., Messing, J., Purice, M.D., Dreyfuss, G., Kim, V.N., and Kataoka, N. (2002). Messenger-RNA-binding pro- Pottier, C., Annu, K., Baker, M., Perkerson, R.B., Kurti, A., et al. (2017). TIA1 teins and the messages they carry. Nat. Rev. Mol. Cell Biol. 3, 195–205. Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia El-Gebali, S., Mistry, J., Bateman, A., Eddy, S.R., Luciani, A., Potter, S.C., Qur- Promote Phase Separation and Alter Stress Granule Dynamics. Neuron 95, eshi, M., Richardson, L.J., Salazar, G.A., Smart, A., et al. (2019). The Pfam pro- 808–816.e9. tein families database in 2019. Nucleic Acids Res. 47 (D1), D427–D432. Maharana, S., Wang, J., Papadopoulos, D.K., Richter, D., Pozniakovsky, A., Feng, H., Bao, S., Rahman, M.A., Weyn-Vanhentenryck, S.M., Khan, A., Wong, Poser, I., Bickle, M., Guillén-boixet, J., Franzmann, T., Jahnel, M., et al. J., Shah, A., Flynn, E.D., Krainer, A.R., and Zhang, C. (2019). Modeling RNA- (2018). RNA buffers the phase separation behavior of prion-like RNA binding Binding Protein Specificity In Vivo by Precisely Registering Protein-RNA proteins. Science 360, 918–921. Crosslink Sites. Mol. Cell 74, 1189–1204.e6. March, Z.M., King, O.D., and Shorter, J. (2016). Prion-like domains as epige- Goldschmidt, L., Teng, P.K., Riek, R., and Eisenberg, D. (2010). Identifying the netic regulators, scaffolds for subcellular organization, and drivers of neurode- amylome, proteins capable of forming amyloid-like fibrils. Proc. Natl. Acad. generative disease. Brain Res. 1647, 9–18. Sci. USA 107, 3487–3492. Marko, M., Vlassis, A., Guialis, A., and Leichter, M. (2012). Domains involved in Gueroussov, S., Weatheritt, R.J., O’Hanlon, D., Lin, Z.Y., Narula, A., Gingras, TAF15 subcellular localisation: dependence on cell type and ongoing tran- A.C., and Blencowe, B.J. (2017). Regulatory Expansion in Mammals of Multi- scription. Gene 506, 331–338. valent hnRNP Assemblies that Globally Control Alternative Splicing. Cell 170, Molliex, A., Temirov, J., Lee, J., Coughlin, M., Kanagaraj, A.P., Kim, H.J., Mit- 324–339.e23. tag, T., and Taylor, J.P. (2015). Phase separation by low complexity domains Cell Reports 30, 1117–1128, January 28, 2020 1127 promotes stress granule assembly and drives pathological fibrillization. Cell Tsuchiya, N., Kamei, D., Takano, A., Matsui, T., and Yamada, M. (1998). Clon- 163, 123–133. ing and characterization of a cDNA encoding a novel heterogeneous nuclear Murray, D.T., Zhou, X., Kato, M., Xiang, S., Tycko, R., and McKnight, S.L. ribonucleoprotein-like protein and its expression in myeloid leukemia cells. (2018). Structural characterization of the D290V mutation site in hnRNPA2 J. Biochem. 123, 499–507. low-complexity-domain polymers. Proc. Natl. Acad. Sci. USA 115, E9782– Vernon, R.M., Chong, P.A., Tsang, B., Kim, T.H., Bah, A., Farber, P., Lin, H., E9791. and Forman-Kay, J.D. (2018). Pi-Pi contacts are an overlooked protein feature Murthy, A.C., Dignon, G.L., Kan, Y., Zerze, G.H., Parekh, S.H., Mittal, J., and relevant to phase separation. eLife 7, e31486. Fawzi, N.L. (2019). Molecular interactions underlying liquid-liquid phase sepa- Vieira, N.M., Naslavsky, M.S., Licinio, L., Kok, F., Schlesinger, D., Vainzof, M., ration of the FUS low-complexity domain. Nat. Struct. Mol. Biol. 26, 637–648. Sanchez, N., Kitajima, J.P., Gal, L., Cavaçana, N., et al. (2014). A defect in the Navarro, S., Marinelli, P., Diaz-Caballero, M., and Ventura, S. (2015). The prion- RNA-processing protein HNRPDL causes limb-girdle muscular dystrophy 1G like RNA-processing protein HNRPDL forms inherently toxic amyloid-like in- (LGMD1G). Hum. Mol. Genet. 23, 4103–4110. clusion bodies in bacteria. Microb. Cell Fact. 14, 102. Wang, J., Choi, J.-M., Holehouse, A.S., Lee, H.O., Zhang, X., Jahnel, M., Ma- Nedelsky, N.B., and Taylor, J.P. (2019). Bridging biophysics and neurology: harana, S., Lemaitre, R., Pozniakovsky, A., Drechsel, D., et al. (2018). A Molec- aberrant phase transitions in neurodegenerative disease. Nat. Rev. Neurol. ular Grammar Governing the Driving Forces for Phase Separation of Prion-like 15, 272–286. RNA Binding Proteins. Cell 174, 688–699.e16. Nigro, V., and Savarese, M. (2014). Genetic basis of limb-girdle muscular dys- Wegmann, S., Eftekharzadeh, B., Tepper, K., Zoltowska, K.M., Bennett, R.E., trophies: the 2014 update. Acta Myol. 33, 1–12. Dujardin, S., Laskowski, P.R., MacKenzie, D., Kamath, T., Commins, C., et al. Patel, A., Lee, H.O., Jawerth, L., Maharana, S., Jahnel, M., Hein, M.Y., Stoy- (2018). Tau protein liquid-liquid phase separation can initiate tau aggregation. nov, S., Mahamid, J., Saha, S., Franzmann, T.M., et al. (2015). A Liquid-to- EMBO J. 37, e98049. Solid Phase Transition of the ALS Protein FUS Accelerated by Disease Muta- tion. Cell 162, 1066–1077. Wu, C., Scott, J., and Shea, J.E. (2012). Binding of Congo red to amyloid pro- tofibrils of the Alzheimer Ab(9-40) peptide probed by molecular dynamics sim- Peskett, T.R., Rau, F., O’Driscoll, J., Patani, R., Lowe, A.R., and Saibil, H.R. ulations. Biophys. J. 103, 550–557. (2018). A Liquid to Solid Phase Transition Underlying Pathological Huntingtin Exon1 Aggregation. Mol. Cell 70, 588–601.e6. Wu, Y.Y., Li, H., Lv, X.Y., Wei, Q., Li, X., Liu, X.Y., Zhou, Q., and Wei, Y.Q. Piñol-Roma, S., and Dreyfuss, G. (1991). Transcription-dependent and tran- (2008). Overexpression of JKTBP1 induces androgen-independent LNCaP scription-independent nuclear transport of hnRNP proteins. Science 253, cell proliferation through activation of epidermal growth factor-receptor 312–314. (EGF-R). Cell Biochem. Funct. 26, 467–477. Piñol-Roma, S., and Dreyfuss, G. (1992). Shuttling of pre-mRNA binding pro- Yang, L., Gal, J., Chen, J., and Zhu, H. (2014). Self-assembled FUS binds teins between nucleus and cytoplasm. Nature 355, 730–732. active chromatin and regulates gene transcription. Proc. Natl. Acad. Sci. Ryan, V.H., Dignon, G.L., Zerze, G.H., Chabata, C.V., Silva, R., Conicella, A.E., USA 111, 17809–17814. Amaya, J., Burke, K.A., Mittal, J., and Fawzi, N.L. (2018). Mechanistic View of Ying, Y., Wang, X.J., Vuong, C.K., Lin, C.H., Damianov, A., and Black, D.L. hnRNPA2 Low-Complexity Domain Structure, Interactions, and Phase Sepa- (2017). Splicing Activation by Rbfox Requires Self-Aggregation through Its ration Altered by Mutation and Arginine Methylation. Mol. Cell 69, 465–479.e7. Tyrosine-Rich Domain. Cell 170, 312–323.e10. Straub, V., Murphy, A., and Udd, B.; LGMD workshop study group (2018). ). Zhang, P., Ji, D., Hu, X., Ni, H., Ma, W., Zhang, X., Liao, S., Zeng, Z., Zhao, Y., 229th ENMC international workshop: Limb girdle muscular dystrophies – and Zhou, H. (2018). Oncogenic heterogeneous nuclear ribonucleoprotein D- Nomenclature and reformed classification Naarden, the Netherlands, 17–19 like promotes the growth of human colon cancer SW620 cells via its regulation March 2017. Neuromuscul. Disord. 28, 702–710. of cell-cycle. Acta Biochim. Biophys. Sin. (Shanghai) 50, 880–887. Su, H., Kodiha, M., Lee, S., and Stochaj, U. (2013). Identification of novel Zhang, T., Delestienne, N., Huez, G., Kruys, V., and Gueydan, C. (2005). Iden- markers that demarcate the nucleolus during severe stress and chemothera- tification of the sequence determinants mediating the nucleo-cytoplasmic peutic treatment. PLoS ONE 8, e80237. shuttling of TIAR and TIA-1 RNA-binding proteins. J. Cell Sci. 118, 5453–5463. Sun, Y., Chen, H., Lu, Y., Duo, J., Lei, L., OuYang, Y., Hao, Y., Da, Y., and Shen, X.M. (2019). Limb girdle muscular dystrophy D3 HNRNPDL related in a Chi- Zhou, H., Ge, Y., Sun, L., Ma, W., Wu, J., Zhang, X., Hu, X., Eaves, C.J., Wu, D., nese family with distal muscle weakness caused by a mutation in the prion- and Zhao, Y. (2014). Growth arrest specific 2 is up-regulated in chronic like domain. J. Neurol. 266, 498–506. myeloid leukemia cells and required for their growth. PLoS ONE 9, e86195. Tannukit, S., Wen, X., Wang, H., and Paine, M.L. (2008). TFIP11, CCNL1 and Zinszner, H., Immanuel, D., Yin, Y., Liang, F.X., and Ron, D. (1997). A topogenic EWSR1 Protein-protein Interactions, and Their Nuclear Localization. Int. J. role for the oncogenic N-terminus of TLS: nucleolar localization when tran- Mol. Sci. 9, 1504–1514. scription is inhibited. Oncogene 14, 451–461. 1128 Cell Reports 30, 1117–1128, January 28, 2020 STAR+METHODS KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal to hnRNPDL Abcam Cat#ab183136 Mouse monoclonal anti G3BP BD Biosciences Cat#611127; RRID: AB_398438 Alexa Fluor 555 Molecular Probes Cat#A31572; RRID: AB_162543 Mouse monoclonal anti GAPDH Santa Cruz Cat#sc47724; RRID: AB_627678 Sheep polyclonal anti tubulin Cytoskeleton Inc Cat#ATN02; RRID:AB_10709401 Mouse monoclonal anti C23 Santa Cruz Cat#sc-8031; RRID: AB_670271 Bacterial and Virus Strains One Shot TOP10 chemically competent E. coli Life Technologies Cat#C404003 HI-Control CL21(DE3) chemically competent Lucigen Corporation Cat#60435-1 cells (SOLOS) Chemicals, Peptides, and Recombinant Proteins 1x TrypLE Express Thermo Fisher Scientific Cat #12604-013 Lipofectamine2000 Thermo Fisher Scientific Cat# 11668027 His-SUMO-hnRNPDL isoform 1 This study N/A His-SUMO-hnRNPDL isoform 2 This study N/A His-SUMO-hnRNPDL isoform 3 This study N/A His-SUMO-hnRNPDL isoform 2 D259N This study N/A His-SUMO-hnRNPDL isoform 2 D259H This study N/A Oregon Green Protein Labeling Kit Molecular Probes Cat #O10241 Texas Red Protein Labeling Kit Molecular Probes Cat#T10244 Q5 site directed mutagenesis kit New England Biolabs Cat#E0554S Expresso T7 SUMO cloning and expression system Lucigen Corporation Cat#49003-2 NEBuilder HiFi DNA Assembly Master Mix New England Biolabs Cat#E2621S Ficoll400 Sigma-Aldrich Cat#F2637 Secure seal imaging spacers 1.9x0.12 mm Grace Biolabs Cat#654002 Proteostat aggresome detection kit Enzo life sciences Cat#ENZ-51035-K100 Experimental Models: Cell Lines HeLa cells ATCC Cat #CCL-2; RRID: CVCL_0030 HeLa hnRNPDL knock out cells This study N/A Experimental Models: Organisms/Strains Drosophila melanogaster: cell line w1118 Bloomington Drosophila Stock Center Cat#3605 Drosophila melanogaster: cell line Mhc-Gal4 Bloomington Drosophila Stock Center Cat#55133 Drosophila melanogaster: cell line UAS-hnRNPDL Bestgene Inc. N/A isoform 2 Drosophila melanogaster: cell line UAS-hnRNPDL Bestgene Inc. N/A isoform 2 D259N Drosophila melanogaster: cell line UAS-hnRNPDL Bestgene Inc. N/A isoform 2 D259H Oligonucleotides See Table S3 for the primer list Recombinant DNA pET28a-hnRNPDL isoform 1 GenScript N/A pETite-His-SUMO Kan vector Lucigen Corporation Cat#49003-1 pETite-His-SUMO-hnRNPDL isoform 1 This study N/A pETite-His-SUMO-hnRNPDL isoform 2 This study N/A (Continued on next page) Cell Reports 30, 1117–1128.e1–e5, January 28, 2020 e1 Continued REAGENT or RESOURCE SOURCE IDENTIFIER pETite-His-SUMO-hnRNPDL isoform 3 This study N/A pETite-His-SUMO-hnRNPDL isoform 2 D259N This study N/A pETite-His-SUMO-hnRNPDL isoform 2 D259H This study N/A pEGFP-C3 vector Clontech Cat#6082-1 pEGFP-C3-hnRNPDL isoform 1 This study N/A pEGFP-C3-hnRNPDL isoform 2 This study N/A pEGFP-C3-hnRNPDL isoform 3 This study N/A pSpCas9(BB)-2A-Puro (PX459) V2.0 Addgene Cat# 62988; RRID:Addgene_62988 pUASTattB Drosophila Genomics Resource Center Cat#1419 Software and Algorithms SigmaPlot 10.0 Systal Software http://www.sigmaplot.co.uk GaphPad Prism 5 GraphPad https://www.graphpad.com/ ImageJ 1.51J Software NIH https://imagej.nih.gov/ij/download.html Igor Pro 7.0 Wavemetrics https://www.wavemetrics.com/ LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagent should be directed to and will be fulfilled by the Lead Contact, S. Ventura (Salvador.ventura@uab.es). All unique/stable reagents generated in this study will be made available on request but we may require a payment and/or a completed Materials Transfer Agreement if there is potential for commercial application. EXPERIMENTAL MODEL AND SUBJECT DETAILS Bacterial Cell Culture cDNA clones were transformed into One Shot TOP10 and BL21(DE3) SOLOS chemically competent E. coli cells (Thermo Fisher Sci- entific). Single colonies were grown overnight at 37 C and 220 rpm in LB media containing kanamycin selection antibiotic at a con- centration of 50 mg/ml. All competent bacterial cells were stored at 80 C in 15% glycerol. Mammalian cell Culture HeLa cells (of female origin) were grown at 37 C and 5% CO2, and maintained in DMEM High Glucose (Hyclone SH30022.01) medium supplemented with 10% fetal bovine serum. Cells were passaged and plated using 1X TrypLE Express (Thermo Fisher Scientific). Cells were authenticated by short tandem repeat (STR) profiling. Drosophila Culture All Drosophila stocks were maintained on standard diet in a 25 C incubator with a 12 h day/night cycle. METHOD DETAILS Isoforms sequence hnRNPDL isoform 1, 2 and 3 (DL1, DL2 and DL3) sequences were obtained from Uniprot (Bateman et al., 2015): MEVPPRLSHVPPPLFPSAPATLASRSLSHWRPRPPRQLAPLLPSLAPSSARQGARRAQRHVTAQQPSRLAGGAAIKGGRRRRP DLFRRHFKSSSIQRSAAAAAATRTARQHPPADSSVTMEDMNEYSNIEEFAEGSKINASKNQQDDGKMFIGGLSWDTSKKDLTEYLSR FGEVVDCTIKTDPVTGRSRGFGFVLFKDAASVDKVLELKEHKLDGKLIDPKRAKALKGKEPPKKVFVGGLSPDTSEEQIKEYFGAFGEIENI ELPMDTKTNERRGFCFITYTDEEPVKKLLESRYHQIGSGKCEIKVAQPKEVYRQQQQQQKGGRGAAAGGRGGTRGRGRGQgqnwnqgf nnyydqgygnynsayggdqnysgyggydytgynygnygygqgyadysgqQSTYGKASRGGGNHQNNYQPY Bold corresponds to the N terminus Arg-rich domain present in DL1; lowercase italic corresponds to the C terminus Tyr-rich domain present in DL1 and DL2. Bacterial molecular cloning pET28a-hnRNPDL vector was purchased from GenScript. This plasmid has assembled the synthetic human gene hnRNPDL isoform 1 (DL1) with optimized codon for bacterial expression. DL1 sequence was amplified by polymerase chain reaction (PCR) using e2 Cell Reports 30, 1117–1128.e1–e5, January 28, 2020 plasmid pET28a-hnRNPDL as a template. The PCR fragment was cloned into pETite-His-SUMO kan vector using the Expresso T7 SUMO cloning and expression system (Lucigen Corporation) obtaining the pETite-SUMO-DL1 vector. The final vector was trans- formed into One Shot TOP10 chemically competent E. coli cells (Thermo Fisher Scientific). pETite-SUMO-DL1 was used as a template for isoform 2 construct (DL2), missing the first 119 amino acids, using the Q5 site directed mutagenesis kit (New England Biolabs). hnRNPDL isoform 2 point mutations D259N/H (DL2N and DL2H) and isoform 3 construct (DL3), missing the first 119 amino acids and residues at position 343-399, were obtained using pETite-SUMO-DL2 plasmid as a template and the Q5 site directed mutagen- esis kit (New England Biolabs). DNA fragments of R/K and Y/F mutated regions were purchased from GenScript in order to obtain pETite-SUMO-DL-R/K, Y/F and R/K+Y/F plasmids. pETite-SUMO-DL1 was used as a template for these constructs using the Q5 site directed mutagenesis kit (New England Biolabs) and DNA fragments were inserted using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). hnRNPDL-Nt was obtained using pETite-SUMO-DL1 plasmid as a template and the Q5 site directed mutagenesis kit (New England Biolabs). Once confirmed the sequences of the final vectors, they were transformed into chemically competent E. coli BL21 DE3 cells from Expresso T7 SUMO cloning and expression system (Lucigen Corporation) for protein expression. Protein expression and purification 100 mL of Luria Broth (LB) with 50 mg/ml kanamycin (kan) were inoculated by single colony of BL21 cells with pETite-SUMO-DL1/2/ 2N/2H/3 and incubated overnight at 37 C and 220 rpm (New Brunswick Innova 44R shaker). 25 mL of saturated overnight culture was transferred into 1 L LB-kan and incubated at 37 C and 220 rpm. The protein expression was induced at OD600 = 0.5 by addition of isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM. The induced culture was incubated for additional 3 h at 37 C and 220 rpm. Cells were then harvested by centrifugation for 15 min at 4,000 g and 4 C (Sorvall LYNX 6000. Thermo Scien- tific). The cell pellet was frosted at 80 C. Pellets from 2 L cell culture were resuspended in 40 mL binding buffer (50 mM HEPES pH 7.5, 1 M NaCl, 5% glycerol, 20 mM imid- azole, 1 mM dithiothreitol (DTT)) supplemented with, 0.2 mg/ml lysozyme, 20 mg/ml DNase and 1 tablet of protease inhibitor cocktail EDTA free (Roche). The suspension was incubated for 20 min at 4 C with slow agitation and then lysed by passing through a LM10 microfluidizer (Microfluidics) at 18.000 psi. Lysate cells were centrifuged for 30 min at 30,000 g and 4 C. The supernatant was filtered through a 0.45 mm PVDF membrane and loaded onto a HisTrap FF Ni-column (GE Healthcare) at a flow rate of 2 ml/min. Protein was eluted by an imidazole gradient over 10 column volumes starting from 0 to 100% of elution buffer (50 mM HEPES pH 7.5, 1 M NaCl, 5% glycerol, 500 mM imidazole, 1 mM DTT). His purified protein was treated with 0.2 mg/ml RNase (Thermo Scientific) for 15 min at 37 C and 1 mM PMSF was added to avoid protein degradation. Then, protein was filtered using 0.22 mm PVDF membrane, concen- trated using 10 K Amicon (Millipore) to 5 ml, filtered again and subjected to size exclusion chromatography on a Superdex 200 16/600 column (GE Healthcare) equilibrated in 50 mM HEPES pH 7.5, 1 M NaCl, 5% glycerol and 1 mM DTT. The fractions were analyzed by SDS-PAGE, pooled, concentrated, filtered and stored in small volume aliquots at 80 C. Absence of RNA was confirmed by 260/280 absorbance ratio using Nanodrop 8000 (Thermo Scientific). Protein concentration was determined by OD280 absorbance using Nanodrop 8000 (Thermo Scientific). For SUMO-hnRNPDL-Nt purification, with a calculated isoelectric point of 10.24, an ion exchange chromatography with a HiTrap SP column (GE Healthcare) using a salt gradient from 50 mM to 1 M NaCl was performed before the size exclusion chromatography. Phase separation Purified proteins were diluted to desired protein and salt concentrations in 50 mM HEPES pH 7,5. Dilution of salt from 1 M NaCl (stor- age buffer) induced phase separation. A secure seal imaging spacer (Grace Biolabs) was used between slide and coverslip to visu- alize protein droplets in a Leica TCS SP8 microscope. Ficoll400 (Sigma) was used in addition to induce phase separation for DL2. For DL2:hnRNPDL-Nt colocalization experiments, DL2 was green labeled with Oregon Green and hnRNPDL-Nt was red label with Texas Red using molecular probes protein labeling kits (Invitrogen) as described in the commercial protocol. A stock at 100 mM in 150 mM NaCl was prepared for each protein and then proteins were mixed at 1:1 ratio making posterior serial dilutions from the 50 mM stock mixture. For RNA experiments, DL1 was used at 6.25 mM final concentration to obtain droplets of 2-6 mm and ensure that the surface was not fully covered with droplets to facilitate imaging. Total RNA was obtained from HeLa cells following the TRIzol Reagent user guide (Invitrogen). RNA diluted in RNase-free water was added at the indicated concentrations and posterior addition of 5 ng/ml of RNase (Thermo Scientific) was used to induce again LLPS. Dynamic light scattering (DLS) hnRNPDL isoforms protein size was determined using a DynaPro NanoStar (Wyatt technologies) in 50 mM HEPES pH 7.5 and 150 mM NaCl at different protein concentrations per duplicate. Protein radius was extracted from mass data. Cell Reports 30, 1117–1128.e1–e5, January 28, 2020 e3 Protein aggregation Right before each experiment, the stock solutions were diluted to 50 mM in 50 mM HEPES buffer pH 7.4 and 150 mM NaCl. For ag- gregation assays the samples were incubated for 4 days at 37 C and 600 rpm. Transmission Electron Microscopy (TEM) The morphology of the aggregated proteins was evaluated by negative staining and using a JEOL TEM-1400Plus Transmission Electron Microscope, 80 KV. 50 mM aggregated protein solution was sonicated for 10 min in an ultrasonic bath (Fisher Scientific FB15052) and then diluted to 1 mM final concentration in H2O. 5 ml of the diluted solution was placed on carbon-coated copper grids and incubated for 5 min. The grids were then washed and stained with 5 ml of 2% w/v uranyl acetate for 2 min. Then, grids were washed again before analysis. Congo Red (CR) binding CR binding to aggregated proteins was analyzed using a Specord 200 Plus spectrophotometer (Analyticjena). The absorbance spectra were recorded from 400 to 650 nm. 10 mL of 50 mM aggregated protein was added to 90 ml of 20 mM CR in 50 mM HEPES buffer pH 7,4 and 150 mM NaCl and was incubated at room temperature for 5 min before the measurement. The same buffer with 20 mM CR and without protein was employed as a control. Thioflavin-T (Th-T) aggregation kinetics assay The fluorescence spectra of Th-T were recorded using a Perkin Elmer EnSpire Multimode plate reader. Reactions were carried out at 50 mM protein concentration in a solution containing 20 mM of Th-T in 50 mM HEPES buffer pH 7,4 and 150 mM NaCl at 37 C. The aggregation kinetics were followed monitoring the changes in Th-T fluorescence intensity at 495 nm every 5 min with prior shaking at 100 rpm for 15 s. The same buffer with 20 mM ThT and without protein was employed as a control. Thioflavin-S (ThS) and proteostat staining 100 mL of aggregates protein was incubated for 1 h in the presence of 150 mM ThS (Sigma) or 1/2000 Proteostat dilutuion (Enzo Life Sciences) in 50 mM HEPES buffer pH 7,4 and 150 mM NaCl. Then, the samples were washed two times with the same buffer. Finally, the precipitated fraction was resuspended in a final volume of 10 mL and placed on a microscope slide and sealed. Images were ob- tained at 20x magnification in an Eclipse 90i fluorescence microscope. Congo Red (CR) birefringence Aggregated protein was first sonicated for 10 min in an ultrasonic bath (Fisher Scientific FB15052). 10 mL sonicated aggregated protein was incubated for 1 h in the presence of 100 mM CR (Sigma). 5 mL sample was placed on a microscope slide and viewed at 10x magnification with a Leica stereoscopic microscope MZFLIII. Fourier transform infrared (FT-IR) spectroscopy 100 mL aggregated protein was centrifuged and washed two times with H2O to remove the presence of salts. The final pellet was resupended in 10 mL H2O. FTIR experiments were performed using a Bruker Tensor 27 FT-IR spectrometer (Bruker Optics Inc) with a Golden Gate MKII ATR accessory. Each spectrum consists of 32 independent scans, measured at a spectral resolution of 4 cm-1 within 1800-1500 cm-1 range. All spectral data were acquired and normalized using the OPUS MIR Tensor 27 software. Data was afterward deconvoluted using the Peak Fit 4.12 program. Mammalian molecular cloning hnRNPDL isoform 1 sequence was amplified by PCR from cDNA extract of U2OS cells and assembled to pEGFP-C3 HindIII and BamHI digested plasmid using the NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs). Similar to bacterial molecular cloning, pEGFP-C3-DL1 plasmid was used as a template for isoform 2 (DL2) construct using the Q5 site directed mutagenesis kit (New England Biolabs). hnRNPDL isoform 3 (DL3) was obtained using pEGFP-C3-DL2 plasmid as a template and the Q5 site directed mutagenesis kit (New England Biolabs). The final vectors were transformed into One Shot TOP10 chemically competent E. coli cells (Thermo Fisher Scientific). Mammalian cell culture and transfection HeLa hnRNPDL KO (HeLaDL-KO) cell line was performed by CRISPR-Cas9 with following gRNA: gRNA1: ATTCTTGCTCGCGTTG ATCT; gRNA2: ACAGAGTACTTGTCTCGATT. Both gRNA target the common exons in all 3 isoforms. pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid # 62988; http://addgene.org/62988; RRID:Addgene_62988). HeLaDL-KO cells were grown and maintained in DMEM High Glucose medium (Hyclone) supplemented with 10% fetal bovine serum (FBS) (Hyclone) at 37 C and 5% CO2. Cells were passaged and plated using 1x TrypLE Express (Thermo Fisher Scientific). For cellular transfection, HeLaDL-KO cells were seeded on 4-well or 8-well glass slides (Millipore) for immunofluorescence or 4-well lab-Tek chambered coverglass (Nunc) for FRAP. Cells were transfected 24h after seeding with 400 ng DNA using Lipofectamine2000 (Invitrogen). After 4 h of transfection, we changed cellular media to fresh one. Cellular stress was applied 24h post transfection. e4 Cell Reports 30, 1117–1128.e1–e5, January 28, 2020 Immunofluorescence Transfected HeLaDL-KO cells were stressed or not with 5 mg/ml actinomycin D for 3 h. Cells were then fixed with 4% parafolmaldehyde (Electron Microscopy Sciences, #15713-S), permeabilized with 0,2% Triton X-100 and blocked with 1% bovine serum albumin (BSA). Primary antibodies used were against G3BP (611127; BD Biosciences), nucleolin (sc-8031; Santa Cruz) and hnRNPDL (ab183136; Abcam). For visualization, the appropriate host-specific Alexa Fluor 488 or 555 (Molecular Probes) secondary antibodies were used. Slides were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen). Images were captured using a Leica TCS SP8 STED 3x confocal microscope (Leica Biosystems) with a 63x oil objective. Fluorescence recovery after photobleaching (FRAP) FRAP experiments were performed with the Opterra II Swept Field Confocal Microscope (Bruker) using Prairie View 5.5 Software. Immediately before imaging, the medium was changed to 500 mL complete phenol red-free DMEM medium (HyClone). During imag- ing, cells were maintained at 37 C and supplied with 5% CO2 using a Bold Line Cage Incubator (Okolabs) and an objective heater (Bioptechs). Imaging was performed using a 60x Plan Apo 1.40NA oil objective and Perfect Focus (Nikon) was engaged for the duration of the capture. For imaging, cells were selected based on level of intensity. Time lapses were taken using the 488-nm imaging laser set at 100 power and 100-ms exposure with acquisition set at max speed (0.5 ms period) for 100 frames. Photobleaching of the nucleus occurred 2 s into capture, using the 488-nm FRAP laser to bleach the green channel. Data was repeated in triplicate for each con- dition, with each replicate having at least n = 10 cells. Data was opened in ImageJ 1.51J (NIH) using the Prairie Reader plugin. ROIs were generated in the photobleach region, a non-photobleached cell, and the background for each timelapse, and the mean intensity of each was extracted. These values were exported into Igor Pro 7.0 (WaveMetrics), where photobleach and background correction were performed, and fit FRAP curves using Hill’s equation were generated. Size exclusion chromatography (SEC) We analyzed individually the elution profile of the contents of cells expressing EGFP-hnRNPDL isoforms, one isoform at a time. A total of 3 dishes of 10 cm per each hnRNPDL isoform transfection were pooled for SEC analysis. Experiments were performed in triplicate for each isoform. HeLaDL-KO cells were transfected 24h after seeding with 3 mg DNA using Lipofectamine2000 (Invitrogen) and media was changed to fresh one after 5 h of transfection. Cells were collected 24 h post transfection, resuspended in 0.5 mL of 1xPBS supplemented with protease inhibitor cocktail (Roche) and lysed using 1 mL syringe (5-10 passes). Protein solubilization was confirmed by Bradford. Supernatant was collected by 5 min centrifugation at 1,000 g and 4 C, filtered with 0.45 mm PVDF membrane and loaded on a Superose 6 Increase 10/300 GL column (GE Healthcare) equilibrated with 1xPBS. The fractions were analyzed by Western Blot using a primary antibody against hnRNPDL (ab183136; Abcam) and the appropriate host-specific secondary antibody (IRDye 680RD Goat anti-Rabbit IgG (H + L), LI-COR, P/N 926-68071). The elution pattern of endogenous hnRNPDL in untransfected HeLa WT cells was also analyzed. Drosophila stocks and culture Gene sequences of hnRNPDL isoform 2 (DL2) WT, DL2 D259H, and DL2 D259N were synthesized and subcloned into the pUASTattB Drosophila expression vector (BioBasic Inc.). Flies carrying the transgenes were generated by performing a standard in- jection through the 4C31 integrase-mediated transgenesis technique (BestGene Inc.). All Drosophila stocks were maintained in a 25 C incubator with a 12 h day/night cycle. Eye phenotypes were imaged by light microscopy. The w1118 line was used as control. To prepare adult fly muscle for immunofluorescence, the Mhc-Gal4 driver was used to express the transgene in muscle at 25 C. Adult flies were embedded in a drop of OCT compound (Sakura Finetek) on a glass slide, frozen with liquid nitrogen and bisected sagitally by a razor blade. After fixing with 4% paraformaldehyde in PBS, hemithoraces were permeabilized with PBS containing 0.2% Triton X-100 and stained with anti-hnRNPDL antibody (ab183136; Abcam). Hemithoraces were additionally stained by Alexa Fluor 647 (Life Technologies) and DAPI according to manufacturer’s instructions. Stained hemi-thoraces were mounted in 80% glycerol, and the musculature was examined by STED (Leica SP8). QUANTIFICATION AND STATISTICAL ANALYSIS Statistical analyses were performed using GraphPad Prism v5. All data are shown as the mean ± standard error of the mean (SEM). The statistical significance of each isoform compared to unfused EGFP was investigated by ordinary two-way ANOVA followed by Dunnett’s multiple comparisons test. The number of samples analyzed per experiment is provided in the corresponding method details section. A p value of less than 0.05 was used to determine significance. DATA AND CODE AVAILABILITY This study did not generate any unique datasets or code. Cell Reports 30, 1117–1128.e1–e5, January 28, 2020 e5