The RNA-binding protein GRSF1 promotes hepatocarcinogenesis via competitively binding to YY1 mRNA with miR-30e-5p.

BACKGROUND: Dysregulation of RNA binding protein (RBP) expression has been confirmed to be causally linked with tumorigenesis. The detailed biological effect and underlying mechanisms of the RBP GRSF1 in hepatocellular carcinoma (HCC) remain unclear. METHODS: HCC cells with stable knockdown of GRSF1 were established using two sh-RNA-encoding lentiviruses. The functions of GRSF1 in HCC were explored using MTT, colony formation, flow cytometry, and Transwell assays and a xenograft model. Transcriptomic sequencing in GRSF1-deficient MHCC-97H cells was carried out to identify the downstream effector of GRSF1. The regulatory mechanisms among GRSF1, YY1 and miR-30e-5p were investigated via RNA immunoprecipitation, luciferase, RNA pull-down and ChIP assays. Several in vivo assays were used to assess the selectivity of the small-molecule compound VE-821 in HCC and to confirm the absence of general toxicity in animal models. RESULTS: GRSF1 was frequently increased in HCC tissue and cells and was associated with worse clinical outcomes. GRSF1 functions as a novel oncogenic RBP by enhancing YY1 mRNA stability, and the GUUU motifs within the YY1 3`UTR 2663-2847 were the specific binding motifs for GRSF1. YY1 feedback promoted GRSF1 expression by binding to the GRSF1 promoter. In addition, YY1 was a critical target of miR-30e-5p, which was confirmed in this study to inhibit HCC hepatocarcinogenesis. GRSF1 and miR-30e-5p competitively regulated YY1 by binding to its 3`UTR 2663-2847 region. Finally, we identified that VE-821 blocked HCC progression by inhibiting the GRSF1/YY1 pathway. CONCLUSION: This study revealed the interaction network among GRSF1, YY1 and miR-30e-5p, providing new insight into HCC pathogenesis, and indicated that VE821 may serve as a novel agent with potential for HCC treatment through inhibition of the GRSF1/YY1 axis.

Characterization of ALBA Family Expression and Localization in Arabidopsis thaliana Generative Organs.

ALBA DNA/RNA-binding proteins form an ancient family, which in eukaryotes diversified into two Rpp25-like and Rpp20-like subfamilies. In most studied model organisms, their function remains unclear, but they are usually associated with RNA metabolism, mRNA translatability and stress response. In plants, the enriched number of ALBA family members remains poorly understood. Here, we studied ALBA dynamics during reproductive development in Arabidopsis at the levels of gene expression and protein localization, both under standard conditions and following heat stress. In generative tissues, ALBA proteins showed the strongest signal in mature pollen where they localized predominantly in cytoplasmic foci, particularly in regions surrounding the vegetative nucleus and sperm cells. Finally, we demonstrated the involvement of two Rpp25-like subfamily members ALBA4 and ALBA6 in RNA metabolism in mature pollen supported by their co-localization with poly(A)-binding protein 3 (PABP3). Collectively, we demonstrated the engagement of ALBA proteins in male reproductive development and the heat stress response, highlighting the involvement of ALBA4 and ALBA6 in RNA metabolism, storage and/or translational control in pollen upon heat stress. Such dynamic re-localization of ALBA proteins in a controlled, developmentally and environmentally regulated manner, likely reflects not only their redundancy but also their possible functional diversification in plants.

RNA-binding and prion domains: the Yin and Yang of phase separation.

Proteins and RNAs assemble in membrane-less organelles that organize intracellular spaces and regulate biochemical reactions. The ability of proteins and RNAs to form condensates is encoded in their sequences, yet it is unknown which domains drive the phase separation (PS) process and what are their specific roles. Here, we systematically investigated the human and yeast proteomes to find regions promoting condensation. Using advanced computational methods to predict the PS propensity of proteins, we designed a set of experiments to investigate the contributions of Prion-Like Domains (PrLDs) and RNA-binding domains (RBDs). We found that one PrLD is sufficient to drive PS, whereas multiple RBDs are needed to modulate the dynamics of the assemblies. In the case of stress granule protein Pub1 we show that the PrLD promotes sequestration of protein partners and the RBD confers liquid-like behaviour to the condensate. Our work sheds light on the fine interplay between RBDs and PrLD to regulate formation of membrane-less organelles, opening up the avenue for their manipulation.

The zinc finger protein DCM1 is required for male meiotic cytokinesis by preserving callose in rice.

Meiotic cytokinesis influences the fertility and ploidy of gametes. However, limited information is available on the genetic control of meiotic cytokinesis in plants. Here, we identified a rice mutant with low male fertility, defective callose in meiosis 1 (dcm1). The pollen grains of dcm1 are proved to be defective in exine formation. Meiotic cytokinesis is disrupted in dcm1, resulting in disordered spindle orientation during meiosis II and formation of pollen grains with varied size and DNA content. We demonstrated that meiotic cytokinesis defect in dcm1 is caused by prematurely dissolution of callosic plates. Furthermore, peripheral callose surrounding the dcm1 pollen mother cells (PMCs) also disappeared untimely around pachytene. The DCM1 protein contains five tandem CCCH motifs and interacts with nuclear poly (A) binding proteins (PABNs) in nuclear speckles. The expression profiles of genes related to callose synthesis and degradation are significantly modified in dcm1. Together, we propose that DCM1 plays an essential role in male meiotic cytokinesis by preserving callose from prematurely dissolution in rice.

Phenotypic Variation in Different Lesions of Mycosis Fungoides Biopsied Within a Short Period of Time From the Same Patient.

Phenotypic variants of mycosis fungoides (MF) include mainly the expression of cytotoxic markers by neoplastic cells (either α/ß or γ/δ cytotoxic). To manage the patient properly, distinction from other cutaneous cytotoxic natural killer/T-cell lymphomas is paramount. Particularly for cutaneous γ/δ T-cell lymphoma, distinction is often based on clinicopathologic correlation (presence of tumors at first diagnosis as opposed to patches only in MF). The authors report a case of cytotoxic MF characterized by expression of TCRγ in two of three biopsies performed within a time frame of 1 week. The patient presented with patches, plaques, and 1 tumor at the time of first diagnosis; thus, distinction from cutaneous γ/δ T-cell lymphoma was not possible on clinical grounds alone. The diagnosis of MF was established, thanks to the phenotypic variations revealed by the three biopsies, with 1 lacking expression of cytotoxic proteins (TIA-1 and granzyme B) and of TCRγ. This case shows the importance to perform several biopsies in cases of cutaneous lymphoma, as morphologic and phenotypic features are variable and information gathered from a single biopsy may result in a wrong diagnosis.

Molecular Characterization of UKp83/68, a Widespread Nuclear Proteins that Bind Poly(A) and Colocalize with a Nuclear Speckle's Component.

We have cloned a gene from a rat liver cDNA library, representing alternatively spliced cDNAs encoding 83-kDa and 68-kDa proteins, which we have designated as UKp83 and UKp68, respectively. Both proteins have a predicted nuclear localization signal and five CCCH motifs (zinc-binding motifs), and share a degree of sequence similarity with Nab2, a yeast protein that contains nucleic acidbinding motifs and tandem CCCH zinc fingers. Nab2 binds homopolymeric RNA and single-stranded DNA and regulates poly(A) tail length and the export of mRNA to the cytosol. The CCCH motifs of UKp83/68 bound poly(A) and ssDNA strongly and other RNA homopolymers and dsDNA less efficiently. The UKp83/68 protein localized within the nucleus with a fibrous or punctate structure that reflected the distribution of SC35, a known marker of nuclear speckles which are nuclear domains enriched in pre-mRNA splicing factors and located in the interchromatin regions of the nucleoplasm of mammalian cells. The distribution of UKp83/68 changed during the different stages of mitosis. During prometaphase, when the nuclear envelope disintegrates, the protein becomes partially localized on the chromosomes; at other times, transiently dispersed over the cytoplasm with the formation of fibrous structure. The transient expression of UKp83 in HEK293T cells had no apparent effect on cellular function, whereas the expression of an antisense sequence or C-terminal domain of UKp83 induced apoptosis. These results suggest that UKp83/68 is probably essential for cell viability and may play important role in mRNA processing.

Comparative in situ topoproteome analysis reveals differences in patch test-induced eczema: cytotoxicity-dominated nickel versus pleiotrope pollen reaction.

A subgroup of patients with atopic eczema develops acute eczematous reactions to type I allergy-inducing agents such as pollen that clinically resemble type IV allergies induced by haptens like metal ions. To clarify the underlying immunologic mechanisms, this study was designed to map the inflammatory in situ topoproteome of eczematous responses to grass/birch pollen and nickel by using atopy patch test (APT) and nickel patch test (NPT) as an appropriate clinical model, respectively. Biopsies from NPT (n = 6) and APT (n = 6) with positive reactions at 72 h were analysed by multiple epitope ligand cartography (MELC), which enabled to investigate coexpression of 49 different epitopes immunohistochemically in a single given tissue section. Colocalisation of IgE and FcepsilonRI was investigated by confocal microscopy. Compared with APT responses, NPT reactions were dominated by cytotoxic TIA-1 + and CD8 + T cells. In contrast, the immune response in APT reactions appeared more pleiotrope - as detected by colocalisation analysis. Multiple combinatorial molecular phenotype (CMP) motifs containing naive, early maturation and memory T cell (CD45RA, CD7, CD44, CD45R0), and general activation markers (CLA, HLA-DR, CD13, CD29, CD58, CD71, CD138) were significantly higher expressed in APT when compared with NPT reactions. APT response was confirmed to be accompanied by IgE bound to FcepsilonRI. In summary, our results demonstrate that the NPT reaction is clearly dominated by cytotoxic events, while the APT reaction to pollen grains is more heterogeneous and elicits a combined humoral and cellular immune reaction.

Modulation of KGF-1 gene expression in oral fibroblasts by ripe areca nut extract.

BACKGROUND: Areca chewing is a common habit of Asians, leading to a high propensity for a variety of oral diseases in this population. This research aimed to study the expression level of genes in oral fibroblast cell lines in response to exposure to ripe areca nut extract (rANE). METHODS: Fifteen oral fibroblast cell lines obtained from individuals aged 20-77 years were established. Treatment of a cell line with 40 micro g/ml rANE for 24 h was performed to achieve RNA for cDNA microarray analysis. RESULTS: Among some 320 genes exhibiting detectable expression levels, 14 were up-regulated and 26 were down-regulated more than 2.5-fold. Semi-quantitative RT-PCR analysis suggested that up-regulation of IL-6 expression and down-regulation of PDGFR, APP-1 and KGF-1 expressions in multiple cell lines assayed, were compatible with the results of the microarray analysis. Using quantitative real-time RT-PCR analysis, a remarkable down-regulation of KGF-1 expression in response to 40 microg/ml rANE, ranging 1.5-ninefold as compared to controls, was found in 60% (9/15) of the cell lines. CONCLUSION: This study established a novel toxicogenomic database for rANE. The down-regulation of KGF-1 expression in oral fibroblast cell lines potentially impairs the proliferation of overlying keratinocytes, which could partially explain the frequent epithelial atrophy observed in chronic areca chewers in vivo.

Refinement of the PARK3 locus on chromosome 2p13 and the analysis of 14 candidate genes.

Parkinson's disease (PD) is a common neurodegenerative disorder with clinical features of bradykinesia, rigidity, resting tremor and postural instability resulting from the deficiency of dopamine in the nigrostriatal system. Previously we mapped a susceptibility gene for an autosomal dominant form of PD to a 10.6 cM region of chromosome 2p (PARK3; OMIM 602404). A common haplotype shared by two North American kindreds (Families B and C) genealogically traced to Southern Denmark and Northern Germany suggested a founder effect. Here we report progress in the refinement of the PARK3 locus and sequence analysis of candidate genes within the region. Members of families B and C were genotyped using polymorphic markers, reducing the minimum common haplotype to eight markers spanning a physical distance of 2.5 Mb. Analysis of 14 genes within the region did not reveal any potentially pathogenic mutations segregating with the disease, implying that none of these genes are likely candidates for PARK3.

Purification and characterization of a 29 kDa poly(A)-binding protein from chickpea (Cicer arietinum) epicotyl.

A poly(A)-binding protein (PABP) with mol wt 29,000 has been purified from chickpea (Cicer arietinum) epicotyl by ammonium sulfate fractionation and Cibacron blue F3-GA chromatography, making a complex with poly(A) and elution of PABP-poly(A) complex at 45 degrees C from oligo d(T)-cellulose. The elution pattern and binding properties show that the purified protein is different from the PABP (mol. wt 72,000) reported earlier from our laboratory.

Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.

Genomic array technologies provide a means for profiling global changes in gene expression under a variety of conditions. However, it has been difficult to assess whether transcriptional or posttranscriptional regulation is responsible for these changes. Additionally, fluctuations in gene expression in a single cell type within a complex tissue like a tumor may be masked by overlapping profiles of all cell types in the population. In this paper, we describe the use of cDNA arrays to identify subsets of mRNAs contained in endogenous messenger ribonucleoprotein complexes (mRNPs) that are cell type specific. We identified mRNA subsets from P19 embryonal carcinoma stem cells by using mRNA-binding proteins HuB, eIF-4E, and PABP that are known to play a role in translation. The mRNA profiles associated with each of these mRNPs were unique and represented gene clusters that differed from total cellular RNA. Additionally, the composition of mRNAs detected in HuB-mRNP complexes changed dramatically after induction of neuronal differentiation with retinoic acid. We suggest that the association of structurally related mRNAs into mRNP complexes is dynamic and may help regulate posttranscriptional events such as mRNA turnover and translation. Recovering proteins specifically associated with mRNP complexes to identify and profile endogenously clustered mRNAs should provide insight into structural and functional relationships among gene transcripts and/or their protein products. We have termed this approach to functional genomics ribonomics and suggest that it will provide a useful paradigm for organizing genomic information in a biologically relevant manner.

Purification and characterization of a poly(A)-binding protein from chickpea (Cicer arietinum) epicotyl.

A poly(A)-binding protein (PABP) with mol wt 72,000 has been purified from chickpea (Cicer arietinum) epicotyls by ammonium sulfate fractionation, Cibacron blue F3-GA and poly(A) agarose chromatography. The binding properties and the specificity of binding show that the purified protein is an analogue of PABPs in other eukaryotes. This PABP is highly susceptible to proteolysis and upon degradation forms a polypeptide fragment of mol wt 21,000 which has an independent poly(A) binding activity.

Unusual sites of arginine methylation in Poly(A)-binding protein II and in vitro methylation by protein arginine methyltransferases PRMT1 and PRMT3.

Arginine methylation is a post-translational modification found mostly in RNA-binding proteins. Poly(A)-binding protein II from calf thymus was shown by mass spectrometry and sequencing to contain NG, NG-dimethylarginine at 13 positions in its amino acid sequence. Two additional arginine residues were partially methylated. Almost all of the modified residues were found in Arg-Xaa-Arg clusters in the C terminus of the protein. These motifs are distinct from Arg-Gly-Gly motifs that have been previously described as sites and specificity determinants for asymmetric arginine dimethylation. Poly(A)-binding protein II and deletion mutants expressed in Escherichia coli were in vitro substrates for two mammalian protein arginine methyltransferases, PRMT1 and PRMT3, with S-adenosyl-L-methionine as the methyl group donor. Both PRMT1 and PRMT3 specifically methylated arginines in the C-terminal domain corresponding to the naturally modified sites.

A pollen-, ovule-, and early embryo-specific poly(A) binding protein from Arabidopsis complements essential functions in yeast.

Poly(A) tails of eukaryotic mRNAs serve as targets for regulatory proteins affecting mRNA stability and translation. Differential mRNA polyadenylation and deadenylation during gametogenesis and early development are now widely recognized as mechanisms of translational regulation in animals, but they have not been observed in plants. Here, we report that the expression of the PAB5 gene encoding one of the poly(A) binding proteins (PABPs) in Arabidopsis is restricted to pollen and ovule development and early embryogenesis. Furthermore, PAB5 is capable of rescuing a PABP-deficient yeast strain by partially restoring both poly(A) shortening and translational initiation functions of PABP. However, PAB5 did not restore the linkage of deadenylation and decapping, thus demonstrating that this function of PABP is not essential for viability. Also, like endogenous PABP, PAB5 expressed in yeast demonstrated genetic interaction with a recently characterized yeast protein SIS1, which is also involved in translational initiation. We propose that PAB5 encodes a post-transcriptional regulatory factor acting through molecular mechanisms similar to those reported for yeast PABP. This factor may have evolved further to post-transcriptionally regulate plant sexual reproduction and early development.