Additional Nuclear Localization Signal Research Articles

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS

Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR-Cas9 components

Enhancing the intracellular delivery and performance of RNA-guided CRISPR-Cas9 nucleases (RGNs) remains in demand. Here, we show that nuclear translocation of commonly used Streptococcus pyogenes Cas9 (SpCas9) proteins is suboptimal. Hence, we generated eCas9.4NLS by endowing the high-specificity eSpCas9(1.1) nuclease (eCas9.2NLS) with additional nuclear localization signals (NLSs). We demonstrate that eCas9.4NLS coupled to prototypic or optimized guide RNAs achieves efficient targeted DNA cleavage and probe the performance of SpCas9 proteins with different NLS compositions at target sequences embedded in heterochromatin versus euchromatin. Moreover, after adenoviral vector (AdV)-mediated transfer of SpCas9 expression units, unbiased quantitative immunofluorescence microscopy revealed 2.3-fold higher eCas9.4NLS nuclear enrichment levels than those observed for high-specificity eCas9.2NLS. This improved nuclear translocation yielded in turn robust gene editing after nonhomologous end joining repair of targeted double-stranded DNA breaks. In particular, AdV delivery of eCas9.4NLS into muscle progenitor cells resulted in significantly higher editing frequencies at defective DMD alleles causing Duchenne muscular dystrophy (DMD) than those achieved by AdVs encoding the parental, eCas9.2NLS, protein. In conclusion, this work provides a strong rationale for integrating viral vector and optimized gene-editing technologies to bring about enhanced RGN delivery and performance.

Preparation and cellular uptake of bicyclic‐peptide cargo clicked to cell penetrating peptides

AbstractCell penetrating peptides (CPPs) are frequently used to deliver peptide cargo into cells where they can exert their biological activity. Their success, however, can be dependent on the cargo, the cell type, and other variables of peptide application. Here we describe the preparation of a bicyclic peptide inhibitor of the Grb7 cancer target and its conjugation via click chemistry to FITC‐labeled derivatives of the classic CPP Penetratin, as well as the equivalent with an additional nuclear localization signal (NLS). We discovered that uptake of the cargo‐Penetratin into the breast cancer cell line MDA‐MB‐231 was slow and led to limited cytosolic distribution. However, the addition of the NLS greatly enhanced the delivery leading to both cytosolic and nuclear distribution.

Multifunctional Nucleus-targeting Nanoparticles with Ultra-high Gene Transfection Efficiency for In Vivo Gene Therapy.

Cancer stem cell-like cells (CSCL) are responsible for tumor recurrence associated with conventional therapy (e.g. surgery, radiation, and chemotherapy). Here, we developed a novel multifunctional nucleus-targeting nanoparticle-based gene delivery system which is capable of targeting and eradicating CSCL. These nanoparticles can facilitate efficient endosomal escape and spontaneously penetrate into nucleus without additional nuclear localization signal. They also induced extremely high gene transfection efficiency (>95%) even in culture medium containing 30% serum, which significantly surpassed that of some commercial transfection reagents, such as Lipofectamine 2000 and Lipofectamine 3000 etc. Especially, when loaded with the TRAIL gene, this system mediated remarkable depletion of CSCL. Upon systemic administration, the nanoparticles accumulated in tumor sites while sparing the non-cancer tissues and significantly inhibited the growth of tumors with no evident systemic toxicity. Taken together, our results suggest that these novel multifunctional, nucleus-targeting nanoparticles are a very promising in vivo gene delivery system capable of targeting CSCL and represent a new treatment candidate for improving the survival of cancer patients.

Artificial Virus Delivers CRISPR-Cas9 System for Genome Editing of Cells in Mice.

CRISPR-Cas9 has emerged as a versatile genome-editing platform. However, due to the large size of the commonly used CRISPR-Cas9 system, its effective delivery has been a challenge and limits its utility for basic research and therapeutic applications. Herein, a multifunctional nucleus-targeting "core-shell" artificial virus (RRPHC) was constructed for the delivery of CRISPR-Cas9 system. The artificial virus could efficiently load with the CRISPR-Cas9 system, accelerate the endosomal escape, and promote the penetration into the nucleus without additional nuclear-localization signal, thus enabling targeted gene disruption. Notably, the artificial virus is more efficient than SuperFect, Lipofectamine 2000, and Lipofectamine 3000. When loaded with a CRISPR-Cas9 plasmid, it induced higher targeted gene disruption efficacy than that of Lipofectamine 3000. Furthermore, the artificial virus effectively targets the ovarian cancer via dual-receptor-mediated endocytosis and had minimum side effects. When loaded with the Cas9-hMTH1 system targeting MTH1 gene, RRPHC showed effective disruption of MTH1 in vivo. This strategy could be adapted for delivering CRISPR-Cas9 plasmid or other functional nucleic acids in vivo.

Nuclear-Cytoplasmic Partitioning of Cucumber Mosaic Virus Protein 2b Determines the Balance between Its Roles as a Virulence Determinant and an RNA-Silencing Suppressor

ABSTRACTThe Cucumber Mosaic Virus (CMV) 2b protein is an RNA-silencing suppressor that plays roles in CMV accumulation and virulence. The 2b proteins of subgroup IA CMV strains partition between the nucleus and cytoplasm, but the biological significance of this is uncertain. We fused an additional nuclear localization signal (NLS) to the 2b protein of subgroup IA strain Fny-CMV to create 2b-NLS and tested its effects on subcellular distribution, silencing, and virulence. The additional NLS enhanced 2b protein nuclear and nucleolar accumulation, but nuclear and nucleolar enrichment correlated with markedly diminished silencing suppressor activity in patch assays and abolished 2b protein-mediated disruption of microRNA activity in transgenic Arabidopsis. Nucleus/nucleolus-localized 2b protein possesses at least some ability to inhibit antiviral silencing, but this was not sufficient to prevent recovery from disease in younger, developing leaves in Arabidopsis. However, enhanced nuclear and nucleolar accumulation of 2b increased virulence and accelerated symptom appearance in older leaves. Experiments with Arabidopsis lines carrying mutant Dicer-like alleles demonstrated that compromised suppressor activity explained the diminished ability of 2b-NLS to enhance virus accumulation. Remarkably, the increased virulence that 2b-NLS engendered was unrelated to effects on microRNA- or short interfering RNA-regulated host functions. Thus, although nucleus- and nucleolus-localized 2b protein is less efficient at silencing suppression than cytoplasm-localized 2b, it enhances CMV virulence. We propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.IMPORTANCE In this work, the main finding is that nucleus/nucleolus-localized 2b protein is strongly associated with CMV virulence, which is independent of its effect on small RNA pathways. Moreover, this work supports the contention that the silencing suppressor activity of CMV 2b protein is predominantly exerted by that portion of the 2b protein residing in the cytoplasm. Thus, we propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.

Dbf4 and Cdc7 Proteins Promote DNA Replication through Interactions with Distinct Mcm2–7 Protein Subunits

The essential cell cycle target of the Dbf4/Cdc7 kinase (DDK) is the Mcm2-7 helicase complex. Although Mcm4 has been identified as the critical DDK phosphorylation target for DNA replication, it is not well understood which of the six Mcm2-7 subunits actually mediate(s) docking of this kinase complex. We systematically examined the interaction between each Mcm2-7 subunit with Dbf4 and Cdc7 through two-hybrid and co-immunoprecipitation analyses. Strikingly different binding patterns were observed, as Dbf4 interacted most strongly with Mcm2, whereas Cdc7 displayed association with both Mcm4 and Mcm5. We identified an N-terminal Mcm2 region required for interaction with Dbf4. Cells expressing either an Mcm2 mutant lacking this docking domain (Mcm2ΔDDD) or an Mcm4 mutant lacking a previously identified DDK docking domain (Mcm4ΔDDD) displayed modest DNA replication and growth defects. In contrast, combining these two mutations resulted in synthetic lethality, suggesting that Mcm2 and Mcm4 play overlapping roles in the association of DDK with MCM rings at replication origins. Consistent with this model, growth inhibition could be induced in Mcm4ΔDDD cells through Mcm2 overexpression as a means of titrating the Dbf4-MCM ring interaction. This growth inhibition was exacerbated by exposing the cells to either hydroxyurea or methyl methanesulfonate, lending support for a DDK role in stabilizing or restarting replication forks under S phase checkpoint conditions. Finally, constitutive overexpression of each individual MCM subunit was examined, and genotoxic sensitivity was found to be specific to Mcm2 or Mcm4 overexpression, further pointing to the importance of the DDK-MCM ring interaction.

Generation of New Cytotoxic Human Ribonuclease Variants Directed to the Nucleus

Ribonucleases are promising agents for use in anticancer therapy. Engineering a nuclear localization signal into the sequence of the human pancreatic ribonuclease has been revealed as a new strategy to endow this enzyme with cytotoxic activity against tumor cells. We previously described a cytotoxic human pancreatic ribonuclease variant, named PE5, which is able to cleave nuclear RNA, inducing the apoptosis of cancer cells and reducing the amount of P-glycoprotein in different multidrug-resistant cell lines. These results open the opportunity to use this ribonuclease in combination with other chemotherapeutics. In this work, we have investigated how to improve the properties of PE5 as an antitumor drug candidate. When attempting to develop a recombinant protein as a drug, two of the main desirable attributes are minimum immunogenicity and maximum potency. The improvements of PE5 have been designed in both senses. First, in order to reduce the potential immunogenicity of the protein, we have studied which residues mutated on PE5 can be reverted to those of the wild-type human pancreatic ribonuclease sequence without affecting its cytotoxicity. Second, we have investigated the effect of introducing an additional nuclear localization signal at different sites of PE5 in an effort to obtain a more cytotoxic enzyme. We show that the nuclear localization signal location is critical for the cytotoxicity. One of these variants, named NLSPE5, presents about a 10-fold increase in cytotoxicity respective to PE5. This variant induces apoptosis and kills the cells using the same mechanism as PE5.

Enhanced Nuclear Import and Transfection Efficiency of TAT Peptide-Based Gene Delivery Systems Modified by Additional Nuclear Localization Signals

Cellular uptake and nuclear localization are two major barriers in gene delivery. In order to evaluate whether additional nuclear localization signals (NLSs) can improve gene transfection efficiency, we introduced different kinds of NLSs to TAT-based gene delivery systems to form three kinds of complexes, including TAT-PV/DNA, TAT/DNA/PV, and TAT/DNA/HMGB1. The DNA binding ability of different vectors was evaluated by agarose gel electrophoresis. The in vitro transfections mediated by different complexes under different conditions were carried out. The cells treated by different complexes were observed by confocal microscopy. The MTT assay showed that all complexes did not exhibit apparent cytotoxicity in both HeLa and Cos7 cell lines even at high N/P ratios. The luciferase reporter gene expression mediated by TAT-PV/DNA complexes exhibited about 200-fold enhancement as compared with TAT/DNA complexes. Confocal study showed that, except TAT/DNA/PV, all other complexes exhibited enhanced nuclear accumulation and cellular uptake in both HeLa and Cos7 cell lines. These results indicated that the introduction of nuclear localization signals could enhance the transfection efficacy of TAT-based peptides, implying that the TAT peptide-based vectors demonstrated here have promising potential in gene delivery.

Regulation of Translesion Synthesis DNA Polymerase η by Monoubiquitination

DNA polymerase eta is a Y family polymerase involved in translesion synthesis (TLS). Its action is initiated by simultaneous interaction between the PIP box in pol eta and PCNA and between the UBZ in pol eta and monoubiquitin attached to PCNA. Whereas monoubiquitination of PCNA is required for its interaction with pol eta during TLS, we now show that monoubiquitination of pol eta inhibits this interaction, preventing its functions in undamaged cells. Identification of monoubiquitination sites within pol eta nuclear localization signal (NLS) led to the discovery that pol eta NLS directly contacts PCNA, forming an extended pol eta-PCNA interaction surface. We name this the PCNA-interacting region (PIR) and show that its monoubiquitination is downregulated by various DNA-damaging agents. We propose that this mechanism ensures optimal availability of nonubiquitinated, TLS-competent pol eta after DNA damage. Our work shows how monoubiquitination can either positively or negatively regulate the assembly of a protein complex, depending on which substrates are targeted by ubiquitin.

Characterization of nuclear import of the domain-specific androgen receptor in association with the importin alpha/beta and Ran-guanosine 5'-triphosphate systems.

Androgen induces androgen receptor (AR) nuclear import, which allows AR to act as a transcriptional factor and ultimately leads to biological activity. However, the mechanism of AR translocation to the nucleus is still unclear. In the present study, we assessed the nuclear import abilities of each domain of AR and their mechanisms related to Ran and importin alpha/beta using green fluorescent protein real-time imaging. The localization of AR to the nucleus in the absence and presence of ligands was dependent upon a complex interplay of the amino terminal transactivation domain (NTD), the DNA binding domain (DBD), and the ligand binding domain (LBD). NTD and DBD showed ligand-independent nuclear import ability, whereas LBD had ligand-dependent transport. In addition, AR deletion mutant lacking DBD was distributed in the cytoplasm regardless of ligand existence, suggesting that the remaining domains, NTD and LBD, are responsible for AR cytoplasmic localization. Cotransfection with a dominant negative form of Ran dramatically inhibited the nuclear import of all AR domains, and a dominant negative form of importin alpha prevented AR and DBD import. Importin beta-knockdown strongly blocked DBD import. These results indicate that there are two additional nuclear localization signals (NLSs) in the NTD and LBD, and there are distinct pathways used to attain domain-specific AR nuclear import: the NLS of DBD is Ran and importin alpha/beta-dependent, whereas the NLSs of NTD and LBD are Ran dependent but importin alpha/beta-independent. Our data suggest that the nuclear import of AR is regulated by the interplay between each domain of the AR.

Degradation of the auxin response factor ARF1

Auxin-mediated gene expression is largely controlled through a family of DNA-binding proteins known as auxin response factors (ARF). Previous studies on the role of proteolytic regulation in auxin signaling have focused on degradation of their interacting partner, the Aux/IAA proteins. Aux/IAA family members with domain II sequences are rapidly degraded, show auxin-enhanced degradation rates, and interact with the related F-box proteins TIR1 and AFB1-3, which indicates that they are ubiquitylated by a CUL1-dependent E3 ligase. To date, limited data have been generated regarding degradation of ARFs. Here, we focus on the degradation rate of one ARF family member, Arabidopsis thaliana ARF1, and find that the half-lives of N-terminally HA-tagged ARF1 and C-terminally luciferase-tagged ARF1 are both approximately 3-4 h. This half-life appears to be conferred by a component of the middle region (MR), and degradation of the luciferase fusion with the MR is more rapid when the fusion includes an additional nuclear localization signal. ARF1 degradation is proteasome-dependent and rates are not altered in a CUL1 mutant background, suggesting that this ARF is targeted for proteasomal degradation via an alternative set of machinery to that used for Aux/IAA degradation. Consistent with this, exogenous indole acetic acid does not affect the degradation of ARF1. Given increasing evidence that the relative ratio of Aux/IAAs to ARFs rather than the absolute quantity within the cell appears to be the mode through which auxin signaling is modulated, this half-life is likely to be biologically relevant.

Characterization of the nuclear localization signal of high risk HPV16 E2 protein

The E2 protein of high risk human papillomavirus type 16 (HPV16) contains an amino-terminal (N) domain, a hinge (H) region and a carboxyl-terminal (C) DNA-binding domain. Using enhanced green fluorescent protein (EGFP) fusions with full length E2 and E2 domains in transfection assays in HeLa cells, we found that the C domain is responsible for the nuclear localization of E2 in vivo, whereas the N and H domains do not contain additional nuclear localization signals (NLSs). Deletion analysis of EGFP-E2 and EGFP-cE2 determined that the C domain contains an α helix cNLS that overlaps with the DNA-binding region. Mutational analysis revealed that the arginine and lysine residues in this cNLS are essential for nuclear localization of HPV16 E2. Interestingly, these basic amino acid residues are well conserved among the E2 proteins of BPV-1 and some high risk HPV types but not in the low risk HPV types, suggesting that there are differences between the NLSs and corresponding nuclear import pathways between these E2 proteins.

Nuclear Export of S6K1 II Is Regulated by Protein Kinase CK2 Phosphorylation at Ser-17

Nuclear Export of S6K1 II Is Regulated by Protein Kinase CK2 Phosphorylation at Ser-17

Regulation of Ci and Su(fu) nuclear import in Drosophila

The Hedgehog (Hh) signal transduction pathway plays a central role in the development of invertebrates and vertebrates. While much is known about the pathway, the role of Suppressor of fused (Su(fu)), a component of the pathway's signaling complex has remained enigmatic. Previous studies have linked Su(fu) to the cytoplasmic sequestration of the zinc finger transcription factor, Cubitus interruptus (Ci), while other studies suggest a role in modulating target gene expression. In examining the cell biology of the pathway, we have found that like its vertebrate homologue, Drosophila Su(fu) enters the nucleus. Furthermore, we find that the nuclear import of Su(fu) occurs in concert with that of Ci in response to Hh signaling. Here, we examine the mechanism by which Su(fu) regulates Ci import by investigating the importance of the Ci nuclear localization signal (NLS) and the effect of adding an additional NLS. Finally, we demonstrate that Ci can bring Su(fu) with it to a multimerized Ci DNA binding site. These results provide a basis for understanding the dual roles played by Su(fu) in the regulation of Ci.

Nuclear localization of the Epstein–Barr virus EBNA3B protein

The Epstein-Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence. By utilizing fusion proteins with EGFP, deletion constructs of EBNA3B and site-directed mutagenesis, three of the four NLSs (aa 160-166, 430-434 and 867-873) were shown to be functional in truncated forms of EBNA3B, whilst an additional NLS (aa 243-246) was identified within the N-terminal region of EBNA3B. Only two of the NLSs were found to be functional in the context of the full-length EBNA3B protein.

Mechanisms of nuclear import and export that control the subcellular localization of class II transactivator.

The presence of the class II transactivator (CIITA) activates the transcription of all MHC class II genes. Previously, we reported that deletion of a carboxyl-terminal nuclear localization signal (NLS) results in the cytoplasmic localization of CIITA and one form of the type II bare lymphocyte syndrome. However, further sequential carboxyl-terminal deletions of CIITA resulted in mutant forms of the protein that localized predominantly to the nucleus, suggesting the presence of one or more additional NLS in the remaining sequence. We identified a 10-aa motif at residues 405-414 of CIITA that contains strong residue similarity to the classical SV40 NLS. Deletion of this region results in cytoplasmic localization of CIITA and loss of transactivation activity, both of which can be rescued by replacement with the SV40 NLS. Fusion of this sequence to a heterologous protein results in its nuclear translocation, confirming the identification of a NLS. In addition to nuclear localization sequences, CIITA is also controlled by nuclear export. Leptomycin B, an inhibitor of export, blocked the nuclear to cytoplasmic translocation of CIITA; however, leptomycin did not alter the localization of the NLS mutant, indicating that this region mediates only the rate of import and does not affect CIITA export. Several candidate nuclear export sequences were also found in CIITA and one affected the export of a heterologous protein. In summary, we have demonstrated that CIITA localization is balanced between the cytoplasm and nucleus due to the presence of NLS and nuclear export signal sequences in the CIITA protein.

Differential expression and subcellular distribution of the mouse metastasis-associated proteins Mta1 and Mta3

The human metastasis-associated gene ( MTA1) is overexpressed in cell lines and tissues representing metastatic tumors. Here we report cloning of the mouse Mta1 as well as a novel structurally related mouse gene, Mta3. The mouse Mta1 protein shares 94 and 59% homology to the human MTA1 and mouse Mta3 proteins, respectively. Northern blotting analysis using an Mta1 cDNA probe revealed a prevalent 3 kb hybridization signal in all mouse tissues except the skeletal muscle while a smaller ∼1.0 kb mRNA product was also detected in the heart. Mta3 transcripts (∼2 kb) were detected in most tissues with an additional ∼6.2 kb signal detected in the brain. In vitro transcription/translation of the full-length Mta1 and Mta3 cDNAs generated products of the expected molecular masses, i.e. 80 and 60 kDa, respectively. To assess subcellular localization, green fluorescence protein (GFP)-tagged expression constructs of Mta1 and Mta3 and various deletion constructs of GFP-Mta1 were transiently expressed in Balb/MK keratinocytes. GFP-Mta1 was found exclusively in the nucleus while GFP-Mta3 was present in both the nucleus and cytoplasm. Compared to Mta3, the carboxy terminal end of Mta1 contains an additional nuclear localization signal (NLS) and a proline-rich Src homology 3 (SH3) ligand. The results of transient expression experiments of various Mta1 fragments containing these domains in different combinations indicated that nuclear localization of Mta1 depended on the presence of at least one NLS and one SH3 binding site. These SH3 ligands appeared to be functional as they facilitated interaction with the adaptor protein, Grb2, and the Src-family tyrosine kinase, Fyn.

RNA-dependent cytoplasmic anchoring of a transcription factor subunit during Xenopus development.

The CCAAT box transcription factor (CBTF) is a multimeric transcription factor that activates expression of the haematopoietic regulatory factor, GATA-2. The 122 kDa subunit of this complex, CBTF(122), is cytoplasmic in fertilized Xenopus eggs and subsequently translocates to the nucleus prior to activation of zygotic GATA-2 transcription at gastrulation. Here we present data suggesting both a role for CBTF(122) prior to its nuclear translocation and the mechanism that retains it in the cytoplasm before the midblastula transition (MBT). CBTF(122) and its variant CBTF(98) are associated with translationally quiescent mRNP complexes. We show that CBTF(122) RNA binding activity is both necessary and sufficient for its cytoplasmic retention during early development. The introduction of an additional nuclear localization signal to CBTF(122) is insufficient to overcome this retention, suggesting that RNA binding acts as a cytoplasmic anchor for CBTF(122). Destruction of endogenous RNA by microinjection of RNase promotes premature nuclear translocation of CBTF(122). Thus, the nuclear translocation of CBTF(122) at the MBT is likely to be coupled to the degradation of maternal mRNA that occurs at that stage.

Regulation of DNA Binding Activity and Nuclear Transport of B-Myb in Xenopus Oocytes

DNA binding activity and nuclear transport of B-Myb in Xenopus oocytes are negatively regulated. Two distinct sequence elements in the C-terminal portion of the protein are responsible for these different inhibitory activities. A C-terminal Xenopus B-Myb protein fragment inhibits the DNA binding activity of the N-terminal repeats in trans, indicating that intramolecular folding may result in masking of the DNA binding function. Xenopus B-Myb contains two separate nuclear localization signals (NLSs), which, in Xenopus oocytes, function only outside the context of the full-length protein. Fusion of an additional NLS to the full-length protein overcomes the inhibition of nuclear import, suggesting that masking of the NLS function rather than cytoplasmic anchoring is responsible for the negative regulation of Xenopus B-Myb nuclear transfer. During Xenopus embryogenesis, when inhibition of nuclear import is relieved, Xenopus B-myb is preferentially expressed in the developing nervous system and neural crest cells. Within the developing neural tube, Xenopus B-myb gene transcription occurs preferentially in proliferating, non-differentiated cells.