State Letter

Eszter E. Najbauer, Sheung Chun Ng, Christian Griesinger, Dirk Görlich & Loren B. Andreas

TL;DR: Perfectly repetitive Nup98 FG sequences form condensates through cohesive interactions between hydrophobic residues, while showing high local rotational mobility. These measurements provide insights into how the FG-rich phase could show both kinetic stability and the dynamic behavior for its permeability barrier and transport functions.

Nuclear pore complexes (NPCs) maintain the nucleocytoplasmic compartmentalization in eukaryotic cells through a selective permeability barrier, formed by a dense phase of intrinsically disordered, Phe-Gly (FG) rich proteins called FG-Nups. In vitro, purified FG-Nups such as Nup98 have been shown to phase separate, forming an FG-rich phase which can recapitulate the transport behavior of the NPC. However, the dynamics and interactions between the FG motifs and how these mediate the transport-relevant properties are not completely understood.

To provide insight into this, Najbauer et al. study engineered, perfectly repetitive versions of Nup98 (prfGLFG_52x12) with a variety of methods like fluorescence recovery after photobleaching and MAS NMR. They show that the prfGLFG_52x12 sequence displays lower critical solution temperature phase behavior, which is often driven by hydrophobic interactions. Moreover, the FG phases showed low translational mobility and high local rotational mobility, thus exhibiting non-Brownian behavior. The local mobility was decreased with increasing the temperature or salt concentration. Additionally, NOESY measurements pointed to proximities between the hydrophobic groups within the FG phase. Guided by these results, the authors also conducted several mutagenesis experiments. Their results emphasized the importance of hydrophobic residues Phe and Leu in the strength of cohesive interactions and the intra-phase dynamics of the FG repeats.

Taken together, these results provide insight into the entropically driven cohesive interactions and dynamics in the FG phase, revealing how it could result in a permeability barrier which is simultaneously kinetically stable and dynamic.

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Yongjia Duan, Aiying Du , Jinge Gu, Gang Duan , Chen Wang, Xinrui Gui, Zhiwei Ma, Beituo Qian, Xue Deng, Kai Zhang, Le Sun, Kuili Tian , Yaoyang Zhang Hong Jiang Cong Liu and Yanshan Fang

TLDR; Post-translational modifications of RNA binding proteins is known to alter phase separation and ribonucleoprotein (RNP) granule formation. Duan et al. demonstrate that poly(ADP-ribosyl)ation (PARylation) of the PAR binding motif of hnRNPA1 regulates its association with stress granules while modification of its K298 PARylation site exhibits alterations to nuclear localization. Interestingly, PARylation of hnRNPA1 promotes co-phase separation of hnRNPA1 and TDP-43 in vitro and interaction in vivo. Genetic and pharmacological inhibition of PAR polymerase results in hnRNPA1 and TDP43 mediated toxicity in ALS models of Drosophila.

RNA binding proteins such as hnRNPA1 contain highly conserved domain structures that are putatively disordered and participate in many crucial cellular processes including, but not limited to, DNA damage repair, transcriptional activation, nuclear localization, and stress granule formation. Familial ALS-associated mutations in hnRNPA1 are known to promote formation of the pathological aggregates that are a hallmark of ALS, yet little is known about how mutations and post-translational modifications such as PARylation of RNA binding proteins impact their stability and ultimately tune phase separation.

Previous literature has identified important hnRNPA1 PAR bindinding sites at K298 and within a ‘PAR binding domain’ by mass-spectrometry. Based on these findings, Duan et al. expressed FLAG-tagged hnRNPA1 in HeLa cells and observed PAR polymerase dependent modulation of stress granule formation. In hnRNPA1 PAR binding motif deletion mutants, nuclear and cytosolic hnRNPA1 was observed that did not colocalize with stress granule reporter TIAR in the absence of stress, whereas the WT and K298A mutants remained entirely nuclear, providing evidence for a regulatory role of PAR binding on nuclear localization. Upon stress, K298A remained entirely nuclear, while the PAR binding domain deletion mutant was recruited to TIAR-labeled stress granules, though to a lesser extent than WT. Interestingly, PARylation was found to not only enhance phase separation of hnRNPA1, but also promoted co-phase separation with TDP43. Finally, it is demonstrated that downregulation of PAR polymerase suppresses TDP43-mediated neurotoxicity in Drosophila.

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Lisa Diez, Larisa E. Kapinos, Janine Hochmair, Sabrina Huebschmann, Alvaro Dominguez-Baquero, Amelie Vogt, Marija Rankovic, Markus Zweckstetter, Roderick Y. H. Lim and Susanne Wegmann

TL;DR: Surface plasmon resonance (SPR) and bio-layer interferometry (BLI) studies by Diez et al. reveal that phosphorylated Tau interacts with nuclear pore complex inner channel protein Nup98, shedding light on possible mechanisms for nucleocytoplasmic defects in Alzheimer’s disease.

Tau is an intrinsically disordered protein well known for its association with neurodegenerative diseases such as Alzheimer's disease (AD). In healthy neurons, Tau is located in the axons and helps stabilize axonal microtubules. In AD and in other neuronal stress conditions, Tau mislocalizes to the neuronal soma, where it has been found to accumulate in cytosolic aggregates. On the other hand, Tau was also found to interact with the nuclear envelope and nuclear pore complexes (NPCs), specifically with FG-Nups. The interaction of Tau with FG-Nups, such as Nup98, leads to defects in nucleocytoplasmic transport, which could be detrimental for the cell. In light of this, Diez et al. set out to investigate the molecular details of Tau:Nup98 interactions and how they relate to pathological changes in Tau which have been associated with its cytoplasmic mislocalization and aggregation. To this end, they use surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to describe how Tau:Nup98 interactions depend on the Tau variant, its post-translational modifications and assembly forms. Their results give several insights into the Tau:Nup98 interactions. The authors show that non-phosphorylated Tau can interact via its repeat domain with Nup98 in a transient and reversible manner. On the other hand, phosphorylated Tau – highly relevant to AD pathology - shows lower affinity but stronger binding to Nup98. On the other hand, Tau oligomers did not bind to Nup98. Therefore, it is possible that the phosphorylated Tau species accumulate at the nuclear envelope and clog NPCs in AD neurons, partially explaining the nucleocytoplasmic defects commonly observed in AD.

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Mainak Bose, Marko Lampe, Julia Mahamid and Anne Ephrussi

TL;DR: Bose et al. show that oskar RNP granules in the developing Drosophila oocyte incorporate mRNA in their liquid state, then transition to solid state which is important for its localization and further embryonic development.

Ribonucleoprotein (RNP) granules are mesoscale RNA-protein assemblies formed through multivalent RNA-RNA, RNA-protein and protein-protein interactions. The majority of RNP granules described to date have liquid-like properties.

In this article, Bose et al. report that oskar granules are RNA-protein condensates with solid-like properties, and show that their liquid-to-solid transition is essential to their function in abdominal patterning and embryo germline formation in Drosophila. By investigating their shape, fusion propensity and sensitivity to 1,6-hexanediol, the authors determined that oskar granules are assembled through liquid-liquid phase separation and have solid-like properties. Minimal oskar RNP condensates could be reconstituted in vitro using known interaction partners of oskar, Bruno and Hrp48, which contain prion-like domains. In vivo, Bruno seeded the granules, while Hrp48 modulated their material properties.

The liquid phase was shown to be essential for the incorporation of oskar mRNA into the granules; oskar mRNA did not localize in the granules in vitro after they had reached the solid state. On the other hand, the solid state of oskar granules was important for client protein (PTB) recruitment as well as their correct localization in the Drosophila cell; as artificial liquid-like oskar granules containing FUS did not localize correctly and were detrimental to embryonic development. The solid-like properties of the oskar granules may support long-distance transport in the cells.

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