CircInteractome: A web tool for exploring circular RNAs and their interacting proteins and microRNAs

<p>Circular RNAs (circRNAs) are widely expressed in animal cells, but their biogenesis and functions are poorly understood. CircRNAs have been shown to act as sponges for miRNAs and may also potentially sponge RNA-binding proteins (RBPs) and are thus predicted to function as robust posttranscriptional regulators of gene expression. The joint analysis of large-scale transcriptome data coupled with computational analyses represents a powerful approach to elucidate possible biological roles of ribonucleoprotein (RNP) complexes. Here, we present a new web tool, CircInteractome (circRNA interactome), for mapping RBP- and miRNA-binding sites on human circRNAs. CircInteractome searches public circRNA, miRNA, and RBP databases to provide bioinformatic analyses of binding sites on circRNAs and additionally analyzes miRNA and RBP sites on junction and junction-flanking sequences. CircInteractome also allows the user the ability to (1) identify potential circRNAs which can act as RBP sponges, (2) design junction-spanning primers for specific detection of circRNAs of interest, (3) design siRNAs for circRNA silencing, and (4) identify potential internal ribosomal entry sites (IRES). In sum, the web tool CircInteractome, freely accessible at http://circinteractome.nia.nih.gov, facilitates the analysis of circRNAs and circRNP biology.</p

Presentation_1_Nicotinamide adenine dinucleotide supplementation drives gut microbiota variation in Alzheimer’s mouse model.pdf

Alzheimer’s disease (AD) is the most common neurodegenerative disease. Growing evidence suggests an important role for gut dysbiosis and gut microbiota-host interactions in aging and neurodegeneration. Our previous works have demonstrated that supplementation with the nicotinamide adenine dinucleotide (NAD+) precursor, nicotinamide riboside (NR), reduced the brain features of AD, including neuroinflammation, deoxyribonucleic acid (DNA) damage, synaptic dysfunction, and cognitive impairment. However, the impact of NR administration on the intestinal microbiota of AD remains unknown. In this study, we investigated the relationship between gut microbiota and NR treatment in APP/PS1 transgenic (AD) mice. Compared with wild type (WT) mice, the gut microbiota diversity in AD mice was lower and the microbiota composition and enterotype were significantly different. Moreover, there were gender differences in gut microbiome between female and male AD mice. After supplementation with NR for 8 weeks, the decreased diversity and perturbated microbial compositions were normalized in AD mice. This included the species Oscillospira, Butyricicoccus, Desulfovibrio, Bifidobacterium, Olsenella, Adlercreutzia, Bacteroides, Akkermansia, and Lactobacillus. Our results indicate an interplay between NR and host-microbiota in APP/PS1 mice, suggesting that the effect of NR on gut dysbiosis may be an important component in its therapeutic functions in AD.</p

DataSheet_1_Homeodomain-only protein suppresses proliferation and contributes to differentiation- and age-related reduced CD8+ T cell expansion.docx

T cell activation is a tightly controlled process involving both positive and negative regulators. The precise mechanisms governing the negative regulators in T cell proliferation remain incompletely understood. Here, we report that homeodomain-only protein (HOPX), a homeodomain-containing protein, and its most abundant isoform HOPXb, negatively regulate activation-induced proliferation of human T cells. We found that HOPX expression progressively increased from naïve (TN) to central memory (TCM) to effector memory (TEM) cells, with a notable upregulation following in vitro stimulation. Overexpression of HOPXb leads to a reduction in TN cell proliferation while HOPX knockdown promotes proliferation of TN and TEM cells. Furthermore, we demonstrated that HOPX binds to promoters and exerts repressive effects on the expression of MYC and NR4A1, two positive regulators known to promote T cell proliferation. Importantly, our findings suggest aging is associated with increased HOPX expression, and that knockdown of HOPX enhances the proliferation of CD8+ T cells in older adults. Our findings provide compelling evidence that HOPX serves as a negative regulator of T cell activation and plays a pivotal role in T cell differentiation and in age-related-reduction in T cell proliferation.</p

Clustering analysis of significant genes in rat UCMSC (rUCMSC) and human UCMSC (hUCMSC) co-cultured with mammary carcinoma cells of their respective species; Mat B III rat breast carcinoma cells or MDA-231 human breast carcinoma cells.

<p>The genes differentially expressed between naïve human or rat UCMSC and those co-cultured with mammary carcinoma cells are listed on the row and the samples are shown on the column. Red are higher expression and black to green are lower expression. This analysis suggests that a short time co-culture with mammary carcinoma cells significantly modifies expression of multiple tumor-suppressor and-promoter genes in both human and rat UCMSC.</p

Treatment with FST-hUCMSC significantly attenuated development of metastatic tumor in the mouse lung.

<p>The numbers of tumor nodules in the mouse lung were counted under a dissection microscope 30 days after the inoculation of the MDA-231 cells. The bar graph represents the average number of tumor nodules in each treatment group. * <i>p</i> < 0.05 compared to LacZ-hUCMSC treated mice.</p

The growth of MDA-231 cells was significantly attenuated by co-culture with FST-hUCMSC (A), the conditioned medium from FST-hUCMSC (B), or by direct over-expression of FST (C).

<p>A, MDA-231 cells were co-cultured with either LacZ-, ADRP- or FST-hUCMSC for 48 hours. Cell growth was measured by MTT assay (panels A and B). Transduction of hUCMSC with adenoviral vector did not affect the cell growth. FST-hUCMSC, but not ADRP-hUCMSC significantly attenuated the growth of co-cultured carcinoma cells to the same level of MDA-231 cells cultured alone. B, the growth of MDA-231 cells was significantly attenuated by culturing with the conditioned medium (CM) from Ad-FST but not-LacZ or-ADRP transduced human UCMSC or defined medium (DM). C, the growth of MDA-231 cells was significantly attenuated by the direct transduction of Ad-FST but not by-LacZ or-ADRP. The colony growth rate of the FST-transduced MDA-231 cells from Day 7 to 12 was 1.02 (no growth), while the other gene-transduced cells colonies grew approximately 1.5 times bigger on Day 12 compared to Day 7. * <i>p</i> < 0.05.</p

Primers used for qRT-PCR.

<p>Primers used for qRT-PCR.</p

Function and expression levels of differentially expressed genes in human and rat UCMSC.

<p>Positive (+) indicates upregulation and negative (-) indicates down regulation.</p><p>¹ PTGS2 (COX2), prostaglandin-endoperoxide synthase 2, SERPINE2, serpin peptidase inhibitor clade E, MIF, macrophage migration inhibitory factor, PDPN, podoplanin, TGFBI, transforming growth factor, beta-induced, 68kDa.</p><p>² SULF1, sulfatase 1, GPI, glucose-6-phosphate isomerase, HTRA1, HtrA serine peptidase 1, ADRP, adipose-differentiation related protein, FST, follistatin, LTBP4, latent transforming growth factor beta binding protein 4, BGN, biglycan, LOXL1, lysyl oxidase-like 1.</p><p>³ P4HA1, prolyl 4-hydroxylase, alpha polypeptide I, COL1A2, collagen, type 1, alpha2, PAM, peptidylglycine alpha-amidating monooxygenase, PDIA5, protein disulfide isomerase family A, member 5.</p><p>Function and expression levels of differentially expressed genes in human and rat UCMSC.</p

Immunohistochemical analysis of cell proliferation (A and B) and apoptosis (A and C) in MDA-231 graft tumors in SCID mouse lungs treated with either PBS, LacZ- or FST-hUCMSC.

<p>A, microscopic images of immunohistochemistry for Ki-67 (top 3 panels) at 20x and TUNEL assay (bottom 3 panels) at 40x. B, treatment with LacZ- or FST-hUCMSC had no significant effect on proliferation of the tumor cells. C, the TUNEL positive cells were significantly increased in the tumors of mice treated with FST-hUCMSC. * <i>p</i> < 0.05 as compared to the level of the PBS-treated control.</p

Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by <i>CircPABPN1</i>

<p>HuR influences gene expression programs and hence cellular phenotypes by binding to hundreds of coding and noncoding linear RNAs. However, whether HuR binds to circular RNAs (circRNAs) and impacts on their function is unknown. Here, we have identified <i>en masse</i> circRNAs binding HuR in human cervical carcinoma HeLa cells. One of the most prominent HuR target circRNAs was hsa_circ_0031288, renamed <i>CircPABPN1</i> as it arises from the <i>PABPN1</i> pre-mRNA. Further analysis revealed that HuR did not influence <i>CircPABPN1</i> abundance; interestingly, however, high levels of <i>CircPABPN1</i> suppressed HuR binding to <i>PABPN1</i> mRNA. Evaluation of <i>PABPN1</i> mRNA polysomes indicated that PABPN1 translation was modulated positively by HuR and hence negatively by <i>CircPABPN1</i>. We propose that the extensive binding of <i>CircPABPN1</i> to HuR prevents HuR binding to <i>PABPN1</i> mRNA and lowers PABPN1 translation, providing the first example of competition between a circRNA and its cognate mRNA for an RBP that affects translation.</p