Purine-rich Element Research Articles

Purine-Rich Element Binding Protein Alpha, a Nuclear Matrix Protein, Has a Role in Prostate Cancer Progression.

Solid tumors as well as leukemias and lymphomas show striking changes in nuclear structure including nuclear size and shape, the number and size of nucleoli, and chromatin texture. These alterations have been used in cancer diagnosis and might be related to the altered functional properties of cancer cells. The nuclear matrix (NM) represents the structural composition of the nucleus and consists of nuclear lamins and pore complexes, an internal ribonucleic protein network, and residual nucleoli. In the nuclear microenvironment, the NM is associated with multi-protein complexes, such as basal transcription factors, signaling proteins, histone-modifying factors, and chromatin remodeling machinery directly or indirectly through scaffolding proteins. Therefore, alterations in the composition of NM could result in altered DNA topology and changes in the interaction of various genes, which could then participate in a cascade of the cancer process. Using an androgen-sensitive prostate cancer cell line, LNCaP, and its androgen-independent derivative, LN96, conventional 2D-proteomic analysis of the NM proteins revealed that purine-rich element binding protein alpha (PURα) was detected in the NM proteins and differentially expressed between the cell lines. In this article, we will review the potential role of the molecule in prostate cancer.

Construction of an exosome-associated miRNA-mRNA regulatory network and validation of FYCO1 and miR-17-5p as potential biomarkers associated with ovarian cancer.

The occurrence and development of several human physiological processes are significantly influenced by the competing endogenous RNA (ceRNA) network. The aim of the present study was to construct a microRNA (miRNA)-mRNA network associated with exosomes in ovarian cancer (OV), and experimental validation of key target genes. By exploring the Gene Expression Omnibus (GEO) database, we analyzed the RNAs from 226 samples to identify differentially expressed miRNAs (DEMs) and genes (DEGs) that showed differential expression as OV progressed. Subsequently, we conducted Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on the DEGs. Furthermore, we constructed a miRNA-mRNA network that pertains to exosomes in OV using DEMs and DEGs. Moreover, we validated the expression levels of mRNAs in the miRNA-mRNA network using Gene Expression Profiling Interactive Analysis (GEPIA2). Ultimately, luciferase reporter assay was used to identify the potential target relationship between FYVE and coiled-coil domain containing 1 (FYCO1) and miRNAs. Our analysis screened a total of 14 DEMs and 101 DEGs, and the DEGs were mainly enriched in DNA replication or repair, amino acid biosynthesis and carbon metabolism. Furthermore, a miRNA-mRNA network was constructed including 3 miRNAs (hsa-miR-17-5p, hsa-miR-20b-5p and hsa-miR-20a-5p) and 2 mRNAs, FYCO1 and purine rich element binding protein A (PURA). Finally, the 2 mRNAs in this miRNA-mRNA network were verified by GEPIA2 using The Cancer Genome Atlas (TCGA) database. Among them, only FYCO1 showed significant different expression of mRNA in OV and normal tissue, while the prognosis of FYCO1 in OV remains controversial due to different database. Interestingly, FYCO1 was identified as the target of hsa-miR-17-5p. By constructing a novel network of miRNA-mRNA, we can gain new understanding of the molecular mechanisms that drive exosomes in OV. Targeting FYCO1, which originates from exosomes, may hold promise as a diagnostic marker for OV.

Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B.

Purine-rich element binding protein B (Purβ) is a single-stranded DNA (ssDNA) and RNA-binding protein that functions as a transcriptional repressor of genes encoding certain muscle-restricted contractile proteins in the setting of cellular stress or tissue injury. A prior report from our laboratory implicated specific basic amino acid residues in the physical and functional interaction of Purβ with the smooth muscle-α actin gene (Acta2) promoter. Independent structural analysis of fruit fly Purα uncovered a role for several aromatic residues in the binding of this related protein to ssDNA. Herein, we examine the functional importance of a comparable set of hydrophobic residues that are positionally conserved in the repeat I (Y59), II (F155), and III (F256) domains of murine Purβ. Site-directed Y/F to alanine substitutions were engineered, and the resultant Purβ point mutants were tested in various biochemical and cell-based assays. None of the mutations affected the cellular expression, structural stability, or dimerization capacity of Purβ. However, the Y59A and F155A mutants demonstrated weaker Acta2 repressor activity in transfected fibroblasts and reduced binding affinity for the purine-rich strand of an Acta2 cis-regulatory element in vitro. Mutation of Y59 and F155 also altered the multisite binding properties of Purβ for ssDNA and diminished the interaction of Purβ with Y-box binding protein 1, a co-repressor of Acta2. Collectively, these findings suggest that some of the same aromatic residues, which govern the specific and high-affinity binding of Purβ to ssDNA, also mediate certain heterotypic protein interactions underlying the Acta2 repressor function of Purβ.

Abstract 2266: Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B

Abstract 2266: Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B

Forming cytoplasmic stress granules PURα suppresses mRNA translation initiation of IGFBP3 to promote esophageal squamous cell carcinoma progression

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal malignancies worldwide. Recently, our group identified purine-rich element binding protein alpha (PURα), a single-stranded DNA/RNA-binding protein, to be significantly associated with the progression of ESCC. Additional immunofluorescence staining demonstrated that PURα forms cytoplasmic stress granules to suppress mRNA translation initiation. The expression level of cytoplasmic PURα in ESCC tumor tissues was significantly higher than that in adjacent epithelia and correlated with a worse patient survival rate by immunohistochemistry. Functionally, PURα strongly preferred to bind to UG-/U-rich motifs and mRNA 3´UTR by CLIP-seq analysis. Moreover, PURα knockout significantly increased the protein level of insulin-like growth factor binding protein 3 (IGFBP3). In addition, it was further demonstrated that PURα-interacting proteins are remarkably associated with translation initiation factors and ribosome-related proteins and that PURα regulates protein expression by interacting with translation initiation factors, such as PABPC1, eIF3B and eIF3F, in an RNA-independent manner, while the interaction with ribosome-related proteins is significantly dependent on RNA. Specifically, PURα was shown to interact with the mRNA 3´UTR of IGFBP3 and inhibit its expression by suppressing mRNA translation initiation. Together, this study identifies cytoplasmic PURα as a modulator of IGFBP3, which could be a promising therapeutic target for ESCC treatment.

PABP/purine-rich motif as an initiation module for cap-independent translation in pattern-triggered immunity

Upon stress, eukaryotes typically reprogram their translatome through GCN2-mediated phosphorylation of the eukaryotic translation initiation factor, eIF2α, to inhibit general translation initiation while selectively translating essential stress regulators. Unexpectedly, in plants, pattern-triggered immunity (PTI) and response to other environmental stresses occur independently of the GCN2/eIF2α pathway. Here, we show that while PTI induces mRNA decapping to inhibit general translation, defense mRNAs with a purine-rich element ("R-motif") are selectively translated using R-motif as an internal ribosome entry site (IRES). R-motif-dependent translation is executed by poly(A)-binding proteins (PABPs) through preferential association with the PTI-activating eIFiso4G over the repressive eIF4G. Phosphorylation by PTI regulators mitogen-activated protein kinase 3 and 6 (MPK3/6) inhibits eIF4G's activity while enhancing PABP binding to the R-motif and promoting eIFiso4G-mediated defense mRNA translation, establishing a link between PTI signaling and protein synthesis. Given its prevalence in both plants and animals, the PABP/R-motif translation initiation module may have a broader role in reprogramming the stress translatome.

Luteolin inhibits viability, migration, angiogenesis and invasion of non-small cell lung cancer vascular endothelial cells via miR-133a-3p/purine rich element binding protein B-mediated MAPK and PI3K/Akt signaling pathways

Luteolin inhibits tumorigenesis of non-small cell lung cancer (NSCLC), but its mechanism still needs to be clarified. We hereby explored the effects of luteolin in vascular endothelial cells of NSCLC (NSCLC-VECs). After extraction and identification of NSCLC-VECs, cells were treated with luteolin and transfected. The viability, migration, angiogenesis and invasion of the cells were measured. The levels of miR-133a-3p, purine rich element binding protein B (PURB), vascular endothelial growth factor (VEGF), phosphatidylinositol 3-kinase (PI3K), Akt, mitogen-activated protein kinases (MAPK), matrix metalloproteinase (MMP)-2/-9 were determined. The interaction relationship of miR-133a-3p and PURB was identified. Luteolin inhibited the viability, migration, angiogenesis and invasion of NSCLC-VECs yet up-regulated miR-133a-3p level, while miR-133a-3p inhibitor counteracted the repressive effect of luteolin on the viability, migration, angiogenesis, and invasion in NSCLC-VECs. Luteolin inhibited the expressions of migration- and invasion-associated proteins (VEGF, MMP-2 and MMP-9), PI3K/Akt and MAPK signaling pathways-related factors, while miR-133a-3p inhibitor reversed the inhibitory effect of Luteolin on NSCLC-VECs. Luteolin decreased the level of PURB, which was targeted by miR-133a-3p. ShPURB promoted miR-133a-3p level in NSCLC-VECs, while reversing the promoting effects of miR-133a-3p inhibitor on the migration, invasion, and levels of migration- and invasion-associated proteins, PI3K/Akt and MAPK pathways-associated factors in NSCLC-VECs. Collectively speaking, luteolin inhibits the migration and invasion of NSCLC-VECs via miR-133a-3p/PURB- mediated MAPK and PI3K/Akt pathways.

Analysis of the role of Pur\u03b1 in the pathogenesis of Alzheimer's disease based on RNA-seq and ChIP-seq

Purine rich element binding protein A (Purα), encoded by the Purα gene, is an important transcriptional regulator that binds to DNA and RNA and is involved in processes such as DNA replication and RNA translation. Purα also plays an important role in the nervous system. To identify the function of Pura, we performed RNA sequence (RNA-seq) analysis of Purɑ-KO mouse hippocampal neuron cell line (HT22) to analyze the effect of Purα deletion on neuronal expression profiles. And combined with ChIP-seq analysis to explore the mechanism of Purα on gene regulation. In the end, totaly 656 differentially expressed genes between HT22 and Purα-KO HT22 cells have been found, which include 7 Alzheimer’s disease (AD)-related genes and 5 Aβ clearance related genes. 47 genes were regulated by Purα directly, the evidence based on CHIP-seq, which include Insr, Mapt, Vldlr, Jag1, etc. Our study provides the important informations of Purα in neuro-development. The possible regulative effects of Purα on AD-related genes consist inthe direct and indirect pathways of Purα in the pathogenesis of AD.

Purine-rich element binding protein B attenuates the coactivator function of myocardin by a novel molecular mechanism of smooth muscle gene repression.

Myocardin is a potent transcriptional coactivator protein, which functions as the master regulator of vascular smooth muscle cell differentiation. The cofactor activity of myocardin is mediated by its physical interaction with serum response factor, a ubiquitously expressed transactivator that binds to CArG boxes in genes encoding smooth muscle-restricted proteins. Purine-rich element binding protein B (Purβ) represses the transcription of the smooth muscle α-actin gene (Acta2) in fibroblasts and smooth muscle cells by interacting with single-stranded DNA sequences flanking two 5' CArG boxes in the Acta2 promoter. In this study, the ability of Purβ to modulate the cofactor activity of myocardin was investigated using a combination of cellular and biochemical approaches. Results of smooth muscle gene promoter-reporter assays indicated that Purβ specifically inhibits the coactivator function of myocardin in a manner requiring the presence of all three single-stranded DNA binding domains in the Purβ homodimer. DNA binding analyses demonstrated that Purβ interacts with CArG-containing DNA elements with a much lower affinity compared to other purine-rich target sequences present in the Acta2 promoter. Co-immunoprecipitation and DNA pull-down assays revealed that Purβ associates with myocardin and serum response factor when free or bound to duplex DNA containing one or more CArG boxes. Functional analysis of engineered Purβ point mutants identified several amino acid residues essential for suppression of myocardin activity. Collectively, these findings suggest an inhibitory mechanism involving direct protein-protein interaction between the homodimeric Purβ repressor and the myocardin-serum response factor-CArG complex.

Comprehensive annotation of the Chinese tree shrew genome by large-scale RNA sequencing and long-read isoform sequencing.

The Chinese tree shrew (Tupaia belangeri chinensis) is emerging as an important experimental animal in multiple fields of biomedical research. Comprehensive reference genome annotation for both mRNA and long non-coding RNA (lncRNA) is crucial for developing animal models using this species. In the current study, we collected a total of 234 high-quality RNA sequencing (RNA-seq) datasets and two long-read isoform sequencing (ISO-seq) datasets and improved the annotation of our previously assembled high-quality chromosome-level tree shrew genome. We obtained a total of 3 514 newly annotated coding genes and 50 576 lncRNA genes. We also characterized the tissue-specific expression patterns and alternative splicing patterns of mRNAs and lncRNAs and mapped the orthologous relationships among 11 mammalian species using the current annotated genome. We identified 144 tree shrew-specific gene families, including interleukin 6 (IL6) and STT3 oligosaccharyltransferase complex catalytic subunit B (STT3B), which underwent significant changes in size. Comparison of the overall expression patterns in tissues and pathways across four species (human, rhesus monkey, tree shrew, and mouse) indicated that tree shrews are more similar to primates than to mice at the tissue-transcriptome level. Notably, the newly annotated purine rich element binding protein A (PURA) gene and the STT3B gene family showed dysregulation upon viral infection. The updated version of the tree shrew genome annotation (KIZ version 3: TS_3.0) is available at http://www.treeshrewdb.org and provides an essential reference for basic and biomedical studies using tree shrew animal models.

MaTAR25 lncRNA regulates the Tensin1 gene to impact breast cancer progression

Misregulation of long non-coding RNA (lncRNA) genes has been linked to a wide variety of cancer types. Here we report on Mammary Tumor Associated RNA 25 (MaTAR25), a nuclear enriched and chromatin associated lncRNA that plays a role in mammary tumor cell proliferation, migration, and invasion, both in vitro and in vivo. MaTAR25 functions by interacting with purine rich element binding protein B (PURB), and associating with a major downstream target gene Tensin1 (Tns1) to regulate its expression in trans. The Tns1 protein product is a critical component of focal adhesions linking signaling between the extracellular matrix and the actin cytoskeleton. Knockout of MaTAR25 results in down-regulation of Tns1 leading to a reorganization of the actin cytoskeleton, and a reduction of focal adhesions and microvilli. We identify LINC01271 as the human ortholog of MaTAR25, and importantly, increased expression of LINC01271 is associated with poor patient prognosis and metastasis. Our findings demonstrate that LINC01271 represents a potential therapeutic target to alter breast cancer progression.

A Click Chemistry Approach to Developing Molecularly Targeted DNA Scissors

Nucleic acid click chemistry was used to prepare a family of chemically modified triplex forming oligonucleotides (TFOs) for application as a new gene-targeted technology. Azide-bearing phenanthrene ligands-designed to promote triplex stability and copper binding-were 'clicked' to alkyne-modified parallel TFOs. Using this approach, a library of TFO hybrids was prepared and shown to effectively target purine-rich genetic elements in vitro. Several of the hybrids provide significant stabilisation toward melting in parallel triplexes (>20 °C) and DNA damage can be triggered upon copper binding in the presence of added reductant. Therefore, the TFO and 'clicked' ligands work synergistically to provide sequence-selectivity to the copper cutting unit which, in turn, confers high stabilisation to the DNA triplex. To extend the boundaries of this hybrid system further, a click chemistry-based di-copper binding ligand was developed to accommodate designer ancillary ligands such as DPQ and DPPZ. When this ligand was inserted into a TFO, a dramatic improvement in targeted oxidative cleavage is afforded.

PURα mediates epithelial-mesenchymal transition to promote esophageal squamous cell carcinoma progression by regulating Snail2.

Esophageal squamous cell carcinoma (ESCC) is one of the most common lethal cancers in the world. Dysregulation of purine-rich element binding protein alpha (PURα), which contributes to the initiation of PURΑ syndrome, is reportedly involved in the progression of multiple cancers, but its function and underlying mechanisms in ESCC progression remain unclear. Here, we first demonstrated that PURα promoted cell growth, migration and invasion in ESCC both in vitro and in vivo. An immunohistochemistry assay was then performed on 225 ESCC tissues, showing that high PURα expression was positively associated with lymph node metastasis and the AJCC stage, and the ESCC patients with positive PURα expression had worse survival. In addition, RNA sequencing implied that PURα induced epithelial-mesenchymal transition (EMT) in ESCC, which was further confirmed by qPCR, Western blotting and immunofluorescence analyses. Mechanistically, PURα enhanced the transcription of Snail2 by binding to its promoter region. Knockdown of Snail2 reversed PURα-induced EMT and inhibited the migration and invasion of ESCC cells. In conclusion, this study indicated that PURα promotes Snail2 transcriptional activity to induce EMT during ESCC progression.

A novel de novo mutation in the PURA gene associated with a new clinical finding: large brainstem

We report the case of a Caucasian Spanish boy, who showed profound neonatal hypotonia, feeding difficulties, apnea, severe developmental delay, epilepsy, bilateral convergent strabismus, poor verbal language development and a large brainstem. Whole-exome sequence uncovered a novel de novo mutation in the purine-rich element binding protein A gene (PURA; NM_005859.4:c.72del:p.(-Gly25AlafsTer53)) that encodes the transcriptional activator protein Pur-alpha (PURA). Mutations in this gene have been identified in patients with PURA syndrome, a rare disorder characterized by an early hypotonia, developmental delay, severe intellectual disability with or without epilepsy, and disability in expressive language development. Although, up to 75 cases have been identified worldwide, to the best of our knowledge, this is the first patient described with a brainstem larger than normal. In conclusion, our data expand both geneticand phenotypic spectrum associated with PURA gene mutations.

Widespread Separation of the Polypyrimidine Tract From 3' AG by G Tracts in Association With Alternative Exons in Metazoa and Plants.

At the end of introns, the polypyrimidine tract (Py) is often close to the 3′ AG in a consensus (Y)20NCAGgt in humans. Interestingly, we have found that they could also be separated by purine-rich elements including G tracts in thousands of human genes. These regulatory elements between the Py and 3′ AG (REPA) mainly regulate alternative 3′ splice sites (3′ SS) and intron retention. Here we show their widespread distribution and special properties across kingdoms. The purine-rich 3′ SS are found in up to about 60% of the introns among more than 1,000 species/lineages by whole genome analysis, and up to 18% of these introns contain the REPA G-tracts (REPAG) in about 0.6 million of 3′ SS in total. In particular, they are significantly enriched over their 3′ SS and genome backgrounds in metazoa and plants, and highly associated with alternative splicing of genes in diverse functional clusters. Cryptic splice sites harboring such G- and the other purine-triplets tend to be enriched (2–9 folds over the disrupted canonical 3′ SS) and aberrantly used in cancer patients carrying mutations of the SF3B1 or U2AF35, factors critical for branch point (BP) or 3′ AG recognition, respectively. Moreover, the REPAGs are significantly associated with reduced occurrences of BP motifs between the −24 and −4 positions, in particular absent between the −7 and −5 positions in several model organisms examined. The more distant BPs are associated with increased occurrences of alternative splicing in humans and zebrafish. The REPAGs appear to have evolved in a species- or phylum-specific way. Thus, there is widespread separation of the Py and 3′ AG by REPAGs that have evolved differentially. This special 3′ SS arrangement likely contributes to the generation of diverse transcript or protein isoforms in biological functions or diseases through alternative or aberrant splicing.

PURB is a positive regulator of amino acid-induced milk synthesis in bovine mammary epithelial cells.

Previous studies have implicated that purine-rich element binding protein B (PURB) is a key regulator of gene transcription and cell physiology. Whether PURB plays a regulatory role in milk synthesis in bovine mammary epithelial cells (BMECs) is not known. We observed that Met and Leu increased PURB expression and nuclear localization. Overexpression of PURB led to increased milk protein and fat synthesis as well as mTOR and SREBP-1c expression whereas PURB knockdown had the opposite effects. The PI3K inhibitor wortmannin totally abolished the stimulation of Met and Leu on PURB expression, and we further confirmed that PURB was required for Met and Leu to stimulate mTOR phosphorylation and SREBP-1c expression. We also demonstrated that PURB binds to the promoters of mTOR and SREBP-1c, and these bindings were increased by Met and Leu stimulation. In summary, our data reveal that PURB is required for amino acids to stimulate mTOR and SREBP-1c gene expression, and PURB is a positive regulator of amino acid-induced PI3K-regulated milk protein and fat synthesis in BMECs.

S14. PURA (Purine-Rich Element Binding Protein A) syndrome

Introduction Burst suppression is an electroencephalographic (EEG) pattern characterized by a brief interval of relatively high voltage activity with intervening periods of generalized suppression. It is commonly seen consequent to anoxic brain injury, deep coma states, encephalopathies or hypothermia. This pattern is differentiated from trace discontinua because of longer interburst interval and higher amplitude during bursts. Myotonic discharges (M.D.) seen in an electromyographic (EMG) study are the abnormal wave of excitation due to the firing of repetitive action potentials. It is characterized by waveforms with alternating frequency and amplitude. This unique pattern is seen in muscular dystrophies, muscle channelopathies, toxic or inflammatory myopathies. We report a case of PURA syndrome with significant EEG findings of burst suppression and EMG pattern of myotonic discharges which has not been described in literature before. Methods A male infant was born at 37-week gestation via spontaneous vaginal delivery to a 35-year-old G1P0 after an uneventful pregnancy. At birth, the infant was observed to have generalized central hypotonia and seizure-like spells of posturing, lip-smacking and blinking lasting for 10–15 s. Family history was unremarkable. A video EEG and EMG were ordered for futher evaluation, in addition to other routine testing. Results Video EEG demonstrated excessive multifocal sharps and periods of burst suppression. EMG study reported myotonic discharges in the first dorsal interosseous and tibialis anterior. Work up for metabolic etiologies were negative. CSF studies, chromosomal studies, infantile epilepsy panel, myotonic dystrophy panel were unremarkable. Whole exome sequencing was significant for a de novo mono-allelic mutation of the PURA gene (variant PR245X, coding DNA c.733 C > T). Conclusion PURA (purine-rich element binding protein A) syndrome is a genetic disorder caused by a gene deletion in 5q31.3, characterized by neonatal hypotonia, seizures and intellectual disability. It leads to an alteration in the PURA protein, affecting the protein’s DNA regulatory function. All reported deletions have been de novo. Therefore the risk of future inheritance of this genetic mutation is low. PURA Syndrome is known to cause epilepsy in 54% of previously reported patients. In our case, the patient’s EEG pattern was consistent with burst suppression, which supports the fact that PURA Syndrome is a state of generalized brain dysfunction. EMG reports were consistent with myotonic discharges. The significance of this finding in our patient is unclear and requires further research to characterize EMG findings in affected patients with PURA syndrome. This case however underscores the importance of considering PURA syndrome in the differential diagnosis of neonatal EEG findings of burst suppression pattern and EMG findings of myotonic discharge.

Gene expression signature for detection of gastric cancer in peripheral blood.

Gastric cancer (stomach cancer) is the fifth most common malignancy and the third leading cause of cancer-associated mortality worldwide. Identifying gastric cancer patients at an early and curable stage of the disease is essential if mortality rates for this disease are to decrease. A non-invasive blood-based test that is an indicator of gastric cancer risk would likely be of benefit in identifying gastric cancer patients at an early stage, and such a test may enhance clinical decision making. This study identified a four-gene expression signature in peripheral blood samples associated with gastric cancer. A total of 216 blood samples were collected, including those from 36 gastric cancer patients, 55 healthy controls and 125 patients with other carcinomas, and gene expression profiles were examined using an Affymetrix Gene Profiling microarray. Blood gene expression profiles were compared between patients with stomach cancer, healthy controls and patients affected with other malignancies. A four-gene panel was identified comprising purine-rich element binding protein B, structural maintenance of chromosomes 1A, DENN/MADD domain containing 1B and programmed cell death 4. The four-gene panel discriminated gastric cancer with an area under the receiver-operating-characteristic curve of 0.99, an accuracy of 95%, sensitivity of 92% and specificity of 96%. The non-invasive nature of the blood test, together with the relatively high accuracy of the four-gene panel may assist physicians in gastric cancer screening decision making.

PURA syndrome: clinical delineation and genotype-phenotype study in 32 individuals with review of published literature

BackgroundDe novo mutations in PURA have recently been described to cause PURA syndrome, a neurodevelopmental disorder characterised by severe intellectual disability (ID), epilepsy, feeding difficulties and neonatal hypotonia.ObjectivesTo delineate the...

112 Identification of Solute Carrier Family 5 Member 9 (SLC5A9) as Biomarker of In Vivo and In Vitro Oocyte Developmental Competence

The transcriptome associated with the ability of oocytes to support embryogenesis and development remains unclear. The aim of this study was to compare the mRNA expression pattern between oocytes collected from fertile heifers (H) and repeat breeders (RB) using RNAseq. Oocytes were collected by ovum pick-up from Italian Simmental (IS) and Holstein Friesian (HF) heifers that became pregnant at the following oestrus, and from cows of the same breeds classified as RB after they failed to become pregnant for a minimum of 3 consecutive AI. Oocytes from each animal (5 for HF_RB, 3 each for all other groups) were pooled and sequenced using Illumina HiSEqn 2000 (Illumina Inc., San Diego, CA, USA). Principal component analysis showed that HF oocytes clustered away from those of IS, irrespective of the fertility status. However, within breeds, H and RB always clustered apart from each other. This suggests that differences in the transcriptional blueprint between breeds was more prominent than differences related to fertility. However, these were still strong when considering breeds separately, therefore we treated breeds as independent groups and performed differential expression analysis on H v. RB in IS and HF. DESEqn 2 identified 135 differentially expressed genes when we compared H and RB in IS and 2124 in HF. This difference in number of DE genes between the 2 breeds reflects the fact that the variance between H and RB transcriptional patterns in HF is larger than in IS. We looked for genes that were differentially and consistently expressed between H and RB in both breeds and identified 10 genes: histone H2B, exo/endonuclease G, solute carrier family 5 member 9, BH3 interacting domain death agonist, TBK1 binding protein 1, purine-rich element binding protein A, tRNA-yW synthesising protein 1 homologue, Jun oncogene, FRA10AC1 homologue, and SKI oncogene. To validate their potential use as biomarkers, we performed a preliminary in vitro validation by RT-qPCR. As described in the literature, oocytes collected from ovaries with more than 10 follicles of 2 to 6 mm were classified as high quality, while those collected from ovaries with fewer than 10 follicles were classified as low quality (Gandolfi et al. 1997 Theriogenology 48, 1153-1160). Gene expression analysis confirmed SLC5A9, a membrane-bound protein involved in sodium-dependent transport of glucose, as a potential biomarker to discriminate between a competent and incompetent oocyte. Altogether, our data provide a detailed overview of the mRNA pattern defining the differences between a competent and incompetent oocyte in vivo, and identified a new potential marker able to differentiate fertile and infertile oocyte both in vivo and in vitro. Study supported by PRIN 08–09, EU-Quantomics no. 222664 and Carraresi Foundation.