NudC guides client transfer between the Hsp40/70 and Hsp90 chaperone systems.

In the eukaryotic cytosol, the Hsp70 and the Hsp90 chaperone machines work in tandem with the maturation of a diverse array of client proteins. The transfer of nonnative clients between these systems is essential to the chaperoning process, but how it is regulated is still not clear. We discovered that NudC is an essential transfer factor with an unprecedented mode of action: NudC interacts with Hsp40 in Hsp40-Hsp70-client complexes and displaces Hsp70. Then, the interaction of NudC with Hsp90 allows the direct transfer of Hsp40-bound clients to Hsp90 for further processing. Consistent with this mechanism, NudC increases client activation in vitro as well as in cells and is essential for cellular viability. Together, our results show the complexity of the cooperation between the major chaperone machineries in the eukaryotic cytosol.

CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5' Capping with NAD.

Nucleoside-containing metabolites such as NAD+ can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

NUDC is critical for rod photoreceptor function, maintenance, and survival.

NUDC (nuclear distribution protein C) is a mitotic protein involved in nuclear migration and cytokinesis across species. Considered a cytoplasmic dynein (henceforth dynein) cofactor, NUDC was shown to associate with the dynein motor complex during neuronal migration. NUDC is also expressed in postmitotic vertebrate rod photoreceptors where its function is unknown. Here, we examined the role of NUDC in postmitotic rod photoreceptors by studying the consequences of a conditional NUDC knockout in mouse rods (rNudC-/- ). Loss of NUDC in rods led to complete photoreceptor cell death at 6 weeks of age. By 3 weeks of age, rNudC-/- function was diminished, and rhodopsin and mitochondria were mislocalized, consistent with dynein inhibition. Levels of outer segment proteins were reduced, but LIS1 (lissencephaly protein 1), a well-characterized dynein cofactor, was unaffected. Transmission electron microscopy revealed ultrastructural defects within the rods of rNudC-/- by 3 weeks of age. We investigated whether NUDC interacts with the actin modulator cofilin 1 (CFL1) and found that in rods, CFL1 is localized in close proximity to NUDC. In addition to its potential role in dynein trafficking within rods, loss of NUDC also resulted in increased levels of phosphorylated CFL1 (pCFL1), which would purportedly prevent depolymerization of actin. The absence of NUDC also induced an inflammatory response in Müller glia and microglia across the neural retina by 3 weeks of age. Taken together, our data illustrate the critical role of NUDC in actin cytoskeletal maintenance and dynein-mediated protein trafficking in a postmitotic rod photoreceptor.

Recent insights into noncanonical 5' capping and decapping of RNA.

The 5' N7-methylguanosine cap is a critical modification for mRNAs and many other RNAs in eukaryotic cells. Recent studies have uncovered an RNA 5' capping quality surveillance mechanism, with DXO/Rai1 decapping enzymes removing incomplete caps and enabling the degradation of the RNAs, in a process we also refer to as "no-cap decay." It has also been discovered recently that RNAs in eukaryotes, bacteria, and archaea can have noncanonical caps (NCCs), which are mostly derived from metabolites and cofactors such as NAD, FAD, dephospho-CoA, UDP-glucose, UDP-N-acetylglucosamine, and dinucleotide polyphosphates. These NCCs can affect RNA stability, mitochondrial functions, and possibly mRNA translation. The DXO/Rai1 enzymes and selected Nudix (nucleotide diphosphate linked to X) hydrolases have been shown to remove NCCs from RNAs through their deNADding, deFADding, deCoAping, and related activities, permitting the degradation of the RNAs. In this review, we summarize the recent discoveries made in this exciting new area of RNA biology.

Structural insights into dpCoA-RNA decapping by NudC.

Various kinds of cap structures, such as m7G, triphosphate groups, NAD and dpCoA, protect the 5' terminus of RNA. The cap structures bond covalently to RNA and affect its stability, translation, and transport. The removal of the caps is mainly executed by Nudix hydrolase family proteins, including Dcp2, RppH and NudC. Numerous efforts have been made to elucidate the mechanism underlying the removal of m7G, triphosphate group, and NAD caps. In contrast, few studies related to the cleavage of the RNA dpCoA cap have been conducted. Here, we report the hydrolytic activity of Escherichia coli NudC towards dpCoA and dpCoA-capped RNA in vitro. We also determined the crystal structure of dimeric NudC in complex with dpCoA at 2.0 Å resolution. Structural analysis revealed that dpCoA is recognized and hydrolysed in a manner similar to NAD. In addition, NudC may also remove other dinucleotide derivative caps of RNA, which comprise the AMP moieties. NudC homologs in Saccharomyces cerevisiae and Arabidopsis thaliana exhibited similar dpCoA decapping (deCoAping) activity. These results together indicate a conserved mechanism underpinning the hydrolysis of dpCoA-capped RNA in both prokaryotes and eukaryotes.

N-Acetyl-D-Glucosamine Kinase Interacts with NudC and Lis1 in Dynein Motor Complex and Promotes Cell Migration.

Recently, we showed that N-acetylglucosamine kinase (NAGK), an enzyme of amino sugar metabolism, interacts with dynein light chain roadblock type 1 (DYNLRB1) and promotes the functions of dynein motor. Here, we report that NAGK interacts with nuclear distribution protein C (NudC) and lissencephaly 1 (Lis1) in the dynein complex. Yeast two-hybrid assays, pull-down assays, immunocytochemistry, and proximity ligation assays revealed NAGK-NudC-Lis1-dynein complexes around nuclei, at the leading poles of migrating HEK293T cells, and at the tips of migratory processes of cultured rat neuroblast cells. The exogenous expression of red fluorescent protein (RFP)-tagged NAGK accelerated HEK293T cell migration during in vitro wound-healing assays and of neurons during in vitro neurosphere migration and in utero electroporation assays, whereas NAGK knockdown by short hairpin RNA (shRNA) delayed migration. Finally, a small NAGK peptide derived from the NudC interacting domain in in silico molecular docking analysis retarded the migrations of HEK293T and SH-SY5Y cells. These data indicate a functional interaction between NAGK and dynein-NudC-Lis1 complex at the nuclear envelope is required for the regulation of cell migration.

NAD in RNA: unconventional headgear.

Although widely assumed to bear a 5'-terminal triphosphate or monophosphate, recent evidence suggests that the 5' end of bacterial RNA can sometimes bear a modification reminiscent of a eukaryotic cap. A new study has now identified Escherichia coli RNAs that begin with a noncanonical cap resembling the redox cofactor nicotinamide adenine dinucleotide (NAD), as well as a cellular enzyme that can remove it. The biological function of such caps remains to be determined.

Corrigendum: NudC L279P mutation destabilizes filamin a by inhibiting the Hsp90 chaperoning pathway and suppresses cell migration.

[This corrects the article DOI: 10.3389/fcell.2021.671233.].

HIF1A-repressed PUS10 regulates NUDC/Cofilin1 dependent renal cell carcinoma migration by promoting the maturation of miR-194-5p.

BACKGROUND: Renal cell carcinoma (RCC) is characterized by a high rate of distant metastasis, which leads to poor prognosis in patients with advanced RCC. PUS10 has been recognized as a member of the pseudouridine synthase family, and recently other functions beyond the synthesis of the RNA modification have been uncovered. However, little is known about its role in diseases such as cancer. METHODS: RT-qPCR, western blot and immunohistochemistry were used to measure the expression of PUS10 in RCC tissues. Transwell assay, wound healing assay, and in vivo metastasis model were conducted to determine the function of PUS10 in RCC progression. MicroRNA sequencing and GEO database were used to screen for the downstream microRNAs of PUS10. RNA immunoprecipitation, dual luciferase reporter assay, immunostaining, and rescue experiments were employed to establish the PUS10/miR-194-5p/nuclear distribution protein C(NUDC)/Cofilin1 axis in RCC migration. Chromatin immunoprecipitation and dual luciferase reporter assay were used to verify its upstream transcriptional regulator. RESULTS: The expression of PUS10 was significantly decreased in RCC tissues, and low expression predicted poor prognosis. In vitro and in vivo experiments showed that PUS10 suppressed RCC migration, which, however, was independent of its classical pseudouridine catalytic function. Mechanically, PUS10 promoted the maturation of miR-194-5p, which sequentially inhibited RCC migration via disrupting NUDC-dependent cytoskeleton. Furthermore, hypoxia and HIF-1 A were found involved in the downregulation of PUS10. CONCLUSION: We unraveled PUS10 restrained RCC migration via the PUS10/miR-194-5p/NUDC/Cofilin1 pathway, which independent of its classical catalytic function. Furthermore, a linkage between the critical tumor microenvironment hallmark with malfunction of the forementioned metastasis inhibition mechanism was presented, as demonstrated by repressed expression of PUS10 due to hypoxia and HIF-1A.

HDAC7 inhibits cell proliferation via NudCD1/GGH axis in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer. In the absence of effective molecular markers for TNBC, there is an urgent clinical need for promising therapeutic target for TNBC. Histone deacetylases (HDACs), key regulators for chromatin remodeling and gene expression, have been suggested to play critical roles in cancer development. However, little is known ~the functions and implications of HDACs in TNBC treatment in the future. By analyzing the expression and prognostic significance of HDAC family members in TNBC through TCGA and METABRIC databases, HDAC7 was found to be downregulated in TNBC samples and the survival of patients with lower expression of HDAC7 was shorter. Furthermore, HDAC7 was negatively associated with NudC domain containing 1 (NudCD1) and γ-glutamyl hydrolase (GGH). Loss of NudCD1 or GGH predicted improved overall survival time (OS) of patients with TNBC. In vitro experiments showed that silencing of HDAC7 enhanced TNBC cell proliferation, while overexpression HDAC7 inhibited TNBC cell proliferation. The results of functional experiments confirmed that HDAC7 negatively modulated GGH and NudCD1 expression. Furthermore, decrease of NudCD1 or GGH inhibited cell proliferation. Notably, the HDAC7-NudCD1/GGH axis was found to be associated with NK cell infiltration. Overall, the present study revealed a novel role of HDAC7-NudCD1/GGH axis in TNBC, which might provide a promising treatment strategy for patients with TNBC.

NudC L279P Mutation Destabilizes Filamin A by Inhibiting the Hsp90 Chaperoning Pathway and Suppresses Cell Migration.

Filamin A, the first discovered non-muscle actin filament cross-linking protein, plays a crucial role in regulating cell migration that participates in diverse cellular and developmental processes. However, the regulatory mechanism of filamin A stability remains unclear. Here, we find that nuclear distribution gene C (NudC), a cochaperone of heat shock protein 90 (Hsp90), is required to stabilize filamin A in mammalian cells. Immunoprecipitation-mass spectrometry and western blotting analyses reveal that NudC interacts with filamin A. Overexpression of human NudC-L279P (an evolutionarily conserved mutation in NudC that impairs its chaperone activity) not only decreases the protein level of filamin A but also results in actin disorganization and the suppression of cell migration. Ectopic expression of filamin A is able to reverse these defects induced by the overexpression of NudC-L279P. Furthermore, Hsp90 forms a complex with filamin A. The inhibition of Hsp90 ATPase activity by either geldanamycin or radicicol decreases the protein stability of filamin A. In addition, ectopic expression of Hsp90 efficiently restores NudC-L279P overexpression-induced protein stability and functional defects of filamin A. Taken together, these data suggest NudC L279P mutation destabilizes filamin A by inhibiting the Hsp90 chaperoning pathway and suppresses cell migration.

Overexpression of the NMig1 Gene Encoding a NudC Domain Protein Enhances Root Growth and Abiotic Stress Tolerance in Arabidopsis thaliana.

The family of NudC proteins has representatives in all eukaryotes and plays essential evolutionarily conserved roles in many aspects of organismal development and stress response, including nuclear migration, cell division, folding and stabilization of other proteins. This study investigates an undescribed Arabidopsis homolog of the Aspergillus nidulans NudC gene, named NMig1 (for Nuclear Migration 1), which shares high sequence similarity to other plant and mammalian NudC-like genes. Expression of NMig1 was highly upregulated in response to several abiotic stress factors, such as heat shock, drought and high salinity. Constitutive overexpression of NMig1 led to enhanced root growth and lateral root development under optimal and stress conditions. Exposure to abiotic stress resulted in relatively weaker inhibition of root length and branching in NMig1-overexpressing plants, compared to the wild-type Col-0. The expression level of antioxidant enzyme-encoding genes and other stress-associated genes was considerably induced in the transgenic plants. The increased expression of the major antioxidant enzymes and greater antioxidant potential correlated well with the lower levels of reactive oxygen species (ROS) and lower lipid peroxidation. In addition, the overexpression of NMig1 was associated with strong upregulation of genes encoding heat shock proteins and abiotic stress-associated genes. Therefore, our data demonstrate that the NudC homolog NMig1 could be considered as a potentially important target gene for further use, including breeding more resilient crops with improved root architecture under abiotic stress.

Tomato SlSAP3, a member of the stress-associated protein family, is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000.

Tomato stress-associated proteins (SAPs) belong to A20/AN1 zinc finger protein family, some of which have been shown to play important roles in plant stress responses. However, little is known about the functions and underlying molecular mechanisms of SAPs in plant immune responses. In the present study, we reported the function of tomato SlSAP3 in immunity to Pseudomonas syringae pv. tomato (Pst) DC3000. Silencing of SlSAP3 attenuated while overexpression of SlSAP3 in transgenic tomato increased immunity to Pst DC3000, accompanied with reduced and increased Pst DC3000-induced expression of SA signalling and defence genes, respectively. Flg22-induced reactive oxygen species (ROS) burst and expression of PAMP-triggered immunity (PTI) marker genes SlPTI5 and SlLRR22 were strengthened in SlSAP3-OE plants but were weakened in SlSAP3-silenced plants. SlSAP3 interacted with two SlBOBs and the A20 domain in SlSAP3 is critical for the SlSAP3-SlBOB1 interaction. Silencing of SlBOB1 and co-silencing of all three SlBOB genes conferred increased resistance to Pst DC3000, accompanied with increased Pst DC3000-induced expression of SA signalling and defence genes. These data demonstrate that SlSAP3 acts as a positive regulator of immunity against Pst DC3000 in tomato through the SA signalling and that SlSAP3 may exert its function in immunity by interacting with other proteins such as SlBOBs, which act as negative regulators of immunity against Pst DC3000 in tomato.

Identification of Candidate Protein Markers in Skeletal Muscle of Laminin-211-Deficient CMD Type 1A-Patients.

Laminin-211 deficiency leads to the most common form of congenital muscular dystrophy in childhood, MDC1A. The clinical picture is characterized by severe muscle weakness, brain abnormalities and delayed motor milestones defining MDC1A as one of the most severe forms of congenital muscular diseases. Although the molecular genetic basis of this neurological disease is well-known and molecular studies of mouse muscle and human cultured muscle cells allowed first insights into the underlying pathophysiology, the definition of marker proteins in human vulnerable tissue such as skeletal muscle is still lacking. To systematically address this need, we analyzed the proteomic signature of laminin-211-deficient vastus muscle derived from four patients and identified 86 proteins (35 were increased and 51 decreased) as skeletal muscle markers and verified paradigmatic findings in a total of two further MDC1A muscle biopsies. Functions of proteins suggests fibrosis but also hints at altered synaptic transmission and accords with central nervous system alterations as part of the clinical spectrum of MDC1A. In addition, a profound mitochondrial vulnerability of the laminin-211-deficient muscle is indicated and also altered abundances of other proteins support the concept that metabolic alterations could be novel mechanisms that underline MDC1A and might constitute therapeutic targets. Intersection of our data with the proteomic signature of murine laminin-211-deficient gastrocnemius and diaphragm allowed the definition of nine common vulnerable proteins representing potential tissue markers.

Genetic Interactions Between BOB1 and Multiple 26S Proteasome Subunits Suggest a Role for Proteostasis in Regulating Arabidopsis Development.

Protein folding and degradation are both required for protein quality control, an essential cellular activity that underlies normal growth and development. We investigated how BOB1, an Arabidopsis thaliana small heat shock protein, maintains normal plant development. bob1 mutants exhibit organ polarity defects and have expanded domains of KNOX gene expression. Some of these phenotypes are ecotype specific suggesting that other genes function to modify them. Using a genetic approach we identified an interaction between BOB1 and FIL, a gene required for abaxial organ identity. We also performed an EMS enhancer screen using the bob1-3 allele to identify pathways that are sensitized by a loss of BOB1 function. This screen identified genetic, but not physical, interactions between BOB1 and the proteasome subunit RPT2a Two other proteasome subunits, RPN1a and RPN8a, also interact genetically with BOB1 Both BOB1 and the BOB1-interacting proteasome subunits had previously been shown to interact genetically with the transcriptional enhancers AS1 and AS2, genes known to regulate both organ polarity and KNOX gene expression. Our results suggest a model in which BOB1 mediated protein folding and proteasome mediated protein degradation form a functional proteostasis module required for ensuring normal plant development.

Development of a Mycobacterium smegmatis transposon mutant array for characterising the mechanism of action of tuberculosis drugs: Findings with isoniazid and its structural analogues.

The development of new drugs is required to control human tuberculosis (TB). This study examined whether drug hypersensitive mutants could be used to reveal novel aspects of the mechanism of action of a TB drug. A transposon mutant collection with an estimated 1.1-fold genome coverage (7680 mutants) was constructed in Mycobacterium smegmatis and screened in high-throughput against isoniazid. Hypersensitive transposants with mutations in genes known to influence the mode of action of isoniazid were isolated. To further investigate the role of one of these genes, nudC, the corresponding mutant was tested for sensitivity towards isoniazid structural analogues. Overexpression of nudC, as well as inhA which encodes a known target of isoniazid, increased M. smegmatis resistance to isoniazid, but failed to increase resistance to three of the analogues, NSC27607, NSC33759, and NSC40350. In contrast, overexpression of katG resulted in increased sensitivity to each of the isoniazid analogues tested including NSC27607, NSC33759, and NSC40350. This provides evidence that the latter isoniazid analogues are activated by KatG in a NudC-independent manner and that InhA may not be their primary target. In summary, characterisation of drug hypersensitive mutants detected genes involved in the mode of action of isoniazid. Furthermore, it identified isoniazid analogues which are resilient to both InhA- and NudC-dependent mechanisms of resistance.

EML4 promotes the loading of NUDC to the spindle for mitotic progression.

Echinoderm microtubule-associated protein (EMAP)-like (EML) family proteins are microtubule-associated proteins that have a conserved hydrophobic EMAP-like protein (HELP) domain and multiple WD40 domains. In this study, we examined the role of EML4, which is a member of the EML family, in cell division. Time-lapse microscopy analysis demonstrated that EML4 depletion induced chromosome misalignment during metaphase and delayed anaphase initiation. Further analysis by immunofluorescence showed that EML4 was required for the organization of the mitotic spindle and for the proper attachment of kinetochores to microtubules. We searched for EML4-associating proteins by mass spectrometry analysis and found that the nuclear distribution gene C (NUDC) protein, which is a critical factor for the progression of mitosis, was associated with EML4. This interaction was mediated by the WD40 repeat of EML4 and by the C-terminus of NUDC. In the absence of EML4, NUDC was no longer able to localize to the mitotic spindle, whereas NUDC was dispensable for EML4 localization. Our results show that EML4 is critical for the loading of NUDC onto the mitotic spindle for mitotic progression.

ASYMMETRIC-LEAVES2 and an ortholog of eukaryotic NudC domain proteins repress expression of AUXIN-RESPONSE-FACTOR and class 1 KNOX homeobox genes for development of flat symmetric leaves in Arabidopsis.

Leaf primordia form around the shoot apical meristem, which consists of indeterminate stem cells. Upon initiation of leaf development, adaxial-abaxial patterning is crucial for appropriate lateral expansion, via cellular proliferation, and the formation of flat symmetric leaves. Many genes that specify such patterning have been identified, but regulation by upstream factors of the expression of relevant effector genes remains poorly understood. In Arabidopsis thaliana, ASYMMETRIC LEAVES2 (AS2) and AS1 play important roles in repressing transcription of class 1 KNOTTED1-like homeobox (KNOX) genes and leaf abaxial-determinant effector genes. We report here a mutation, designated enhancer of asymmetric leaves2 and asymmetric leaves1 (eal), that is associated with efficient generation of abaxialized filamentous leaves on the as2 or as1 background. Levels of transcripts of many abaxial-determinant genes, including ETTIN (ETT)/AUXIN RESPONSE FACTOR3 (ARF3), and all four class 1 KNOX genes were markedly elevated in as2 eal shoot apices. Rudimentary patterning in as2 eal leaves was suppressed by the ett mutation. EAL encodes BOBBER1 (BOB1), an Arabidopsis ortholog of eukaryotic NudC domain proteins. BOB1 was expressed in plant tissues with division potential and bob1 mutations resulted in lowered levels of transcripts of some cell-cycle genes and decreased rates of cell division in shoot and root apices. Coordinated cellular proliferation, supported by BOB1, and repression of all class 1 KNOX genes, ETT/ARF3 by AS2 (AS1) and BOB1 might be critical for repression of the indeterminate state and of aberrant abaxialization in the presumptive adaxial domain of leaf primordia, which might ensure the formation of flat symmetric leaves.