Cellular Nucleic Acid Binding Protein Research Articles

The transcription of the main gene associated with Treacher–Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP)

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

Cellular nucleic acid binding protein facilitates cardiac repair after myocardial infarction by activating β-catenin signaling

The regenerative capacity of the adult mammalian heart is limited, while the neonatal heart is an organ with regenerative and proliferative ability. Activating adult cardiomyocytes (CMs) to re-enter the cell cycle is an effective therapeutic method for ischemic heart disease such as myocardial infarction (MI) and heart failure. Here, we aimed to reveal the role and potential mechanisms of cellular nucleic acid binding protein (CNBP) in cardiac regeneration and repair after heart injury. CNBP is highly expressed within 7 days post-birth while decreases significantly with the loss of regenerative ability. In vitro, overexpression of CNBP promoted CM proliferation and survival, whereas knockdown of CNBP inhibited these processes. In vivo, knockdown of CNBP in CMs robustly hindered myocardial regeneration after apical resection in neonatal mice. In adult MI mice, CM-specific CNBP overexpression in the infarct border zone ameliorated myocardial injury in acute stage and facilitated CM proliferation and functional recovery in the long term. Quantitative proteomic analysis with TMT labeling showed that CNBP overexpression promoted the DNA replication, cell cycle progression, and cell division. Mechanically, CNBP overexpression increased the expression of β-catenin and its downstream target genes CCND1 and c-myc; Furthermore, Luciferase reporter and Chromatin immunoprecipitation (ChIP) assays showed that CNBP could directly bind to the β-catenin promoter and promote its transcription. CNBP also upregulated the expression of G1/S-related cell cycle genes CCNE1, CDK2, and CDK4. Collectively, our study reveals the positive role of CNBP in promoting cardiac repair after injury, providing a new therapeutic option for the treatment of MI.

Cellular nucleic acid-binding protein restricts SARS-CoV-2 by regulating interferon and disrupting RNA-protein condensates.

A detailed understanding of the innate immune mechanisms involved in restricting SARS-CoV-2 infection and how the virus disrupts these processes could reveal new strategies to boost antiviral mechanisms and develop therapeutics for COVID-19. Here, we identify cellular nucleic acid-binding protein (CNBP) as a key host factor controlling SARS-CoV-2 infection. In response to RNA-sensing pathways, CNBP is phosphorylated and translocates from the cytosol to the nucleus where it binds to the interferon-β enhancer to initiate transcription. Because SARS-CoV-2 evades immune detection by the host's RNA-sensing pathways, CNBP is largely retained in the cytosol where it restricts SARS-CoV-2 directly, leading to a battle between the host and SARS-CoV-2 that extends beyond antiviral immune signaling pathways. We further demonstrated that CNBP binds SARS-CoV-2 viral RNA directly and competes with the viral nucleocapsid protein to prevent viral RNA and nucleocapsid protein from forming liquid-liquid phase separation (LLPS) condensates critical for viral replication. Consequently, cells and animals lacking CNBP have higher viral loads, and CNBP-deficient mice succumb rapidly to infection. Altogether, these findings identify CNBP as a key antiviral factor for SARS-CoV-2, functioning both as a regulator of antiviral IFN gene expression and a cell-intrinsic restriction factor that disrupts LLPS to limit viral replication and spread. In addition, our studies also highlight viral condensates as important targets and strategies for the development of drugs to combat COVID-19.

Unraveling the structural aspects of the G-quadruplex in SMO promoter and elucidating its contribution in transcriptional regulation

We located a 25 nt G-rich sequence in the promoter region of SMO oncogene. We performed an array of biophysical and biochemical assays and confirmed the formation of a parallel G quadruplex (SMO1-GQ) by the identified sequence. SMO1-GQ is highly conserved in primates. For a comprehensive characterization of the SMO quadruplex structure, we have performed spectroscopic and in silico analysis with established GQ binder small molecules TMPyP4 and BRACO-19. We observed comparatively higher stable interaction of BRACO-19 with SMO1-GQ. Structure-based, rational drug design against SMO1-GQ to target SMO oncogene requires a detailed molecular anatomy of the G-quadruplex. We structurally characterised the SMO1-GQ using DMS footprinting assay and molecular modelling, docking, and MD simulation to identify the probable atomic regions that interact with either of the small molecules. We further investigated SMO1-GQ in vivo by performing chromatin immunoprecipitation (ChIP) assay. ChIP data revealed that this gene element functions as a scaffold for a number of transcription factors: specificity protein (Sp1), nucleolin (NCL), non-metastatic cell 2 (NM23-H2), cellular nucleic acid binding protein (CNBP), and heterogeneous nuclear ribonucleoprotein K (hnRNPK) which reflects the SMO1-P1 G-quadruplex to be the master regulator of SMO1 transcriptional activity. The strong binding interaction detected between SMO1-GQ and BRACO-19 contemplates the potential of the G quadruplex as a promising anti-cancer druggable target to downregulate SMO1 oncogene driven cancers. Communicated by Ramaswamy H. Sarma

Therapeutic targeting of CNBP phase separation inhibits ribosome biogenesis and neuroblastoma progression via modulating SWI/SNF complex activity

Neuroblastoma (NB) is the most common extracranial malignancy in childhood; however, the mechanisms underlying its aggressive characteristics still remain elusive. Integrative data analysis was performed to reveal tumour-driving transcriptional regulators. Co-immunoprecipitation and mass spectrometry assays were applied for protein interaction studies. Real-time reverse transcription-polymerase chain reaction, western blotting, sequential chromatin immunoprecipitation and dual-luciferase reporter assays were carried out to explore gene expression regulation. The biological characteristics of NB cell lines were examined via gain- and loss-of-function assays. For survival analysis, the Cox regression model and log-rank tests were used. Cellular nucleic acid-binding protein (CNBP) was found to be an independent factor affecting NB outcome, which exerted oncogenic roles in ribosome biogenesis, tumourigenesis and aggressiveness. Mechanistically, karyopherin subunit beta 1 (KPNB1) was responsible for nuclear transport of CNBP, whereas liquid condensates of CNBP repressed the activity of switch/sucrose-nonfermentable (SWI/SNF) core subunits (SMARCC2/SMARCC1/SMARCA4) via interaction with SMARCC2, leading to alternatively increased activity of SMARCC1/SMARCA4 binary complex in facilitating gene expression essential for 18S ribosomal RNA (rRNA) processing in tumour cells, extracellular vesicle-mediated delivery of 18S rRNA and subsequent M2 macrophage polarisation. A cell-penetrating peptide blocking phase separation and interaction of CNBP with SMARCC2 inhibited ribosome biogenesis and NB progression. High KPNB1, CNBP, SMARCC1 or SMARCA4 expression or low SMARCC2 levels were associated with poor survival of NB patients. These findings suggest that CNBP phase separation is a target for inhibiting ribosome biogenesis and tumour progression in NB via modulating SWI/SNF complex activity.

Identification and characterization of a flexile G-quadruplex in the distal promoter region of stemness gene REX1

We have identified a parallel G-quadruplex (R1WT) in the distal promoter region (−821 base-pairs upstream of the TSS) of the pluripotent gene REX1. Through biophysical and biochemical approach, we have characterized the G-quadruplex (GQ) as a potential molecular switch that may control REX1 promoter activity to determine the transcriptional fate. Small- molecule interactive study of the monomeric form of R1WT (characterized as R1mut2) with TMPyP4 and BRACO-19 revealed GQ destabilization upon interaction with TMPyP4 and stabilization upon interaction with BRACO-19. This distinctive drug interactivity suggests the in cellulo R1WT to be a promising drug target. The endogenous existence of R1WT was confirmed by BG4 antibody derived chromatin immunoprecipitation experiment. Here in, we also report the endogenous interaction of GQ specific transcription factors (TFs) with R1WT region in the human chromatin of cancer cell. The wild-type G-quadruplex was found to interact with four important transcription factors, (i) specificity protein (Sp1) (ii) non-metastatic cell 2 (NM23-H2): a diphosphatase (iii) cellular nucleic acid binding protein (CNBP) and (iv) heterogenous nuclear ribonucleoprotein K (hnRNPK) in the REX1 promoter. In contrast, nucleolin protein (NCL) binding was found to be low to the said G-quadruplex. The flexibility of R1WT between folded and unfolded states, obtained from experimental and computational analysis strongly suggests R1WT to be an important gene regulatory element in the genome. It controls promoter DNA relaxation with the coordinated interaction of transcription factors, the deregulation of which seeds stemness characteristic in cancer cells for further metastatic progression.

CircPACRGL promoted cell proliferation, migration and invasion as well as inhibited cell apoptosis in colorectal cancer via regulation of the miR-330-3p/CNBP axis.

CircRNAs are a member of noncoding RNAs and have been verified to play an important regulatory role in cancers. In CRC, the regulatory mechanisms of various circRNAs have not been elucidated. The expression of circPACRGL and miR-330-3p was detected with qRT-PCR. The protein expression of CDK4, MMP-9, Bcl-2, Bax, cellular nucleic acid-binding protein (CNBP) and β-actin was measured with western blot. Cell proliferation was analyzed using MTT assay, colony formation assay, and EDU assay. Cell apoptosis was detected using flow cytometry. Cell migration and invasion were measured with wound healing and transwell invasion assay. Luciferase reporter assay and RIP assay was used to determine the relationship of among miR-330-3p, circPACRGL and CNBP in CRC cells. In this study, we found that circPACRGL and CNBP expressed high and miR-330-3p expressed low in CRC tissues and cells. Functional experiments showed that inhibition of circPACRGL reduced cell proliferation, migration and invasion in CRC. In addition, knockdown of circPACRGL contributed to cell apoptosis in CRC. Dual-luciferase report assay determined that circPACRGL was a miR-330-3p sponge molecular and CNBP was a target of miR-330-3p. Reversed experiments showed that the effects of sh-circPACRGL transfection on CRC cells were rescued by up-regulating CNBP expression. In this study, we for the first time found a novel regulatory network of circPACRGL in CRC. The results manifested that circPACRGL affected tumor growth by targeting miR-330-3p/CNBP axis in CRC, highlighting the potential of circPACRGL as a therapeutic target for colorectal cancer.

Myotonic Dystrophies: A Genetic Overview.

Myotonic dystrophies (DM) are the most common muscular dystrophies in adults, which can affect other non-skeletal muscle organs such as the heart, brain and gastrointestinal system. There are two genetically distinct types of myotonic dystrophy: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2), both dominantly inherited with significant overlap in clinical manifestations. DM1 results from CTG repeat expansions in the 3′-untranslated region (3′UTR) of the DMPK (dystrophia myotonica protein kinase) gene on chromosome 19, while DM2 is caused by CCTG repeat expansions in intron 1 of the CNBP (cellular nucleic acid-binding protein) gene on chromosome 3. Recent advances in genetics and molecular biology, especially in the field of RNA biology, have allowed better understanding of the potential pathomechanisms involved in DM. In this review article, core clinical features and genetics of DM are presented followed by a discussion on the current postulated pathomechanisms and therapeutic approaches used in DM, including the ones currently in human clinical trial phase.

Immobilized Titanium (IV) Ion Affinity Chromatography Contributes to Efficient Proteomics Analysis of Cellular Nucleic Acid-Binding Proteins.

Cellular nucleic acid-binding proteins (NABPs), namely, DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in many biological processes. However, extracting NABPs with high efficiency in living cells is challenging, which greatly limited their proteomics analysis and comprehensive characterization. Here, we discovered that titanium (IV) ion-immobilized metal affinity chromatography (Ti4+-IMAC) material could enrich DNA and RNA with high efficiency (96.82 ± 2.67 and 85.75 ± 2.99%, respectively). We therefore developed a Ti4+-IMAC method for the joint extraction of DBPs and RBPs. Through utilizing formaldehyde (FA) cross-linking, DBPs and RBPs were covalently linked to nucleic acids (NAs) and further denatured by organic solvents. After Ti4+-IMAC capture, 2000 proteins were identified in 293T cells, among which 417 DBPs and 999 RBPs were revealed, showing promising selectivity for NABPs. We further applied the Ti4+-IMAC capture method to lung cancer cell lines 95C and 95D, which have different tumor progression abilities. The DNA- and RNA-binding capabilities of many proteins have been dysregulated in 95D. Under our conditions, Ti4+-IMAC can be used as a selective and powerful tool for the comprehensive characterization of both DBPs and RBPs, which might be utilized to study their dynamic interactions with nucleic acids.

Translational control of polyamine metabolism by CNBP is required for Drosophila locomotor function.

Microsatellite expansions of CCTG repeats in the cellular nucleic acid-binding protein (CNBP) gene leads to accumulation of toxic RNA and have been associated with myotonic dystrophy type 2 (DM2). However, it is still unclear whether the dystrophic phenotype is also linked to CNBP decrease, a conserved CCHC-type zinc finger RNA-binding protein that regulates translation and is required for mammalian development. Here, we show that depletion of Drosophila CNBP in muscles causes ageing-dependent locomotor defects that are correlated with impaired polyamine metabolism. We demonstrate that the levels of ornithine decarboxylase (ODC) and polyamines are significantly reduced upon dCNBP depletion. Of note, we show a reduction of the CNBP-polyamine axis in muscles from DM2 patients. Mechanistically, we provide evidence that dCNBP controls polyamine metabolism through binding dOdc mRNA and regulating its translation. Remarkably, the locomotor defect of dCNBP-deficient flies is rescued by either polyamine supplementation or dOdc1 overexpression. We suggest that this dCNBP function is evolutionarily conserved in vertebrates with relevant implications for CNBP-related pathophysiological conditions.

Characterisation of Non-Pathogenic Premutation-Range Myotonic Dystrophy Type 2 Alleles.

Myotonic dystrophy type 2 (DM2) is caused by expansion of a (CCTG)n repeat in the cellular retroviral nucleic acid-binding protein (CNBP) gene. The sequence of the repeat is most commonly interrupted and is stably inherited in the general population. Although expanded alleles, premutation range and, in rare cases, also non-disease associated alleles containing uninterrupted CCTG tracts have been described, the threshold between these categories is poorly characterised. Here, we describe four families with members reporting neuromuscular complaints, in whom we identified altogether nine ambiguous CNBP alleles containing uninterrupted CCTG repeats in the range between 32 and 42 repeats. While these grey-zone alleles are most likely not pathogenic themselves, since other pathogenic mutations were identified and particular family structures did not support their pathogenic role, they were found to be unstable during intergenerational transmission. On the other hand, there was no observable general microsatellite instability in the genome of the carriers of these alleles. Our results further refine the division of CNBP CCTG repeat alleles into two major groups, i.e., interrupted and uninterrupted alleles. Both interrupted and uninterrupted alleles with up to approximately 30 CCTG repeats were shown to be generally stable during intergenerational transmission, while intergenerational as well as somatic instability seems to gradually increase in uninterrupted alleles with tract length growing above this threshold.

Cellular nucleic acid–binding protein is essential for type I interferon–mediated immunity to RNA virus infection

Type I interferons (IFNs) are innate immune cytokines required to establish cellular host defense. Precise control of IFN gene expression is crucial to maintaining immune homeostasis. Here, we demonstrated that cellular nucleic acid-binding protein (CNBP) was required for the production of type I IFNs in response to RNA virus infection. CNBP deficiency markedly impaired IFN production in macrophages and dendritic cells that were infected with a panel of RNA viruses or stimulated with synthetic double-stranded RNA. Furthermore, CNBP-deficient mice were more susceptible to influenza virus infection than were wild-type mice. Mechanistically, CNBP was phosphorylated and translocated to the nucleus, where it directly binds to the promoter of IFNb in response to RNA virus infection. Furthermore, CNBP controlled the recruitment of IFN regulatory factor (IRF) 3 and IRF7 to IFN promoters for the maximal induction of IFNb gene expression. These studies reveal a previously unrecognized role for CNBP as a transcriptional regulator of type I IFN genes engaged downstream of RNA virus-mediated innate immune signaling, which provides an additional layer of control for IRF3- and IRF7-dependent type I IFN gene expression and the antiviral innate immune response.

Transcriptome analysis of resistance mechanism to potato wart disease.

To understand the molecular mechanism of the resistance to potato wart disease, we used the potato cultivar Qingshu 9 as the experimental material and prepared potato samples with different levels of disease through inoculation. The RNAs of the samples were extracted, and transcriptome analysis was performed on the samples not infected by the disease (control group) and also on the samples with different levels of disease, with the aid of high-throughput sequencing. Next, the differentially expressed genes (DEGs) associated with the resistance to potato wart disease were identified based on the analysis results. Using bioinformatic tools, the DEGs were functionally annotated, classified, and enriched in related metabolic pathways. The main results are as follows: Compared with the control group, 4 DEGs were identified in the samples with light disease, 52 were found in the samples with medium disease, and 214 were discovered in the samples with heavy disease. Potato mainly resists the wart disease by suppressing its gene expression, and the degree of suppression depends on the level of the disease; the disease resistance might be dominated by cellular nucleic acid-binding protein, AP2-like transcription factor, and E3 ubiquitin-protein ligase. This research provides new insights into the molecular mechanism of potato resistance against wart disease.

The SARS-CoV-2 RNA\u2013protein interactome in infected human cells

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.

Zinc-finger protein CNBP alters the 3-D structure of lncRNA Braveheart in solution

Long non-coding RNAs (lncRNAs) constitute a significant fraction of the transcriptome, playing important roles in development and disease. However, our understanding of structure-function relationships for this emerging class of RNAs has been limited to secondary structures. Here, we report the 3-D atomistic structural study of epigenetic lncRNA, Braveheart (Bvht), and its complex with CNBP (Cellular Nucleic acid Binding Protein). Using small angle X-ray scattering (SAXS), we elucidate the ensemble of Bvht RNA conformations in solution, revealing that Bvht lncRNA has a well-defined, albeit flexible 3-D structure that is remodeled upon CNBP binding. Our study suggests that CNBP binding requires multiple domains of Bvht and the RHT/AGIL RNA motif. We show that RHT/AGIL, previously shown to interact with CNBP, contains a highly flexible loop surrounded by more ordered helices. As one of the largest RNA-only 3-D studies, the work lays the foundation for future structural studies of lncRNA-protein complexes.

CNBP controls tumor cell biology by regulating tumor-promoting gene expression.

Cellular nucleic acid-binding protein (CNBP) is associated with cell proliferation, and its expression is elevated in human tumors, but the molecular mechanisms of CNBP in tumor cell biology have not been fully elucidated. In this study, we report that CNBP is a transcription factor essential for regulating matrix metalloproteinases mmp-2, mmp-14, and transcription factor e2f2 gene expression by binding to their promoter regions via a sequence-specific manner. Importantly, epidermal growth factorstimulation is required to induce CNBP phosphorylation and nuclear transport, thereby promoting the expression of mmp-2, mmp-14, and e2f2 genes. As a consequence, loss of cnbp attenuates the ability of tumor cell growth, invasion, and migration. Conversely, overexpression of cnbp is associated with tumor cell biology. Collectively, our findings reveal CNBP as a key transcriptional regulator of tumor-promoting target genes to control tumor cell biology.

Sensing danger through a “finger”

In this issue of JEM, the study by Chen et al. (https://doi.org/10.1084/jem.20181031) reveals a previously unrecognized role of cellular nucleic acid-binding protein (Cnbp) as a novel transcriptional regulator of interleukin-12β (IL-12β) transcription and IL-12-driven, Th1-mediated immune responses, which has important implications for both host defense and inflammatory disease.

CNBP controls IL-12 gene transcription and Th1 immunity.

An inducible program of inflammatory gene expression is a hallmark of antimicrobial defenses. Recently, cellular nucleic acid-binding protein (CNBP) was identified as a regulator of nuclear factor-kappaB (NF-κB)-dependent proinflammatory cytokine gene expression. Here, we generated mice lacking CNBP and found that CNBP regulates a very restricted gene signature that includes IL-12β. CNBP resides in the cytosol of macrophages and translocates to the nucleus in response to diverse microbial pathogens and pathogen-derived products. Cnbp-deficient macrophages induced canonical NF-κB/Rel signaling normally but were impaired in their ability to control the activation of c-Rel, a key driver of IL-12β gene transcription. The nuclear translocation and DNA-binding activity of c-Rel required CNBP. Lastly, Cnbp-deficient mice were more susceptible to acute toxoplasmosis associated with reduced production of IL-12β, as well as a reduced T helper type 1 (Th1) cell IFN-γ response essential to controlling parasite replication. Collectively, these findings identify CNBP as important regulator of c-Rel-dependent IL-12β gene transcription and Th1 immunity.

Phosphoglucomutase 1 inhibits hepatocellular carcinoma progression by regulating glucose trafficking.

Glycogen metabolism commonly altered in cancer is just beginning to be understood. Phosphoglucomutase 1 (PGM1), the first enzyme in glycogenesis that catalyzes the reversible conversion between glucose 1-phosphate (G-1-P) and glucose 6-phosphate (G-6-P), participates in both the breakdown and synthesis of glycogen. Here, we show that PGM1 is down-regulated in hepatocellular carcinoma (HCC), which is associated with the malignancy and poor prognosis of HCC. Decreased PGM1 expression obstructed glycogenesis pathway, which leads to the increased flow of glucose into glycolysis, thereby promoting tumor cell proliferation and HCC development. The loss of forkhead box protein J2 (FOXJ2), at least partly due to low genomic copy number in HCC, releases cellular nucleic acid-binding protein (CNBP), a nucleic acid chaperon, to bind to and promote G-quadruplex formation in PGM1 promoter and therefore decreases PGM1 expression. In addition, integrated analyses of PGM1 and FOXJ2 expression provide a better prediction for the malignance and prognosis of HCC. This study establishes a tumor-suppressive role of PGM1 by regulating glucose trafficking and uncovers a novel regulatory mechanism of PGM1 expression.

CNBP Homologues Gis2 and Znf9 Interact with a Putative G-Quadruplex-Forming 3' Untranslated Region, Altering Polysome Association and Stress Tolerance in Cryptococcus neoformans.

In Cryptococcusneoformans, mRNAs encoding ribosomal proteins (RP) are rapidly and specifically repressed during cellular stress, and the bulk of this repression is mediated by deadenylation-dependent mRNA decay. A motif-finding approach was applied to the 3' untranslated regions (UTRs) of RP transcripts regulated by mRNA decay, and a single, significant motif, GGAUG, was identified. Znf9, a small zinc knuckle RNA binding protein identified by mass spectrometry, was found to interact specifically with the RPL2 3'-UTR probe. A second, homologous protein, Gis2, was identified in the genome of C.neoformans and also bound the 3'-UTR probe, and deletion of both genes resulted in loss of binding in cell extracts. The RPL2 3' UTR contains four G-triplets (GGG) that have the potential to form a G-quadruplex, and temperature gradient gel electrophoresis revealed a potassium-dependent structure consistent with a G-quadruplex that was abrogated by mutation of G-triplets. However, deletion of G-triplets did not abrogate the binding of either Znf9 or Gis2, suggesting that these proteins either bind irrespective of structure or act to prevent structure formation. Deletion of both GIS2 and ZNF9 resulted in a modest increase in basal stability of the RPL2 mRNA which resulted in an association with higher-molecular-weight polysomes under unstressed conditions. The gis2Δ mutant and gis2Δ znf9Δ double mutant exhibited sensitivity to cobalt chloride, fluconazole, and oxidative stress, and although transcriptional induction of ERG25 was similar to that of the wild type, analysis of sterol content revealed repressed levels of sterols in the gis2Δ and gis2Δ znf9Δ double mutant, suggesting a role in translational regulation of sterol biosynthesis.IMPORTANCE Stress adaptation is fundamental to the success of Cryptococcus neoformans as a human pathogen and requires a reprogramming of the translating pool of mRNA. This reprogramming begins with the regulated degradation of mRNAs encoding the translational machinery. The mechanism by which these mRNAs are specified has not been determined. This study has identified a cis element within a G-quadruplex structure that binds two C. neoformans homologues of cellular nucleic acid binding protein (CNBP). These proteins regulate the polysome association of the target mRNA but perform functions related to sterol homeostasis which appear independent of ribosomal protein mRNAs. The presence of two CNBP homologues in C. neoformans suggests a diversification of function of these proteins, one of which appears to regulate sterol biosynthesis and fluconazole sensitivity.