Putative Nuclear Localization Signal Research Articles

Plant-specific calreticulin is localized in the nuclei of highly specialized cells in the pistil-new observations for an old hypothesis.

One of the first cellular locations of the calreticulin (CRT) chaperone in eukaryotic cells, apart from its obvious localization in the endoplasmic reticulum (ER), was the cell nucleus (Opas et al. 1991). The presence of CRT has been detected inside the nucleus and in the nuclear envelope of animal and plant cells, and a putative nuclear localization signal (NLS) in the CRT amino acid sequence has been mapped in several animal and plant species. Over the last 30years, other localization sites of this protein outside the ER and cell nucleus have also been discovered, suggesting that CRT is a multifunctional Ca2+-binding protein widely found in various cell types. In our previous studies focusing on plant developmental biology, we have demonstrated the presence of CRT inside and outside the ER in highly specialized plant cells, as well as the possibility of CRT localization in the cell nucleus. In this paper, we present a detailed analysis of immunocytochemical localization of CRT inside nuclei of the pistil transmission tract somatic cells before and after pollination. We show a similar pattern of the nuclear CRT localization in relation to exchangeable Ca2+ for two selected species of angiosperms, dicotyledonous Petunia and monocot Haemanthus, that differ in anatomical structure of the pistil and discuss the potential role of CRT in the cell nucleus.

Serotype-Specific Regulation of Dengue Virus NS5 Protein Subcellular Localization.

Dengue virus (DENV) nonstructural protein 5 (NS5), consisting of methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, is critical for viral RNA synthesis within endoplasmic reticulum-derived replication complexes in the cytoplasm. However, a significant proportion of NS5 is localized to the nucleus of infected cells for DENV2, 3, and 4, whereas DENV1 NS5 is localized diffusely in the cytoplasm. We still have an incomplete understanding of how the DENV NS5 subcellular localization is regulated. Within NS5, two putative nuclear localization signal (NLS) sequences have been identified: NLSCentral residing in the palm of the RdRp domain as well as the recently discovered NLSC-term residing in the flexible region at the C-terminal of the RdRp domain. We have previously shown that DENV2 NS5 nuclear localization can be significantly reduced by single-point mutations to the NLSC-term. Here, we present biochemical, virological, and structural data demonstrating that the relative importance of either NLS in NS5 nuclear localization is unique to each of the four DENV serotypes. DENV1 NS5's cytoplasmic localization appears to be due to a functionally weak interaction between its NLSCentral and importin-α (IMPα), while DENV2 NS5 is almost exclusively nuclear through its NLSC-term's strong interaction with IMPα. Both NLSs of DENV3 NS5 appear to contribute to directing its nuclear localization. Lastly, in the case of DENV4, the regulation of its NS5 nuclear localization remains an enigma but appears to be associated with its NLSC-term.

Identification of RNA silencing suppressor encoded by citrus chlorotic dwarf-associated virus

IntroductionCitrus chlorotic dwarf-associated virus (CCDaV) is an economically important citrus virus associated with leaf curling, deformation, and chlorosis found in China. Plants have evolved RNA silencing to defend against viral infections; however, the mechanism by which CCDaV suppresses RNA silencing in citrus remains unknown.MethodsSix proteins encoded by CCDaV were ectopically expressed in Nicotiana benthamiana 16c using the pCHF3 vector to identify RNA-silencing suppression activities.ResultsV2 protein encoded by CCDaV suppressed local RNA silencing and systemic RNA silencing triggered by GFP RNA, but did not impede short-distance movement of the RNA silencing signal in N. benthamiana 16c. GFP fluorescence observations showed that the ability of V2 protein to suppress RNA silencing was weaker than tomato bushy stunt virus P19. Deletion analysis showed that the putative nuclear localization signal (NLS, 25–54 aa) was involved in the RNA silencing suppression activity of V2 protein. Furthermore, V2 protein cannot block dsRNA-triggered RNA silencing. The subcellular localization assay suggested that V2 protein was localized to nucleus of N. benthamiana.ConclusionOverall, the results of this study demonstrate that CCDaV-V2 acts as an activity of silencing suppression. This is the first reported RNA-silencing suppressor encoded by Citlodavirus and will be valuable in revealing the molecular mechanism of CCDaV infection.

ZBTB34 is a hepatocellular carcinoma-associated protein with a monopartite nuclear localization signal.

ZBTB34 is a novel zinc finger protein with an unknown function. In this study, the gene expression and survival prognosis of ZBTB34 were analyzed across tumors based on the TCGA datasets. According to the bioinformatics analysis and qPCR results, liver hepatocellular carcinomas exhibit a high level of ZBTB34 expression. Additionally, the experiment supported the bioinformatics analysis findings that ZBTB34 expression was regulated by miR-125b-5p and that ZBTB34 affected ZBTB10, POLR1B, and AUH expression in HepG2 cells. Biological software analysis further revealed that ZBTB34 contains a monopartite nuclear localization signal (NLS). Arginine and lysine inside the putative NLS were substituted using the alanine-scanning mutagenesis method. The findings showed that the ability of ZBTB34 to enter the nucleus was abolished by the alanine substitution of the sequence 320RGGRARQKRA329 and the mutation of Lys327 and Arg328 residues. ZBTB34 was co-immunoprecipitated with importin α1, importin α3, importin α4, and importin β1, according to the results of the co-immunoprecipitation assay. In conclusion, ZBTB34 is a hepatocellular carcinoma-associated protein with a monopartite NLS. The nuclear import of ZBTB34 is mediated by importin α1, importin α3, importin α4, and importin β1. ZBTB34 performs its biological functions via a putative miR-125b-5p/ZBTB34/(ZBTB10, POLR1B, and AUH) signaling axis in HepG2 cells.

Experience-dependent Tip60 nucleocytoplasmic transport is regulated by its NLS/NES sequences for neuroplasticity gene control

Nucleocytoplasmic transport (NCT) in neurons is critical for enabling proteins to enter the nucleus and regulate plasticity genes in response to environmental cues. Such experience-dependent (ED) neural plasticity is central for establishing memory formation and cognitive function and can influence the severity of neurodegenerative disorders like Alzheimer's disease (AD). ED neural plasticity is driven by histone acetylation (HA) mediated epigenetic mechanisms that regulate dynamic activity-dependent gene transcription profiles in response to neuronal stimulation. Yet, how histone acetyltransferases (HATs) respond to extracellular cues in the in vivo brain to drive HA-mediated activity-dependent gene control remains unclear. We previously demonstrated that extracellular stimulation of rat hippocampal neurons in vitro triggers Tip60 HAT nuclear import with concomitant synaptic gene induction. Here, we focus on investigating Tip60 HAT subcellular localization and NCT specifically in neuronal activity-dependent gene control by using the learning and memory mushroom body (MB) region of the Drosophila brain as a powerful in vivo cognitive model system. We used immunohistochemistry (IHC) to compare the subcellular localization of Tip60 HAT in the Drosophila brain under normal conditions and in response to stimulation of fly brain neurons in vivo either by genetically inducing potassium channels activation or by exposure to natural positive ED conditions. Furthermore, we found that both inducible and ED condition-mediated neural induction triggered Tip60 nuclear import with concomitant induction of previously identified Tip60 target genes and that Tip60 levels in both the nucleus and cytoplasm were significantly decreased in our well-characterized Drosophila AD model. Mutagenesis of a putative nuclear localization signal (NLS) sequence and nuclear export signal (NES) sequence that we identified in the Drosophila Tip60 protein revealed that both are functionally required for appropriate Tip60 subcellular localization. Our results support a model by which neuronal stimulation triggers Tip60 NCT via its NLS and NES sequences to promote induction of activity-dependent neuroplasticity gene transcription and that this process may be disrupted in AD.

Henipavirus Matrix Protein Employs a Non-Classical Nuclear Localization Signal Binding Mechanism.

Nipah virus (NiV) and Hendra virus (HeV) are highly pathogenic species from the Henipavirus genus within the paramyxovirus family and are harbored by Pteropus Flying Fox species. Henipaviruses cause severe respiratory disease, neural symptoms, and encephalitis in various animals and humans, with human mortality rates exceeding 70% in some NiV outbreaks. The henipavirus matrix protein (M), which drives viral assembly and budding of the virion, also performs non-structural functions as a type I interferon antagonist. Interestingly, M also undergoes nuclear trafficking that mediates critical monoubiquitination for downstream cell sorting, membrane association, and budding processes. Based on the NiV and HeV M X-ray crystal structures and cell-based assays, M possesses a putative monopartite nuclear localization signal (NLS) (residues 82KRKKIR87; NLS1 HeV), positioned on an exposed flexible loop and typical of how many NLSs bind importin alpha (IMPα), and a putative bipartite NLS (244RR-10X-KRK258; NLS2 HeV), positioned within an α-helix that is far less typical. Here, we employed X-ray crystallography to determine the binding interface of these M NLSs and IMPα. The interaction of both NLS peptides with IMPα was established, with NLS1 binding the IMPα major binding site, and NLS2 binding as a non-classical NLS to the minor site. Co-immunoprecipitation (co-IP) and immunofluorescence assays (IFA) confirm the critical role of NLS2, and specifically K258. Additionally, localization studies demonstrated a supportive role for NLS1 in M nuclear localization. These studies provide additional insight into the critical mechanisms of M nucleocytoplasmic transport, the study of which can provide a greater understanding of viral pathogenesis and uncover a potential target for novel therapeutics for henipaviral diseases.

START domain-mediated nuclear translocation of Them1 regulates the expression of metabolic genes relative to thermogenesis in brown adipocytes

Thioesterase superfamily member 1 (Them1) is a long-chain fatty acyl-CoA thioesterase that is highly expressed in brown adipose tissue (BAT), where it limits energy expenditure. Them1 is comprised of tandem enzymatic domains and a C-terminal lipid-binding steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. Fatty acid binding to the START domain stabilizes the enzymatic domains and increases activity, whereas lysophosphatidylcholine (LPC) acts in the opposite manner. In brown adipocytes, Them1 localizes to puncta within the cytoplasm, but diffuses in response to β-adrenergic stimulation after which it is also found in the nucleus. The aim of this study was to determine the mechanism of Them1 nuclear trafficking and the metabolic consequences. METHODS: Identification of a putative nuclear localization signal (NLS) was determined by computational analysis. Full-length, NLS-deleted, or truncated Them1 cDNA constructs were cloned and linked to EGFP. Plasmid or adenoviral vectors were transfected into immortalized brown adipose cells (iBAs) that do not express Them1. Them1 was visualized by live cell confocal imaging via EGFP and with NucSpot nuclear staining. Signal quantification was performed with Imaris software. Them1 nuclear localization was evaluated in the presence or absence of norepinephrine (NE), leptomycin B, cycloheximide, or lipids that bind the START domain. Them1 nuclear localization was also quantified in BAT from mice treated with a β3-adrenergic receptor agonist, CL316,243. mRNA expression in iBAs expressing full-length or NLS-mutant Them1 in response to NE was determined by RNA-Seq. RESULTS: In the diffuse state, Them1 localized to the nucleus in a time-dependent manner. The NLS resides in a hinge region between the thioesterase domains and the START domain. Deletion of either the NLS or the START domain blocked nuclear targeting. Conversely, truncated Them1 containing only the START domain was targeted to the nucleus. Entry into the nucleus was balanced by trafficking out of the nucleus and new protein synthesis was not required, as evidenced by using leptomycin B to block nuclear efflux and cycloheximide to block protein synthesis, respectively. Incubation of NE-stimulated iBAs with LPC promoted nuclear Them1 translocation. Them1 protein was also detected in the nucleus of BAT from wild-type mice in response to CL administration. Nuclear Them1 localization in iBAs was associated with down-regulated mRNA expression of genes that control lipid and glucose metabolism during cold adaptation. CONCLUSIONS: Our data suggest that LPC generated during thermogenesis binds and destabilizes the START domain, leading to exposure of the NLS and nuclear trafficking of Them1 in response to β-adrenergic stimulation. Once inside the nucleus, Them1 regulates the transcription of lipid and glucose metabolic enzymes that are required for chronic cold adaptation. This work was supported by NIH DK103046 to DEC, SJH, and EAO This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

RKIP localizes to the nucleus through a bipartite nuclear localization signal and interaction with importin α to regulate mitotic progression

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin α for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression.

The Terminal Extensions of Dbp7 Influence Growth and 60S Ribosomal Subunit Biogenesis in Saccharomyces cerevisiae

Ribosome synthesis is a complex process that involves a large set of protein trans-acting factors, among them DEx(D/H)-box helicases. These are enzymes that carry out remodelling activities onto RNAs by hydrolysing ATP. The nucleolar DEGD-box protein Dbp7 is required for the biogenesis of large 60S ribosomal subunits. Recently, we have shown that Dbp7 is an RNA helicase that regulates the dynamic base-pairing between the snR190 small nucleolar RNA and the precursors of the ribosomal RNA within early pre-60S ribosomal particles. As the rest of DEx(D/H)-box proteins, Dbp7 has a modular organization formed by a helicase core region, which contains conserved motifs, and variable, non-conserved N- and C-terminal extensions. The role of these extensions remains unknown. Herein, we show that the N-terminal domain of Dbp7 is necessary for efficient nuclear import of the protein. Indeed, a basic bipartite nuclear localization signal (NLS) could be identified in its N-terminal domain. Removal of this putative NLS impairs, but does not abolish, Dbp7 nuclear import. Both N- and C-terminal domains are required for normal growth and 60S ribosomal subunit synthesis. Furthermore, we have studied the role of these domains in the association of Dbp7 with pre-ribosomal particles. Altogether, our results show that the N- and C-terminal domains of Dbp7 are important for the optimal function of this protein during ribosome biogenesis.

Identification of a Novel Solinvivirus with Nuclear Localization Associated with Mass Mortalities in Cultured Whiteleg Shrimp (Penaeus vannamei).

The emergence and spread of disease-causing viruses in shrimp aquaculture is not uncommon. Since 2016, unusual mortalities have been affecting the Brazilian shrimp industry and we have associated these unusual mortalities with a novel variant of infectious myonecrosis virus (IMNV). The transcriptome analysis of these diseased shrimp showed an additional divergent viral sequence that we have assigned to the family Solinviviridae. The novel virus has been tentatively termed Penaeus vannamei solinvivirus (PvSV) (GenBank accession: OP265432). The full-length genome of the PvSV is 10.44 kb (excluding the poly A tail) and codes for a polyprotein of 3326 aa. Five conserved domains coding for a helicase, RdRp, calicivirus coat protein, G-patch and tegument protein were identified. The genome organization of the PvSV is similar to other (Nylan deria fulva virus 1) solinvivirus. A unique feature of this virus that differs from other members of the Solinviviridae is the presence of putative nuclear localization signals. The tissue tropism of this virus is wide, infecting cells of the hepatopancreas, gastrointestinal tract, lymphoid organ and muscle tissue. Another unique feature is that it is the only RNA virus of penaeid shrimp that shows a nuclear localization by in situ hybridization. The PvSV has a wide distribution in Brazil and has been found in the states of Maranhão State (Perizes de Baixo), Piaui State (Mexeriqueira), Ceará State (Camocim, Jaguaruana, Aracati and Alto Santo) and Pará State where it has been detected in coinfections with IMNV. The diagnostic methods developed here (real-time RT-PCR and in situ hybridization) are effective for the detection of the pathogen and should be employed to limit its spread. Furthermore, the identification of the PvSV shows the increasing host range of the relatively new family Solinviviridae.

AC5 protein encoded by squash leaf curl China virus is an RNA silencing suppressor and a virulence determinant.

Squash leaf curl China virus (SLCCNV) is a bipartite Begomovirus. The function of the protein AC5, which is encoded by SLCCNV, is unknown. Here, we confirmed that the 172-amino acids (aa) long AC5 protein of SLCCNV could suppress single-stranded RNA but not double-stranded RNA-induced post-transcriptional gene silencing (PTGS). Furthermore, we determined that the C-terminal domain (96–172 aa) of the AC5 protein was responsible for RNA silencing suppressor (RSS) activity via deletion mutant analysis. The AC5 protein can reverse GFP silencing and inhibit systemic silencing of GFP by interfering with the systemic spread of the GFP silencing signal. The SLCCNV AC5 protein was localized to both the nucleus and cytoplasm of Nicotiana benthamiana cells. Furthermore, deletion analysis showed that the putative nuclear localization signal (NLS, 102–155 aa) was crucial for the RNA silencing suppression activity of AC5. In addition, the AC5 protein elicited a hypersensitive response and enhanced potoao virus X (PVX) RNA accumulation in infected N. benthamiana plants. Using the infectious clones of the SLCCNV and SLCCNV-AC5 null mutants, mutational analysis confirmed that knockout of the AC5 gene abolished SLCCNV-induced leaf curl symptoms, showing SLCCNV AC5 is also a virulence determinant.

PcWRKY11, an II-d WRKY Transcription Factor from Polygonum cuspidatum, Enhances Salt Tolerance in Transgenic Arabidopsis thaliana.

Being an invasive plant, Polygonum cuspidatum is highly resilient and can survive in unfavorable environments for long periods; however, its molecular mechanisms associated with such environmental resistance are largely unknown. In this study, a WRKY transcription factor (TF) gene, PcWRKY11, was identified from P. cuspidatum by analyzing methyl jasmonate (MeJA)-treated transcriptome data. It showed a high degree of homology with WRKY11 from Arabidopsis thaliana, containing a WRKY domain and a zinc finger structure and II-d WRKY characteristic domains of HARF, a calmodulin-binding domain (C-motif), and a putative nuclear localization signal (NLS) through sequence alignment and functional element mining. qPCR analysis showed that the expression of PcWRKY11 can be induced by NaCl, osmotic stress, and UV-C. In this study, we also found that overexpression of PcWRKY11 in A. thaliana could significantly increase salt tolerance. To explore its possible molecular mechanism, further investigations showed that compared with the wild type (WT), under salt stress, the transgenic plants showed a lower malondialdehyde (MDA) content, higher expression of ascorbate peroxidase (APX) and superoxide dismutase (SOD), and higher enzyme activity of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). Moreover, the transgenic plants also showed higher expression of Δ1-pyrroline-5-carboxylate synthase (AtP5CS), and higher contents of proline and soluble sugar. Taken together, these results indicate that PcWRKY11 may have a positive role in plants’ adaptation to salinity conditions by reducing reactive oxygen species (ROS) levels and increasing osmosis substance synthesis.

Rpn4p without the DNA-Binding Domain Provides Saccharomyces cerevisiae Resistance to Oxidative Stress and Cycloheximide

The ubiquitin-proteasome system is involved in the control of all essential molecular processes under normal conditions and the response of cells to stress. Rpn4p serves as a key transcriptional regulator of the proteasome in Saccharomycetes yeast and is also involved in the cellular response to various stresses. In addition to proteasomal genes, Rpn4 affects the expression of several hundred other genes, including genes involved in DNA repair and oxidative stress response. At the same time, the molecular mechanisms used by Rpn4 in controlling target genes and its functioning as a regulator of the cellular response to stress remain largely unclear. The aim of this work was to determine the Rpn4 domains required to ensure cell resistance to stress. It was shown that the N-terminal and central regions of the protein contain sites required for resistance to all types of stresses. The putative nuclear localization signal does not affect the functioning of Rpn4. Unexpectedly, a protein with the deletion of both zinc finger motifs that form the DNA-binding domain provides yeast resistance to oxidative stress and cycloheximide. Moreover, we showed that Rpn4 can be recruited to the promoter regions of the regulated genes even if they do not contain its binding sites. Based on these data, it can be assumed that Rpn4 is involved in gene regulation and the cellular response to stress due to protein-protein interactions.

Recombinant expression and biophysical characterization of Mrt4 protein that involved in mRNA turnover and ribosome assembly from Saccharomyces cerevisiae

ABSTRACT The mRNA turnover and ribosome assembly are facilitated by Mrt4 protein from Saccharomyces cerevisiae. In present study, we are reporting the cloning, expression and homogeneous purification of recombinant Mrt4. Mrt4 is a 236-amino-acid-long nuclear protein that plays a very crucial role in mRNA turnover and ribosome assembly during the translation process. mrt4 gene was amplified by polymerase chain reaction and cloned in expression vector pET23a (+) under the bacteriophage T7-inducible promoter and lac operator. Furthermore, protein was purified to homogeneity using immobilized metal affinity chromatography (IMAC) and its homogeneous purification was further validated by immunoblotting with anti-His antibody. The far-UV CD spectra represent that Mrt4 has a typical α helix with characteristic negative minima at 222 and 208 nm. At physiological pH, the fluorescence spectra and CD spectra showed properly folded tertiary and secondary structures of Mrt4, respectively. Saccharomyces Mrt4 protein possesses putative bipartite NLS (nuclear localization signal) at the N-terminal part followed by two well-conserved domains, rRNA-binding domains and translation factor (TF) binding domain. PIPSA analysis evaluates electrostatic interaction properties of proteins and concluded that Mrt4 protein can be used as a fingerprint for classifying Mrt4-like mRNA turnover protein from various species. The availability of an ample amount of protein may help in its biochemical and biophysical characterization, crystallization and identification of new interacting partners of Mrt4.

Nuclear Exportin 1 (XPO1) Binds to the Nuclear Localization/Export Signal of the Turnip Mosaic Virus NIb to Promote Viral Infection.

The nuclear localization signal (NLS) and nuclear export signal (NES) are key signatures of proteins for controlling nuclear import and export. The NIb protein of turnip mosaic virus (TuMV) is an RNA-dependent RNA polymerase (RdRP) that is absolutely required for viral genome replication. Previous studies have shown that NIb is a nucleocytoplasmic shuttling protein and contains four putative NES and four putative NLS motifs. Here, we analyzed the function of these NESs and NLSs, and identified two functional NESs and one functional NLS. Mutation of the identified functional NESs or NLS inhibited viral RNA accumulation and systemic infection. Exportin 1 (XPO1) is a nuclear export receptor that binds directly to cargo proteins harboring a leucine-rich NES and translocates them to the cytoplasm. We found that XPO1 contains two NIb-binding domains, which recognize the NLS and NES of NIb, respectively, to mediate the nucleocytoplasmic transport of NIb and promote viral infection. Taken together, these data suggest that the nucleocytoplasmic transport of NIb is modulated by XPO1 through its interactions with the functional NLS and NES of NIb to promote viral infection.

Mammalian acetate-dependent acetyl CoA synthetase 2 contains multiple protein destabilization and masking elements

Besides contributing to anabolism, cellular metabolites serve as substrates or cofactors for enzymes and may also have signaling functions. Given these roles, multiple control mechanisms likely ensure fidelity of metabolite-generating enzymes. Acetate-dependent acetyl CoA synthetases (ACS) are de novo sources of acetyl CoA, a building block for fatty acids and a substrate for acetyltransferases. Eukaryotic acetate-dependent acetyl CoA synthetase 2 (Acss2) is predominantly cytosolic, but is also found in the nucleus following oxygen or glucose deprivation, or upon acetate exposure. Acss2-generated acetyl CoA is used in acetylation of Hypoxia-Inducible Factor 2 (HIF-2), a stress-responsive transcription factor. Mutation of a putative nuclear localization signal in endogenous Acss2 abrogates HIF-2 acetylation and signaling, but surprisingly also results in reduced Acss2 protein levels due to unmasking of two protein destabilization elements (PDE) in the Acss2 hinge region. In the current study, we identify up to four additional PDE in the Acss2 hinge region and determine that a previously identified PDE, the ABC domain, consists of two functional PDE. We show that the ABC domain and other PDE are likely masked by intramolecular interactions with other domains in the Acss2 hinge region. We also characterize mice with a prematurely truncated Acss2 that exposes a putative ABC domain PDE, which exhibits reduced Acss2 protein stability and impaired HIF-2 signaling. Finally, using primary mouse embryonic fibroblasts, we demonstrate that the reduced stability of select Acss2 mutant proteins is due to a shortened half-life, which is a result of enhanced degradation via a nonproteasome, nonautophagy pathway.

RNA methyltransferase METTL16: Targets and function.

The N6‐methyladenosine (m6A) RNA methyltransferase METTL16 is an emerging player in the RNA modification landscape of the human cell. Originally thought to be a ribosomal RNA methyltransferase, it has now been shown to bind and methylate the MAT2A messenger RNA (mRNA) and U6 small nuclear RNA (snRNA). It has also been shown to bind the MALAT1 long noncoding RNA and several other RNAs. METTL16's methyltransferase domain contains the Rossmann‐like fold of class I methyltransferases and uses S‐adenosylmethionine (SAM) as the methyl donor. It has an RNA methylation consensus sequence of UACAGARAA (modified A underlined), and structural requirements for its known RNA interactors. In addition to the methyltransferase domain, METTL16 protein has two other RNA binding domains, one of which resides in a vertebrate conserved region, and a putative nuclear localization signal. The role of METTL16 in the cell is still being explored, however evidence suggests it is essential for most cells. This is currently hypothesized to be due to its role in regulating the splicing of MAT2A mRNA in response to cellular SAM levels. However, one of the more pressing questions remaining is what role METTL16's methylation of U6 snRNA plays in splicing and potentially cellular survival. METTL16 also has several other putative coding and noncoding RNA interactors but the definitive methylation status of those RNAs and the role METTL16 plays in their life cycle is yet to be determined. Overall, METTL16 is an intriguing RNA binding protein and methyltransferase whose important functions in the cell are just beginning to be understood.This article is categorized under:RNA Processing > RNA Editing and ModificationRNA Interactions with Proteins and Other Molecules > RNA‐Protein Complexes

Abstract 2877: LUZP1 is an understudied transcriptional regulator in triple negative breast cancer (TNBC)

Abstract Introduction: TNBC is associated with poorer prognosis compared to other types of breast cancer due to poorly understood aggressive metastatic behavior. Proteomic profiling prioritized Leucine-Zipper Protein 1 (LUZP1) as an understudied TNBC-specific protein from an analysis of breast cancer patient-derived xenografts (PDXs) [PMC5379071]. Approach: RNA-seq data and isobaric tags for relative and absolute quantitation (iTRAQ) data from 16 PDXs were used to identify differentially expressed genes in basal-like and claudin-low breast cancers, compared to luminal and HER2-enriched subtypes. Multiple datasets (DepMap, METABRIC, and TCGA) were used to validate the enrichment of LUZP1 in TNBC. T-test was applied to identify differential genes/proteins. Pearson correlation analysis was used to identify RNAs that are correlated with LUZP1 protein level in CTPAC proteogenomic clinical sample data [PMC5102256 and Cell in press]. Gene Set Enrichment Analysis (GSEA) was used to identify enriched pathways. Cell fractionation was used to determine the subcellular expression of LUZP1. qPCR was used to validate differential expression observed in RNA-seq. Results: Leucine-Zipper Protein 1 (LUZP1) was differentially elevated in basal-like and claudin-low PDX models versus luminal and HER2-enriched at the mRNA and protein levels across 16 PDXs. Furthermore, LUZP1 mRNA was significantly enriched in TNBCs, relative to luminal and HER2-enriched subtypes, in breast cancer cell lines (data from DepMap), and human breast tumors (data from METABRIC and TCGA). LUZP1 has three putative nuclear localization signals and three leucine-zipper motifs, thought to mediate its protein dimerization and DNA-binding properties. Fractionation of TNBC cells showed that LUZP1 was predominately present in the nucleus. RNA-sequencing of control and MDA-MB-231 LUZP1 knockdown cells revealed that several extracellular matrix (ECM)-related pathways and epithelial-to-mesenchymal transition (EMT) were regulated by LUZP1. In particular, expression of migration, invasion, and metastasis genes such as matrix metalloproteinases (MMPs), Bone Morphogenetic Protein 2 (BMP2), and Transforming Growth Factor Beta 2 (TGFB2) were found to be LUZP1 regulated. Additionally, in CPTAC breast cancer proteogenomic data, LUZP1-correlated proteins and transcripts were enriched in ECM-related pathways and EMT, confirming the pathophysiological relevance of the cell-line knock-down data. Conclusion: LUZP1 is a novel TNBC-enriched protein in breast cancer PDXs, cell lines, and human tumors data. Gene silencing experiments demonstrated that LUZP1 transcriptionally regulates metastasis-related genes, and thus may drive a transcriptional program responsible for invasion, metastasis and EMT in TNBC. Functional data will be presented. Citation Format: Junkai Wang, Doug Chan, Beom-Jun Kim, Gloria V. Echeverria, Eric Jaehnig, Bing Zhang, Matthew J. Ellis. LUZP1 is an understudied transcriptional regulator in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2877.

Prp19-associated splicing factor Cwf15 regulates fungal virulence and development in the rice blast fungus.

The splicing factor Cwf15 is an essential component of the Prp19-associated component of the spliceosome and regulates intron splicing in several model species, including yeasts and human cells. However, the roles of Cwf15 remain unexplored in plant pathogenic fungi. Here, we report that MoCWF15 in the rice blast fungus Magnaporthe oryzae is non-essential to viability and important to fungal virulence, growth and conidiation. MoCwf15 contains a putative nuclear localization signal (NLS) and is localized into the nucleus. The NLS sequence but not the predicted phosphorylation site or two sumoylation sites was essential for the biological functions of MoCwf15. Importantly, MoCwf15 physically interacted with the Prp19-associated splicing factors MoCwf4, MoSsa1 and MoCyp1, and negatively regulated protein accumulations of MoCyp1 and MoCwf4. Furthermore, with the deletion of MoCWF15, aberrant intron splicing occurred in near 400 genes, 20 of which were important to the fungal development and virulence. Taken together, MoCWF15 regulates fungal growth and infection-related development by modulating the intron splicing efficiency of a subset of genes in the rice blast fungus.

USP42 protects ZNRF3/RNF43 from R-spondin-dependent clearance and inhibits Wnt signalling.

The tumour suppressors RNF43 and ZNRF3 play a central role in development and tissue homeostasis by promoting the turnover of the Wnt receptors LRP6 and Frizzled (FZD). The stem cell growth factor R‐spondin induces auto‐ubiquitination and membrane clearance of ZNRF3/RNF43 to promote Wnt signalling. However, the deubiquitinase stabilising ZNRF3/RNF43 at the plasma membrane remains unknown. Here, we show that the USP42 antagonises R‐spondin by protecting ZNRF3/RNF43 from ubiquitin‐dependent clearance. USP42 binds to the Dishevelled interacting region (DIR) of ZNRF3 and stalls the R‐spondin‐LGR4‐ZNRF3 ternary complex by deubiquitinating ZNRF3. Accordingly, USP42 increases the turnover of LRP6 and Frizzled (FZD) receptors and inhibits Wnt signalling. Furthermore, we show that USP42 functions as a roadblock for paracrine Wnt signalling in colon cancer cells and mouse small intestinal organoids. We provide new mechanistic insights into the regulation R‐spondin and conclude that USP42 is crucial for ZNRF3/RNF43 stabilisation at the cell surface.