Nucleolar Localization Signal Research Articles

Myosin VI in the nucleolus of neurosecretory PC12 cells: its involvement in the maintenance of nucleolar structure and ribosome organization.

We have previously shown that unconventional myosin VI (MVI), a unique actin-based motor protein, shuttles between the cytoplasm and nucleus in neurosecretory PC12 cells in a stimulation-dependent manner and interacts with numerous proteins involved in nuclear processes. Among the identified potential MVI partners was nucleolin, a major nucleolar protein implicated in rRNA processing and ribosome assembly. Several other nucleolar proteins such as fibrillarin, UBF (upstream binding factor), and B23 (also termed nucleophosmin) have been shown to interact with MVI. A bioinformatics tool predicted the presence of the nucleolar localization signal (NoLS) within the MVI globular tail domain, and immunostaining confirmed the presence of MVI within the nucleolus. Depletion of MVI, previously shown to impair PC12 cell proliferation and motility, caused disorganization of the nucleolus and rough endoplasmic reticulum (rER). However, lack of MVI does not affect nucleolar transcription. In light of these data, we propose that MVI is important for nucleolar and ribosome maintenance but not for RNA polymerase 1-related transcription.

Fragile X Messenger Ribonucleoprotein Protein and Its Multifunctionality: From Cytosol to Nucleolus and Back.

Silencing of the fragile X messenger ribonucleoprotein 1 (FMR1) gene and a consequent lack of FMR protein (FMRP) synthesis are associated with fragile X syndrome, one of the most common inherited intellectual disabilities. FMRP is a multifunctional protein that is involved in many cellular functions in almost all subcellular compartments under both normal and cellular stress conditions in neuronal and non-neuronal cell types. This is achieved through its trafficking signals, nuclear localization signal (NLS), nuclear export signal (NES), and nucleolar localization signal (NoLS), as well as its RNA and protein binding domains, and it is modulated by various post-translational modifications such as phosphorylation, ubiquitination, sumoylation, and methylation. This review summarizes the recent advances in understanding the interaction networks of FMRP with a special focus on FMRP stress-related functions, including stress granule formation, mitochondrion and endoplasmic reticulum plasticity, ribosome biogenesis, cell cycle control, and DNA damage response.

Abstract 6271: Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer

Abstract Prostate cancer (PC) stands as the most frequently diagnosed and second leading cause of cancer death among American men. PC mortality is predominantly attributed to castration-resistant prostate cancer (CRPC), often emerging within 2-3 years following the initiation of androgen deprivation therapy. This resistance primarily arises from sustained androgen receptor (AR) signaling, ultimately resulting in a compromised response to AR-directed therapies. AR splice variant expression is a key contributor to the persistent AR signaling in CRPC. AR-variant 7 (AR-V7) is the most clinically prevalent variant, expressed in about 75% of patients with CRPC and confers resistance to standard of care (SoC) treatments. AR-V7 differs from the full-length AR (AR-fl) because it lacks the ligand binding and hinge domains, rendering it resistant to SoC treatments. Currently, there are no selective inhibitors for AR-V7. Therefore, we sought to identify unique biological features of AR-V7, distinct from AR-fl, to exploit therapeutic options for targeting AR-V7. Previous studies have demonstrated high sequence homology, largely overlapping cistromes, and gene transactivation profiles between AR-V7 and AR-FL. Our published mechanistic studies revealed that AR-V7 utilizes a unique nuclear import pathway, not shared by AR-fl, allowing for constitutive import and activation of target genes in the nucleus. Herein, we report an additional novel subnuclear phenotype of AR-V7 in LNCAP95 and 22RV1 cells modeling CRPC. Staining with AR-V7- and AR-fl- specific antibodies revealed that unlike AR-fl, AR-V7 exhibited prominent nucleolar localization. To validate AR-V7 nucleolar localization, CRPC cells stained with fibrillarin (nucleolar marker), and subsequent fluorescence intensity analysis revealed that AR-V7 was predominantly localized in fibrillarin positive nucleoli, whereas AR-fl protein was solely localized in the nucleoplasm. This suggests a novel functional role of AR-V7 in the nucleus. To further confirm AR-V7 nucleolar localization, Actinomycin D (ActD) was used as a tool compound to disrupt nucleolar integrity. Treatment with ActD for 4 hours in CRPC cells significantly reduced the area of fibrillarin positive nucleoli and prompted AR-V7 re-distribution out of the nucleoli and into the nucleoplasm. Ongoing efforts to further investigate AR-V7 nucleolar function in CRPC cells include mutating the predicted nucleolar localization signal located in AR-V7 and identifying nucleolar-interacting proteins through proximity-labeling assays. In conclusion, we unveiled a novel nuclear compartmentalization of AR-V7 within the nucleolus, the primary hub for ribosomal biogenesis. These observations suggest that AR-V7 could regulate the high levels of protein synthesis occurring in CRPC cells, creating a window of a potential therapeutic opportunity for selective inhibition. Citation Format: Michelle K. Naidoo, Paraskevi Giannakakou. Novel insights into androgen receptor-variant 7 subnuclear localization and function in castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6271.

NPM1c Mutations Are Associated with Reduced Levels of Multiple Ribosome Biogenesis Factors Leading to Therapeutic Vulnerabilities

Objectives: Mutations in the NPM1 gene are found in more than 30% of acute myeloid leukaemia (AML) cases. The mutations disrupt a nucleolar localization signal (NoLS) and create a novel nuclear export signal (NES), leading to cytoplasmic displacement of the protein (NPM1c). NPM1cmutations prime haematopoietic progenitors to leukaemic transformation, but their precise molecular consequences remain elusive. Here, we first examine the effects of isolated NPM1c mutations on mouse haematopoietic cells and explore their impact on their transcriptome, including at single-cell resolution, and their proteome. Then, using several murine and human AML models and datasets we investigate the role of such NPM1c-associated changes in fully transformed AML cells and explore their potential as therapeutic vulnerabilities. Methods: We performed RNA-seq, CITE-seq and TMT global quantitative proteomics using lineage-depleted bone marrow progenitor cells from NPM1 flox-cA/+;Mx1-cre mice ( Vassiliou et al, Nat Genet, 2011). Proteomic changes were contrasted to those identified in a recently published dataset of 44 human AMLs, including 8 NPM1-mutant AMLs. Candidate vulnerabilities were identified using datasets from CRISPR-Cas9 drop-out screens and were validated in NPM1 cA/+-harbouring cell lines and primary mouse NPM1 cA/+;FLT3 ITD/+ AMLs. Results: We found a global increase in the protein abundance of several members of the KPN family in pre-leukaemic Npm1 cA/+ cells, an effect also observed in human NPM1c+ AMLs. Moreover, we observed a decrease in the relative abundance of several ribosome biogenesis-related proteins in pre-leukaemic Npm1 cA/+ cells and confirmed that this effect persists in human NPM1c+ AML patient samples. We go on to show that, when compared to NPM1 +/+ cells, pre-leukaemic NPM1 cA/+cells show high sensitivity to Actinomycin D and other RNA pol I inhibitors, but not to the translation inhibitor cycloheximide. Combination treatment with Actinomycin D and Venetoclax inhibited the growth and colony forming ability of both pre-leukaemic NPM1 cA/+cells and primary mouse Npm1 cA/+;Flt3 ITD/+AMLs. By contrasting our findings with data from CRISPR drop-out screens, including a genome-wide screen in primary mouse Npm1 cA/+;Flt3 ITD/+/Cas9AMLcells, we identified and validated ribosome biogenesis factors, such as Tsr3, whose knock-out selectively affected cell growth, survival and clonogenicity of NPM1 +/+AML cells, therefore representing novel potential therapeutic targets that could be exploited in the clinic. Conclusions: We show that NPM1c mutations are associated with reduced levels in several ribosome biogenesis factors resulting in therapeutic vulnerabilities of NPM1-mutant pre-leukaemic and leukaemic (AML) cells. The development of novel therapeutics to target these factors may help improve the survival of patients with NPM1-mutant AML. Figure Legend: (A) Volcano plot showing protein abundance in mouse NPM1 cA/+vs NPM1 +/+ pre-leukaemic haematopoetic progenitors. (B) Relative abundance of ribosome-related proteins in human NPM1 cA/+vs NPM1 +/+AML samples (dataset from Kramer et al., 2022).

Cationic Oligopeptides with Amino Groups as Synthetic Nucleolar Localization Signals for the Rational Design of Nucleolus-Staining Probes.

Cationic oligopeptides with amino groups were found to function as synthetic nucleolar localization signals for directing various fluorophores to the nucleolus with high selectivity in the cells with a view toward the development of nucleolus-staining probes.

Abstract PR001: Mutant NPM1 hijacks transcriptional hub to maintain pathogenic gene programs and block differentiation in acute myeloid leukemia

Abstract Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of functions including ribosome biogenesis, mRNA processing, and maintenance of genomic stability. In acute myeloid leukemia (AML), the terminal exon of NPM1 is often mutated (~30% of adult AMLs), changing the nucleolar localization signal into a nuclear export signal, which results in a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have an aberrant expression of the HOXA genes, whose overexpression leads to leukemogenic transformation. The gene regulatory role of NPM1c has long been an enigma. Recently, it was shown that depletion or re-localization of the NPM1c protein into the nucleus [PG1] causes downregulation of the HOXA genes, leading to the speculation that NPM1c modulates transcription of HOXA genes. Here, we found NPM1c binds to chromatin and is located genome-wide at gene promoters and colocalized at active regions marked by H3K27ac in NPM1c leukemia cell lines and primary leukemia blasts. NPM1c clearly binds to known leukemia-driving genes - posterior HOXA, HOXB, and MEIS1/PBX3 genes as well as novel targets -IRX5 and NKX2-3. After PROTAC degradation of NPM1c, we observed a significant RNA polymerase II elongation defect genome-wide. The dramatic elongation defect is caused by the loss of the Super Elongation Complex (SEC) chromatin binding. Further tests suggest that loss of NPM1c leads to disruption of RNA polymerase II condensates as well as other direct transcriptional regulators of HOX genes - Menin and AFF4 through IDR interactions. We found that pulse PROTAC degradation of NPM1c leads to sustained differentiation with the loss of histone acetylation at NPM1c target loci. Further, NPM1c maintains the active chromatin state of target genes by antagonizing HDAC activity. Additionally, we found that NPM1c’s activation of the leukemia driving genes depends on the pre-established active chromatin state in the transformed hematopoietic stem and progenitor cells. NPM1c alone is not sufficient to activate these leukemia driving genes with heterochromatin marks accumulated. We also found that NPM1c binds to genomic regions that have been pre-bound by its export partner, XPO1. The XPO1 inhibitors Selinexor and Eltanexor can displace NPM1c from chromatin. The combination of clinical-grade XPO1 inhibitor eltanexor and Menin inhibitor MI-3454 displayed a strong synergy in inducing differentiation and reducing leukemia driving gene expression in the NPM1 mutated leukemia cell lines and PDX model, which promises to be an effective combination therapy for NPM1c leukemia patients. Citation Format: Xue Qing Wang, Dandan Fan, Yiman Liu, Qinyu Han, Jolanta Grembecka, Tomasz Cierpicki, Jianzhong Su, Shasha Chong, Liling Wan, Xiaotian Zhang. Mutant NPM1 hijacks transcriptional hub to maintain pathogenic gene programs and block differentiation in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR001.

Rad17 Translocates to Nucleolus upon UV Irradiation through Nucleolar Localization Signal in the Central Basic Domain.

The nucleolus is a non-membranous structure in the nucleus and forms around ribosomal DNA repeats. It plays a major role in ribosomal biogenesis through the transcription of ribosomal DNA and regulates mRNA translation in response to cellular stress including DNA damage. Rad17 is one of the proteins that initiate and maintain the activation of the ATR pathway, one of the major DNA damage checkpoints. We have recently reported that the central basic domain of Rad17 contains a nuclear localization signal and that the nuclear translocation of Rad17 promotes its proteasomal degradation. Here, we show that the central basic domain contains the nucleolar localization signal as well as the nuclear localization signal. The nucleolar localization signal overlaps with the nuclear localization signal and is capable of transporting an exogenous protein into the nucleolus. Phosphomimetic mutations of the central basic domain inhibit nucleolar accumulation, suggesting that the post-translational modification sites regulate the nucleolar localization. Nucleolar accumulation of Rad17 is promoted by proteasome inhibition and UV irradiation. Our data show the nucleolar localization of Rad17 and suggest a possible role of Rad17 in the nucleolus upon UV irradiation.

Nuclear dynamics: Formation of bodies and trafficking in plant nuclei.

The existence of the nucleus distinguishes prokaryotes and eukaryotes. Apart from containing most of the genetic material, the nucleus possesses several nuclear bodies composed of protein and RNA molecules. The nucleus is separated from the cytoplasm by a double membrane, regulating the trafficking of molecules in- and outwards. Here, we investigate the composition and function of the different plant nuclear bodies and molecular clues involved in nuclear trafficking. The behavior of the nucleolus, Cajal bodies, dicing bodies, nuclear speckles, cyclophilin-containing bodies, photobodies and DNA damage foci is analyzed in response to different abiotic stresses. Furthermore, we research the literature to collect the different protein localization signals that rule nucleocytoplasmic trafficking. These signals include the different types of nuclear localization signals (NLSs) for nuclear import, and the nuclear export signals (NESs) for nuclear export. In contrast to these unidirectional-movement signals, the existence of nucleocytoplasmic shuttling signals (NSSs) allows bidirectional movement through the nuclear envelope. Likewise, nucleolar signals are also described, which mainly include the nucleolar localization signals (NoLSs) controlling nucleolar import. In contrast, few examples of nucleolar export signals, called nucleoplasmic localization signals (NpLSs) or nucleolar export signals (NoESs), have been reported. The existence of consensus sequences for these localization signals led to the generation of prediction tools, allowing the detection of these signals from an amino acid sequence. Additionally, the effect of high temperatures as well as different post-translational modifications in nuclear and nucleolar import and export is discussed.

Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage

BackgroundN-acetyltransferase 10 (NAT10), an abundant nucleolar protein with both lysine and RNA cytidine acetyltransferase activities, has been implicated in Hutchinson-Gilford progeria syndrome and human cancer. We and others recently demonstrated that NAT10 is translocated from the nucleolus to the nucleoplasm after DNA damage, but the underlying mechanism remains unexplored.MethodsThe NAT10 and PARP1 knockout (KO) cell lines were generated using CRISPR-Cas9 technology. Knockdown of PARP1 was performed using specific small interfering RNAs targeting PARP1. Cells were irradiated with γ-rays using a 137Cs Gammacell-40 irradiator and subjected to clonogenic survival assays. Co-localization and interaction between NAT10 and MORC2 were examined by immunofluorescent staining and immunoprecipitation assays, respectively. PARylation of NAT10 and translocation of NAT10 were determined by in vitro PARylation assays and immunofluorescent staining, respectively.ResultsHere, we provide the first evidence that NAT10 underwent covalent PARylation modification following DNA damage, and poly (ADP-ribose) polymerase 1 (PARP1) catalyzed PARylation of NAT10 on three conserved lysine (K) residues (K1016, K1017, and K1020) within its C-terminal nucleolar localization signal motif (residues 983–1025). Notably, mutation of those three PARylation residues on NAT10, pharmacological inhibition of PARP1 activity, or depletion of PARP1 impaired NAT10 nucleoplasmic translocation after DNA damage. Knockdown or inhibition of PARP1 or expression of a PARylation-deficient mutant NAT10 (K3A) attenuated the co-localization and interaction of NAT10 with MORC family CW-type zinc finger 2 (MORC2), a newly identified chromatin-remodeling enzyme involved in DNA damage response, resulting in a decrease in DNA damage-induced MORC2 acetylation at lysine 767. Consequently, expression of a PARylation-defective mutant NAT10 resulted in enhanced cellular sensitivity to DNA damage agents.ConclusionCollectively, these findings indicate that PARP1-mediated PARylation of NAT10 is key for controlling its nucleoplasmic translocation and function in response to DNA damage. Moreover, our findings provide novel mechanistic insights into the sophisticated paradigm of the posttranslational modification-driven cellular response to DNA damage.7RTHzWMYUwTGweyv2z3-hVVideo

NPM1 exon 5 mutations in acute myeloid leukemia: Implications in diagnosis and minimal residual monitoring.

NPM1 exon 5 mutations in acute myeloid leukemia: Implications in diagnosis and minimal residual monitoring.

Contribution of DEAD-Box RNA Helicase 21 to the Nucleolar Localization of Porcine Circovirus Type 4 Capsid Protein.

Porcine circovirus type 4 (PCV4) is a newly emerging pathogen which might be associated with diverse clinical signs, including respiratory and gastrointestinal distress, dermatitis, and various systemic inflammations. The host cellular proteins binding to PCV4 capsid (Cap) protein are still not clear. Herein, we found that the PCV4 Cap mediated translocation of DEAD-box RNA helicase 21 (DDX21) to the cytoplasm from the nucleolus and further verified that the nucleolar localization signal (NoLS) of the PCV4 Cap bound directly to the DDX21. The NoLS of PCV4 Cap and 763GSRSNRFQNK772 residues at the C-terminal domain (CTD) of DDX21 were required for this PCV4 Cap/DDX21 interaction. Further studies indicated that the PCV4 Cap NoLS exploited DDX21 to facilitate its nucleolar localization. In summary, our results firstly demonstrated that DDX21 binds directly to the NoLS of the PCV4 Cap thereby contributing to the nucleolar localization of the PCV4 Cap protein.

The Nucleolar Localization Signal of Porcine Circovirus Type 4 Capsid Protein Is Essential for Interaction With Serine-48 Residue of Nucleolar Phosphoprotein Nucleophosmin-1.

Porcine circovirus type 4 (PCV4) is an emerging etiological agent which was first detected in 2019. The nucleolar localization signal (NoLS) of PCV4 Cap protein and its binding host cellular proteins are still not elucidated. In the present study, we discovered a distinct novel NoLS of PCV4 Cap, which bound to the nucleolar phosphoprotein nucleophosmin-1 (NPM1). The NoLS of PCV4 Cap and serine-48 residue at the N-terminal oligomerization domain of NPM1 were necessary for PCV4 Cap/NPM1 interaction. Furthermore, the charge property of serine residue at position 48 of the NPM1 was crucial for its oligomerization and interaction with PCV4 Cap. In summary, our findings show for the first time that the PCV4 Cap NoLS and the NPM1 oligomerization determine the interaction of Cap/NPM1.

Molecular Coevolution of Nuclear and Nucleolar Localization Signals inside the Basic Domain of HIV-1 Tat

During evolution, viruses had to adapt to an increasingly complex environment of eukaryotic cells. Viral proteins that need to enter the cell nucleus or associate with nucleoli possess nuclear localization signals (NLSs) and nucleolar localization signals (NoLSs) for nuclear and nucleolar accumulation, respectively. As viral proteins are relatively small, acquisition of novel sequences seems to be a more complicated task for viruses than for eukaryotes. Here, we carried out a comprehensive analysis of the basic domain (BD) of HIV-1 Tat to show how viral proteins might evolve with NLSs and NoLSs without an increase in protein size. The HIV-1 Tat BD is involved in several functions, the most important being the transactivation of viral transcription. The BD also functions as an NLS, although it is substantially longer than a typical NLS. It seems that different regions in the BD could function as NLSs due to its enrichment with positively charged amino acids. Additionally, the high positive net charge inevitably causes the BD to function as an NoLS through a charge-specific mechanism. The integration of NLSs and NoLSs into functional domains enriched with positively charged amino acids might be a mechanism that allows the condensation of different functional sequences in small protein regions and, as a result, reduces protein size, influencing the origin and evolution of NLSs and NoLSs in viruses. IMPORTANCE Here, we investigated the molecular mechanism of nuclear localization signal (NLS) and nucleolar localization signal (NoLS) integration into the basic domain of HIV-1 Tat (49RKKRRQRRR57) and found that these two supplementary functions (i.e., function of NLS and function of NoLS) are embedded in the basic domain amino acid sequence. The integration of NLSs and NoLSs into functional domains of viral proteins enriched with positively charged amino acids is a mechanism that allows the concentration of different functions within small protein regions. Integration of NLS and NoLS into functional protein domains might have influenced the viral evolution, as this could prevent an increase in the protein size.

PiggyBac transposase and transposon derivatives for gene transfer targeting the ribosomal DNA loci of CHO cells

Integrative non-viral vectors such as transposons engineered to mediate targeted gene transfer into safe harbor sites in the genome may be a promising approach for the production of therapeutic proteins or for gene therapy in an efficient and secure way. In this context, we designed and evaluated two strategies for targeting the nuclear ribosomal DNA (rDNA) loci. One approach relied on the co-location of the transposase and transposon near transcriptionally active rDNA copies using a nucleolar localization signal (NoLS). Another one consisted of targeting the 18S-coding region in the rDNA loci using a NoLS-FokI-dCas9 endonuclease to perform targeted transgene knock-in. We show that integration into the rDNA of Chinese hamster ovary (CHO) cells can be achieved at a high frequency using the piggyBac transposon system, indicating that the rDNA is highly accessible for transposition. Consistently, rDNA-targeted transposition events were most frequently obtained when both the piggyBac transposon DNA and the transposase were nucleoli-targeted, yielding cells displaying stable and homogeneous expression of the transgene. This approach thus provides an alternative strategy to improve targeted transgene delivery and protein expression using CHO cells.

The interaction between PDCD4 and YB1 is critical for cervical cancer stemness and cisplatin resistance.

Cancer multidrug resistance (MDR) is existence in stem cell-like cancer cells characterized by stemness including high-proliferation and self-renewal. Programmed cell death 4 (PDCD4), as a proapoptotic gene, whether it engaged in cancer stemness and cisplatin resistance is still unknown. Here we showed that PDCD4 expressions in Hela/DDP (cisplatin resistance) cells were lower than in parental Hela cells. Moreover, the levels of drug resistance genes and typical stemness markers were markedly elevated in Hela/DDP cells. In vivo, xenograft tumor assay confirmed that knockdown of PDCD4 accelerated the grafted tumor growth. In vitro, colony formation and MTT assay demonstrated that PDCD4 overexpression inhibited cells proliferation in conditions with or without cisplatin. By contrast, PDCD4 deficiency provoked cell proliferation and cisplatin resistance. On mechanism, PDCD4 decreased the protein levels of pAKT and pYB1, accompanied by reduced MDR1 expression. Correspondingly, luciferase reporter assay showed PDCD4 regulated MDR1 promoter activity entirely relied on YB1. Furthermore, Ch-IP, GST-pulldown, and Co-IP assays provided novel evidence that PDCD4 could directly bind with YB1 by the nucleolar localization signal (NOLS) segment, causing the reduced YB1 binding into the MDR1 promoter region through blocking YB1 nucleus translocation, triggering the decreased MDR1 transcription. Taken together, PDCD4-pAKT-pYB1 forms the integrated molecular network to regulate MDR1 transcription during the process of stemness-associated cisplatin resistance.

DNA Damaging Agents Cause a Sub-Cellular Re-Localisation of Nucleophosmin in an npm1 Mutated Acute Myeloid Leukemia (AML) Cell Line and in Primary AML Cells Carrying an npm1 Mutation

Nucleophosmin (NPM) is a ubiquitously expressed phosphoprotein involved in many cellular functions including ribosome biogenesis, p53 activation, cell proliferation and genetic stability and is considered essential for cellular activity and survival. NPM contains a number of motifs which mediate interactions with a range of binding partners as well as influencing its cellular localisation. Primarily, NPM is located to the nucleolus due to a C-terminal nucleolar localisation signal (NoLS), however it is able to translocate through the nucleoplasm into the cytoplasm due to the presence of a bipartite nuclear localisation signal (NLS) and two nuclear export signals (NES).The most commonly mutated gene in Acute Myeloid Leukemia (AML) is npm1, occurring in approximately 35% of all AML patients. All npm1 mutations characterised to date occur within the C-terminal domain and alter the normal nucleo-cytoplasmic shuttling of NPM resulting in an abnormal cytoplasmic localisation of the protein. The mutations are always heterozygous, however mutant NPM oligomerises with wild type NPM resulting in an accumulation of both forms of the protein within the cytoplasm, leading to abnormal cellular functioning.Utilising an AML cell line containing a heterozygous npm1 mutation (OCI-AML3), we have shown that nucleolar localisation of NPM can be restored following short term exposure (4 - 24 hours) of the cells to the DNA damaging agent etoposide. Western blot analysis of the levels of NPM within cytoplasmic and nuclear fractions isolated from OCI-AML3 cells showed a significant decrease in cytoplasmic NPM following treatment relative to untreated cells (p=0.0045; N=3). Further analysis of OCI-AML3 cells using confocal microscopy identified an increase in nucleolar NPM following treatment relative to untreated controls (Figure 1). This post treatment increase of nucleolar NPM was also observed in primary leukemic cells isolated from patients with npm1 mutated AML.Similar results were observed with the DNA damaging agent cytarabine, however the potent anti-leukemic multi-kinase inhibitor, SB1317 had no effect on the sub-cellular localisation of NPM suggesting that the response is specific to DNA damage.In order to identify whether the nucleolar re-localisation of NPM following DNA damage could be abrogated by inhibiting the cellular response to DNA damage, OCI-AML3 cells were co-treated with etoposide and the PARP-1 DNA repair inhibitor, olaparib for 24 hours. Confocal microscope analysis of co-treated cells revealed that the sub-cellular distribution of NPM was predominantly cytoplasmic, with very little NPM-nucleolus co-localisation, i.e. resembling that of untreated cells.In summary, our data show that DNA damage caused by chemotherapeutic drugs results in a sub-cellular re-localisation of NPM in npm1 mutated AML cells, with the post-treatment NPM localisation resembling that of npm1 wildtype cells. In a chemotherapeutic context utilising DNA-damaging agents however, this re-localisation of NPM may not be desirable considering the involvement of NPM in DNA repair pathways. Re-localisation of NPM can be prevented however, by inhibiting the activity of other molecules involved in DNA damage response pathways, specifically PARP-1. Therefore these results suggest that the treatment of npm1 mutated AML with DNA damaging drugs should be considered within this context. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nucleolar Phosphoprotein NPM1 Interacts With Porcine Circovirus Type 3 Cap Protein and Facilitates Viral Replication

Porcine circovirus type 3 (PCV3) is a recently discovered virus with potentially significant implications on the global swine industry. PCV3 replication involves the entry of the viral capsid (Cap) protein with nucleolar localization signals into the nucleus. Using liquid chromatography-mass spectrometry analysis, nucleolar phosphoprotein NPM1 was identified as one of the cellular proteins bound to PCV3 Cap. Co-immunoprecipitation demonstrated that PCV3 Cap interacts directly with NPM1, where the region binding with NPM1 is mapped to amino acid residues 1–38 of Cap. Upon co-transfection, the expression of Cap protein promoted the redistribution of NPM1, which translocated from the nucleus to the cytoplasm and colocalized with Cap in cultured PK15 cells. NPM1 expression was upregulated and translocated from the nucleus to the cytoplasm in PCV3-infected cells, upon siRNA-mediated depletion, or upon treatment with NPM1 inhibitor in PK15 cells with impaired PCV3 replication, as evidenced by decreased levels of viral DNA synthesis and protein expression. By contrast, the replication of PCV3 was enhanced in stably NPM1-expressing cells via a lentivirus-delivered system. Taken together, these findings indicate that NPM1 interacts with PCV3 Cap and plays a crucial role in PCV3 replication.

A Role for the Autophagic Receptor, SQSTM1/p62, in Trafficking NF-κB/RelA to Nucleolar Aggresomes.

Elevated NF-κB activity is a contributory factor in many hematologic and solid malignancies. Nucleolar sequestration of NF-κB/RelA represses this elevated activity and mediates apoptosis of cancer cells. Here, we set out to understand the mechanisms that control the nuclear/nucleolar distribution of RelA and other regulatory proteins, so that agents can be developed that specifically target these proteins to the organelle. We demonstrate that RelA accumulates in intranucleolar aggresomes in response to specific stresses. We also demonstrate that the autophagy receptor, SQSTM1/p62, accumulates alongside RelA in these nucleolar aggresomes. This accumulation is not a consequence of inhibited autophagy. Indeed, our data suggest nucleolar and autophagosomal accumulation of p62 are in active competition. We identify a conserved motif at the N-terminus of p62 that is essential for nucleoplasmic-to-nucleolar transport of the protein. Furthermore, using a dominant-negative mutant deleted for this nucleolar localization signal (NoLS), we demonstrate a role for p62 in trafficking RelA and other aggresome-related proteins to nucleoli, to induce apoptosis. Together, these data identify a novel role for p62 in trafficking nuclear proteins to nucleolar aggresomes under conditions of cell stress, thus maintaining cellular homeostasis. They also provide invaluable information on the mechanisms that regulate the nuclear/nucleolar distribution of RelA that could be exploited for therapeutic purpose. IMPLICATIONS: The data open up avenues for the development of a unique class of therapeutic agents that act by targeting RelA and other aberrantly active proteins to nucleoli, thus killing cancer cells.

The serine-48 residue of nucleolar phosphoprotein nucleophosmin-1 plays critical role in subcellular localization and interaction with porcine circovirus type 3 capsid protein

The transport of circovirus capsid protein into nucleus is essential for viral replication in infected cell. However, the role of nucleolar shuttle proteins during porcine circovirus 3 capsid protein (PCV3 Cap) import is still not understood. Here, we report a previously unidentified nucleolar localization signal (NoLS) of PCV3 Cap, which hijacks the nucleolar phosphoprotein nucleophosmin-1 (NPM1) to facilitate nucleolar localization of PCV3 Cap. The NoLS of PCV3 Cap and serine-48 residue of N-terminal oligomerization domain of NPM1 are essential for PCV3 Cap/NPM1 interaction. In addition, charge property of serine-48 residue of NPM1 is critical for nucleolar localization and interaction with PCV3 Cap. Taken together, our findings demonstrate for the first time that NPM1 interacts with PCV3 Cap and is responsible for its nucleolar localization.

Nuclear and Nucleolar Localization of Bovine Adenovirus-3 Protein V.

The L2 region of bovine adenovirus-3 (BAdV-3) encodes a Mastadenovirus genus-specific protein, designated as pV, which is important for the production of progeny viruses. Here, we demonstrate that BAdV-3 pV, expressed as 55 kDa protein, localizes to the nucleus and specifically targets nucleolus of the infected cells. Analysis of deletion mutants of pV suggested that amino acids 81–120, 190–210, and 380–389 act as multiple nuclear localization signals (NLS), which also appear to serve as the binding sites for importin α-3 protein, a member of the importin α/β nuclear import receptor pathway. Moreover, pV amino acids 21–50 and 380–389 appear to act as nucleolar localization signals (NoLs). Interestingly, amino acids 380–389 appear to act both as NLS and as NoLS. The presence of NoLS is essential for the production of infectious progeny virions, as deletion of both NoLs are lethal for the production of infectious BAdV-3. Analysis of mutant BAV.pVd1d3 (isolated in pV completing CRL cells) containing deletion/mutation of both NoLS in non-complementing CRL cells not only revealed the altered intracellular localization of mutant pV but also reduced the expression of some late proteins. However, it does not appear to affect the incorporation of viral proteins, including mutant pV, in BAV.pVd1d3 virions. Further analysis of CsCl purified BAV.pVd1d3 suggested the presence of thermo-labile virions with disrupted capsids, which appear to affect the infectivity of the progeny virions. Our results suggest that pV contains overlapping and non-overlapping NoLS/NLS. Moreover, the presence of both NoLS appear essential for the production of stable and infectious progeny BAV.pVd1d3 virions.