USP42 drives nuclear speckle mRNA splicing via directing dynamic phase separation to promote tumorigenesis

Liquid–liquid phase separation is considered a generic approach to organize membrane-less compartments, enabling the dynamic regulation of phase-separated assemblies to be investigated and pivotal roles of protein posttranslational modifications to be demonstrated. By surveying the subcellular localizations of human deubiquitylases, USP42 was identified to form nuclear punctate structures that are associated with phase separation properties. Bioinformatic analysis demonstrated that the USP42 C-terminal sequence was intrinsically disordered, which was further experimentally confirmed to confer phase separation features. USP42 is distributed to SC35-positive nuclear speckles in a positively charged C-terminal residue- and enzymatic activity-dependent manner. Notably, USP42 directs the integration of the spliceosome component PLRG1 into nuclear speckles, and its depletion interferes with the conformation of SC35 foci. Functionally, USP42 downregulation deregulates multiple mRNA splicing events and leads to deterred cancer cell growth, which is consistent with the impact of PLRG1 repression. Finally, USP42 expression is strongly correlated with that of PLRG1 in non-small-cell lung cancer samples and predicts adverse prognosis in overall survival. As a deubiquitylase capable of dynamically guiding nuclear speckle phase separation and mRNA splicing, USP42 inhibition presents a novel anticancer strategy by targeting phase separation.Subject terms: Non-small-cell lung cancer, Deubiquitylating enzymes     

Analysis of P-body assembly in Saccharomyces cerevisiae

Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), that contain untranslating mRNAs in conjunction with proteins involved in translation repression and mRNA decapping and degradation. However, the order of protein assembly into P-bodies and the interactions that promote P-body assembly are unknown. To gain insight into how yeast P-bodies assemble, we examined the P-body accumulation of Dcp1p, Dcp2p, Edc3p, Dhh1p, Pat1p, Lsm1p, Xrn1p, Ccr4p, and Pop2p in deletion mutants lacking one or more P-body component. These experiments revealed that Dcp2p and Pat1p are required for recruitment of Dcp1p and of the Lsm1-7p complex to P-bodies, respectively. We also demonstrate that P-body assembly is redundant and no single known component of P-bodies is required for P-body assembly, although both Dcp2p and Pat1p contribute to P-body assembly. In addition, our results indicate that Pat1p can be a nuclear-cytoplasmic shuttling protein and acts early in P-body assembly. In contrast, the Lsm1-7p complex appears to primarily function in a rate limiting step after P-body assembly in triggering decapping. Taken together, these results provide insight both into the function of individual proteins involved in mRNA degradation and the mechanisms by which yeast P-bodies assemble.     

CPEB interacts with an ovary-specific eIF4E and 4E-T in early Xenopus oocytes

CPEB (cytoplasmic polyadenylation element-binding protein) is an important regulator of translation in oocytes and neurons. Although previous studies of CPEB in late Xenopus oocytes involve the eIF4E-binding protein maskin as the key factor for the repression of maternal mRNA, a second mechanism must exist, since maskin is absent earlier in oogenesis. Using co-immunoprecipitation and gel filtration assays, we show that CPEB specifically interacts, via protein/protein interactions, with the RNA helicase Xp54, the RNA-binding proteins P100(Pat1) and RAP55, the eIF4E-binding protein 4E-T, and an eIF4E protein. Remarkably, these CPEB complex proteins have been characterized, in one or more organism, as P-body, maternal, or neuronal granule components. We do not detect interactions with eIF4E1a, the canonical cap-binding factor, eIF4G, or eIF4A or with proteins expressed late in oogenesis, including maskin, PARN, and 4E-BP1. The eIF4E protein was identified as eIF4E1b, a close homolog of eIF4E1a, whose expression is restricted to oocytes and early embryos. Although eIF4E1b possesses all residues required for cap and eIF4G binding, it binds m(7)GTP weakly, and in pull-down assays, rather than binding eIF4G, it binds 4E-T, in a manner independent of the consensus eIF4E-binding site, YSKEELL. Wild type and Y-A mutant 4E-T (which binds eIF4E1b but not eIF4E1a), when tethered to a reporter mRNA, represses its translation in a cap-dependent manner, and injection of eIF4E1b antibody accelerates meiotic maturation. Altogether, our data suggest that CPEB, partnered with several highly conserved RNA-binding partners, inhibits protein synthesis in oocytes using a novel pairing of 4E-T and eIF4E1b.     

Pat1 contains distinct functional domains that promote P-body assembly and activation of decapping

The control of mRNA degradation and translation are important aspects of gene regulation. Recent results suggest that translation repression and mRNA decapping can be intertwined and involve the formation of a quiescent mRNP, which can accumulate in cytoplasmic foci referred to as P bodies. The Pat1 protein is a key component of this complex and an important activator of decapping, yet little is known about its function. In this work, we analyze Pat1 in Saccharomyces cerevisiae function by deletion and functional analyses. Our results identify two primary functional domains in Pat1: one promoting translation repression and P-body assembly and a second domain promoting mRNA decapping after assembly of the mRNA into a P-body mRNP. In addition, we provide evidence that Pat1 binds RNA and has numerous domain-specific interactions with mRNA decapping factors. These results indicate that Pat1 is an RNA binding protein and a multidomain protein that functions at multiple stages in the process of translation repression and mRNA decapping.     

Differential dynamics of splicing factor SC35 during the cell cycle

Pre-mRNA splicing factors are enriched in nuclear domains termed interchromatin granule clusters or nuclear speckles. During mitosis, nuclear speckles are disassembled by metaphase and reassembled in telophase in structures termed mitotic interchromatin granules (MIGs). We analysed the dynamics of the splicing factor SC35 in interphase and mitotic cells. In HeLa cells expressing green fluorescent protein (GFP)-SC35, this was localized in speckles during interphase and dispersed in metaphase. In telophase, GFP-SC35 was highly enriched within telophase nuclei and also detected in MIGs. Fluorescence recovery after photobleaching (FRAP) experiments revealed that the mobility of GFP-SC35 was distinct in different mitotic compartments. Interestingly, the mobility of GFP-SC35 was 3-fold higher in the cytoplasm of metaphase cells compared with interphase speckles, the nucleoplasm or MIGs. Treatment of cells with inhibitors of cyclin-dependent kinases (cdks) caused changes in the organization of nuclear compartments such as nuclear speckles and nucleoli, with corresponding changes in the mobility of GFP-SC35 and GFP-fibrillarin. Our results suggest that the dynamics of SC35 are significantly influenced by the organization of the compartment in which it is localized during the cell cycle.     

Shuttling components of nuclear import machinery involved in nuclear translocation of steroid receptors exit nucleus via exportin-1/CRM-1* independent pathway

The nucleocytoplasmic transport processes are mediated by soluble transport factors constantly navigating between nuclear and cytoplasmic compartments. Our understanding about nuclear export of general 'nuclear import factors' that deliver the cargo to the nucleus is still fragmentary. Utilizing green fluorescent protein tagged glucocorticoid receptor (GR) and relA as our working model and with judicious use of LMB, we show in living cells that all the soluble components of the nuclear import machinery exit nucleus via exportin1/CRM1 independent pathway(s).     

Nuclear staining for the small heat shock protein alphaB-crystallin colocalizes with splicing factor SC35

AlphaB-Crystallin has for a long time been considered a specific eye lens protein. Later on it appeared that this protein belongs to the family of the small heat shock proteins and that it occurs also extra-lenticularly in many different cell types. AlphaB-Crystallin is mainly present in the cytoplasm, but there are some indications that it might have a function in the nucleus too. However, till now its presence in the nucleus is uncertain. We therefore compared the localization of alphaB-crystallin in nine cell lines cultured under normal conditions using four different antisera. All four antisera gave a diffuse staining for alphaB-crystallin in the cytoplasm, but one of the antibodies consistently showed nuclear staining in eight of the cell types, in the form of distinct speckles. These speckles are equally pronounced in the different cell types, whether or not cytoplasmic alphaB-crystallin is present. Preabsorption of the antiserum with alphaB-crystallin abolished the staining. Furthermore we demonstrate that if only minor amounts of alphaB-crystallin are present, the protein seems to be located exclusively in the nucleus. However, in case of higher amounts of protein, alphaB-crystallin is distributed between cytoplasm and nucleus. The nuclear alphaB-crystallin exists, like the cytoplasmic alphaB-crystallin, in non-phosphorylated and phosphorylated forms, is Triton-insoluble but can be extracted by 2 M NaCl. These data suggest that alphaB-crystallin might be bound to the nuclear matrix per se or to nuclear matrix proteins via other proteins. In agreement with other nuclear matrix proteins, nuclear alphaB-crystallin staining turns diffuse upon mitosis and leaves the chromosomes unstained. Double staining experiments revealed colocalization of alphaB-crystallin with the splicing factor SC35 in nuclear speckles, suggesting a role for alphaB-crystallin in splicing or protection of the splicing machinery.     

The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

The mammalian cell nucleus is functionally compartmentalized into various substructures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, we performed a phenotypic screen of HeLa cells treated with a series of small interfering RNAs. Depletion of Ran-binding protein 2 induced cytoplasmic intermediates of nuclear speckles in G1 phase. Detailed analyses of these structures suggested that a late step in the sequential nuclear entry of mitotic interchromatin granule components was disrupted and that phosphorylated SR proteins were sequestered in an SR protein kinase-dependent manner. As a result, the cells had an imbalanced subcellular distribution of phosphorylated and hypophosphorylated SR proteins, which affected alternative splicing patterns. This study demonstrates that the speckled distribution of phosphorylated pre-mRNA processing factors is regulated by the nucleocytoplasmic transport system in mammalian cells and that it is important for alternative splicing.     

Mimicking phosphorylation of the small heat-shock protein alphaB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles.

The mammalian small heat shock protein alphaB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild-type, nonphosphorylatable and all possible pseudophosphorylation mutants of alphaB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/S59D) sites induced the partial translocation of alphaB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The alphaB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated alphaB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against alphaB-crystallin endogenously phosphorylated at Ser45, our findings suggest that alphaB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.     

蚕豆细胞核仁和核质中剪接因子SC35的定位研究

SC35 is a non-snRNP spliceosome component purified from mammalian cells by Fu and Maniatis in 1990. In vitro splicing assays showed that SC35 plays a key role in splicing site selection and ATP-dependent pre-spliceosome assembly. In the mammalian nucleus, SC35 has been localized to distinct and dynamic nuclear domains: immunofluorescence observations revealed the presence of SC35 in speckles distributed in various regions throughout the nucleoplasm, which, as identified with immunoelectron microscopy, correspond to the interchromatin granules (IGs) and perichromatin fibrils (PFs). However, there has been no report regarding the presence and distribution pattern of SC35 in higher plant nuclei. Engage in such studies will surely contribute to our understanding of RNA processing and the spatial organization or structure basis of this process in higher plant. In this article, we studied the distribution pattern of SC35 in the nucleus of the root meristematic cells of Vicia faba by immunoelectron microscopy. After immunolabeling with anti-SC35 mAb and protein A-colloidal gold, IGs and PFs in the nucleoplasm and dense fibrillar component (DFC) of the nucleolus were heavily labeled with gold particles, while only a few of the gold particles were found in fibrillar centers (FC) and nucleolar vacuoles (NV) of the nucleolus and the central domains of the condensed chromatin. Densities of gold particles in the areas of DFC and the area of IGs plus PFs were 65.89/microns 2 and 36.28/microns 2 respectively, much higher than that of the central domain of condensed chromatin and that of FC plus NV, which were only 5.90/microns 2 and 6.26/microns 2 respectively. This indicates that DFC of the nucleolus and the area of IGs plus PFs of the nucleoplasm are enriched with SC35 or SC35-like protein. The distribution pattern of SC35 or SC35-like protein in the nucleoplasm of Vicia faba is similar to that of the mammalian nuclei. To the authors' knowledge, it is a new finding that SC35 or SC35-like protein exists in the nucleolus.     

P-body components, Dhh1 and Pat1, are involved in tRNA nuclear-cytoplasmic dynamics

The nuclear-cytoplasmic distribution of tRNA depends on the balance between tRNA nuclear export/re-export and retrograde tRNA nuclear import in Saccharomyces cerevisiae. The distribution of tRNA is sensitive to nutrient availability as cells deprived of various nutrients exhibit tRNA nuclear accumulation. Starvation induces numerous events that result in translational repression and P-body formation. This study investigated the possible coordination of these responses with tRNA nuclear-cytoplasmic distribution. Dhh1 and Pat1 function in parallel to promote translation repression and P-body formation in response to starvation. Loss of both, Dhh1 and Pat1, results in a failure to repress translation and to induce P-body formation in response to glucose starvation. This study reports that nutrient deprived dhh1 pat1 cells also fail to accumulate tRNA within nuclei. Conversely, inhibition of translation initiation and induction of P-body formation by overproduction of Dhh1 or Pat1 cause tRNA nuclear accumulation in nutrient-replete conditions. Also, loss of the mRNA decapping activator, Lsm1, causes tRNA nuclear accumulation. However, the coordination between P-body formation, translation repression, and tRNA distribution is limited to the early part of the P-body formation/translation repression pathway as loss of mRNA decapping or 5' to 3' degradation does not influence tRNA nuclear-cytoplasmic dynamics. The data provide the first link between P-body formation/translation initiation and tRNA nuclear-cytoplasmic dynamics. The current model is that Dhh1 and Pat1 function in parallel to promote starvation-induced tRNA nuclear accumulation.     

A novel shuttling protein, 4E-T, mediates the nuclear import of the mRNA 5' cap-binding protein, eIF4E

The eukaryotic translation initiation factor 4E (eIF4E) plays an important role in the control of cell growth. eIF4E binds to the mRNA 5' cap structure m(7)GpppN (where N is any nucleotide), and promotes ribosome binding to the mRNA in the cytoplasm. However, a fraction of eIF4E localizes to the nucleus. Here we describe the cloning and functional characterization of a new eIF4E-binding protein, referred to as 4E-T (eIF4E-Transporter). We demonstrate that 4E-T is a nucleocytoplasmic shuttling protein that contains an eIF4E-binding site, one bipartite nuclear localization signal and two leucine-rich nuclear export signals. eIF4E forms a complex with the importin alphabeta heterodimer only in the presence of 4E-T. Overexpression of wild-type 4E-T, but not of a mutant defective for eIF4E binding, causes the nuclear accumulation of HA-eIF4E in cells treated with leptomycin B. Taken together, these results demonstrate that the novel nucleocytoplasmic shuttling protein 4E-T mediates the nuclear import of eIF4E via the importin alphabeta pathway by a piggy-back mechanism.