Publications

Silver nanoparticles (AgNPs) are among the most extensively used nanoparticles and are found in a variety of products. This ubiquity leads to inevitable exposure to these particles in everyday life. However, the effects of AgNPs on neuron and astrocyte networks are still largely unknown. In this study, we used neurons and astrocytes derived from human embryonic stem cells as a cellular model to study the neurotoxicity that is induced by citrate-coated AgNPs (AgSCs). Immunostaining with the astrocyte and neuron markers, glial fibrillary acidic protein and microtubule-associated protein-2 (MAP2), respectively, showed that exposure to AgSCs at the concentration of 0.1 µg/mL increased the astrocyte/neuron ratio. In contrast, a higher concentration of AgSCs (5.0 µg/ml) significantly changed the morphology of astrocytes. These results suggest that astrocytes are sensitive to AgSC exposure and that low concentrations of AgSCs promote astrogenesis. Furthermore, our results showed that AgSCs reduced neurite outgrowth, decreased the expression of postsynaptic density protein 95 and synaptophysin, and induced neurodegeneration in a concentration-dependent manner. Our findings additionally suggest that the expression and phosphorylation status of MAP2 isoforms, as modulated by the activation of the Akt/glycogen synthase kinase-3/caspase-3 signaling pathway, may play an important role in AgSC-mediated neurotoxicity. We also found that AgNO3 exposure only slightly reduced neurite outgrowth and had little effect on MAP2 expression, suggesting that AgSCs and AgNO3 have different neuronal toxicity mechanisms. In addition, most of these effects were reduced when the cell culture was co-treated with AgSCs and the antioxidant ascorbic acid, which implies that oxidative stress is the major cause of AgSC-mediated astrocytic/neuronal toxicity and that antioxidants may have a neuroprotective effect.

Pre-mRNA alternative splicing and alternative polyadenylation have been implicated to play important roles during eukaryotic gene expression. However, much remains unknown regarding the regulatory mechanisms and the interactions of these two processes in plants. Here we focus on an Arabidopsis gene OXT6 (Oxidative Tolerant-6) that has been demonstrated to encode two proteins through alternative splicing and alternative polyadenylation. Specifically, alternative polyadenylation at Intron-2 of OXT6 produces a transcript coding for AtCPSF30, an Arabidopsis ortholog of 30 kDa subunit of the Cleavage and Polyadenylation Specificity Factor. On the other hand, alternative splicing of Intron-2 generates a longer transcript encoding a protein named AtC30Y, a polypeptide including most part of AtCPSF30 and a YT521B domain. To investigate the expression outcome of OXT6 in plants, a set of mutations were constructed to alter the splicing and polyadenylation patterns of OXT6. Analysis of transgenic plants bearing these mutations by quantitative RT-PCR revealed a competition relationship between these two processes. Moreover, when both splice sites and poly(A) signals were mutated, polyadenylation became the preferred mode of OXT6 processing. These results demonstrate the interplay between alternative splicing and alternative polyadenylation, and it is their concerted actions that define a gene's expression outcome.

BACKGROUND: The goal of this project was to characterize the molecular and cellular roles of various gene targets regulated by miRNAs identified in differentiating and stimulating avian macrophages. Once a monocyte arrives to a site of infection, local signals induce a redistribution of resources into a macrophage phenotype. This may involve upregulating pathogen pattern recognizing receptors and increasing the efficiency of lysosomal biogenesis, while simultaneously recycling components involved in circulatory migration and leukocyte extravasation. a monocyte tooled with chemokine surface receptors and an internal cytoskeletal structure geared towards mobility may efficiently sense, react, and migrate toward a site of infection.

METHODS: Peripheral blood derived monocytes were purified and cultured from young chickens. RNA sequencing was performed on both peripheral blood monocytes during differentiation into macrophages and on mature macrophages following stimulation with interferon gamma. A set of microRNAs were identified and investigated using bioinformatics methods to ascertain their potential role in avian macrophage biology.

RESULTS: Among a number of miRNAs that are found to be expressed in avian macrophages, we focused on eight specific miRNAs (miR-1618, miR-1586, miR-1633, miR-1627, miR-1646, miR-1649, miR-1610, miR-1647) associated with macrophage differentiation and activation. Expression profiles of microRNAs were characterized during differentiation and activation. Candidate miRNA targets were implicated in processes including Wnt signaling, ubiquitination, PPAR mediated macrophage function, vesicle mediated cytokine trafficking, and WD40 domain protein functions.

CONCLUSION: A global theme for macrophage function that may be modulated by microRNAs is the comprehensive redistribution of the cell's protein repertoire. This redistribution involves two processes: 1) the degradation and recycling of unneeded cytoplasmic and membrane components and 2) the mobilization of newly synthesized cellular components via vesicular trafficking. Generally, it appears that macrophages need to closely regulate gene expression for differentiation to be able to activate successfully in response to a pathogen. This is a process in which miRNAs participate by affecting several pathways critical for both, differentiation and activation.

CPSF100 is a core component of the cleavage and polyadenylation specificity factor (CPSF) complex for 3'-end formation of mRNA, but it still has no clear functional assignment. CPSF100 was reported to play a role in RNA silencing and promote flowering in Arabidopsis. However, the molecular mechanisms underlying these phenomena are not fully understood. Our genetics analyses indicate that plants with a hypomorphic mutant of CPSF100 (esp5) show defects in embryogenesis, reduced seed production or altered root morphology. To unravel this puzzle, we employed a poly(A) tag sequencing protocol and uncovered a different poly(A) profile in esp5. This transcriptome-wide analysis revealed alternative polyadenylation of thousands of genes, most of which result in transcriptional read-through in protein-coding genes. AtCPSF100 also affects poly(A) signal recognition on the far-upstream elements; in particular it prefers less U-rich sequences. Importantly, AtCPSF100 was found to exert its functions through the change of poly(A) sites on genes encoding binding proteins, such as nucleotide-binding, RNA-binding and poly(U)-binding proteins. In addition, through its interaction with RNA Polymerase II C-terminal domain (CTD) and affecting the expression level of CTD phosphatase-like 3 (CPL3), AtCPSF100 is shown to potentially ensure transcriptional termination by dephosphorylation of Ser2 on the CTD. These data suggest a key role for CPSF100 in locating poly(A) sites and affecting transcription termination.

Miniature inverted repeat transposable elements (MITEs) are prevalent in eukaryotic genomes. They are known to critically influence the process of genome evolution and play a role in gene regulation. As the first study concentrated in the transposition activities of MITEs among different ecotype accessions within a species, we conducted a genome-wide comparative analysis by characterizing and comparing MITEs in 19 Arabidopsis thaliana accessions. A total of 343485 MITE putative sequences, including canonical, diverse and partial ones, were delineated from all 19 accessions. Within the entire population of MITEs sequences, 80.7% of them were previously unclassified MITEs, demonstrating a different genomic distribution and functionality compared to the classified MITEs. The interactions between MITEs and homologous genes across 19 accessions provided a fine source for analyzing MITE transposition activities and their impacts on genome evolution. Moreover, a significant proportion of MITEs were found located in the last exon of genes besides the ordinary intron locality, thus potentially modifying the end of genes. Finally, analysis of the impact of MITEs on gene expression suggests that migrations of MITEs have no detectable effect on the expression level for host genes across accessions.

Alternative polyadenylation has been recognized as a key contributor of gene expression regulation by generating different transcript isoforms with altered 3' ends. Although polyadenylation is well known for marking the end of a 3' UTR, an increasing number of studies have reported previously less-addressed polyadenylation events located in other parts of genes in many eukaryotic organisms. These other locations include 5' UTRs, introns and coding sequences (termed herein as non-3UTR), as well as antisense and intergenic polyadenlation. Focusing on the non-3UTR polyadenylation sites (n3PASs), we detected and characterized more than 11000 n3PAS clusters in the Arabidopsis genome using poly(A)-tag sequencing data (PAT-Seq). Further analyses suggested that the occurrence of these n3PASs were positively correlated with certain characteristics of their respective host genes, including the presence of spliced, diminutive or diverse beginning of 5' UTRs, number of introns and whether introns have extreme lengths. The interaction of the host genes with surrounding genetic elements, like a convergently overlapped gene and associated transposable element, may contribute to the generation of a n3PAS as well. Collectively, these results provide a better understanding of n3PASs, and offer some new insights of the underlying mechanisms for non-3UTR polyadenylation and its regulation in plants.

Alternative polyadenylation (APA), in which a transcript uses one of the poly(A) sites to define its 3'-end, is a common regulatory mechanism in eukaryotic gene expression. However, the potential of APA in determining crop agronomic traits remains elusive. This study systematically tallied poly(A) sites of 14 different rice tissues and developmental stages using the poly(A) tag sequencing (PAT-seq) approach. The results indicate significant involvement of APA in developmental and quantitative trait loci (QTL) gene expression. About 48% of all expressed genes use APA to generate transcriptomic and proteomic diversity. Some genes switch APA sites, allowing differentially expressed genes to use alternate 3' UTRs. Interestingly, APA in mature pollen is distinct where differential expression levels of a set of poly(A) factors and different distributions of APA sites are found, indicating a unique mRNA 3'-end formation regulation during gametophyte development. Equally interesting, statistical analyses showed that QTL tends to use APA for regulation of gene expression of many agronomic traits, suggesting a potential important role of APA in rice production. These results provide thus far the most comprehensive and high-resolution resource for advanced analysis of APA in crops and shed light on how APA is associated with trait formation in eukaryotes.

Alternative polyadenylation (APA) is an important layer of gene regulation that produces mRNAs that have different 3' ends and/or encode diverse protein isoforms. Up to 70% of annotated genes in plants undergo APA. Increasing numbers of poly(A) sites collected in various plant species demand new methods and tools to access and mine these data. We have created an open-access web service called PlantAPA (http://bmi.xmu.edu.cn/plantapa) to visualize and analyze genome-wide poly(A) sites in plants. PlantAPA provides various interactive and dynamic graphics and seamlessly integrates a genome browser that can profile heterogeneous cleavage sites and quantify expression patterns of poly(A) sites across different conditions. Particularly, through PlantAPA, users can analyze poly(A) sites in extended 3' UTR regions, intergenic regions, and ambiguous regions owing to alternative transcription or RNA processing. In addition, it also provides tools for analyzing poly(A) site selections, 3' UTR lengthening or shortening, non-canonical APA site switching, and differential gene expression between conditions, making it more powerful for the study of APA-mediated gene expression regulation. More importantly, PlantAPA offers a bioinformatics pipeline that allows users to upload their own short reads or ESTs for poly(A) site extraction, enabling users to further explore poly(A) site selection using stored PlantAPA poly(A) sites together with their own poly(A) site datasets. To date, PlantAPA hosts the largest database of APA sites in plants, including Oryza sativa, Arabidopsis thaliana, Medicago truncatula, and Chlamydomonas reinhardtii. As a user-friendly web service, PlantAPA will be a valuable addition to the community of biologists studying APA mechanisms and gene expression regulation in plants.

Polyadenylation [poly(A)] is an essential posttranscriptional processing step in the maturation of eukaryotic mRNA. The advent of next-generation sequencing (NGS) technology has offered feasible means to generate large-scale data and new opportunities for intensive study of polyadenylation, particularly deep sequencing of the transcriptome targeting the junction of 3'-UTR and the poly(A) tail of the transcript. To take advantage of this unprecedented amount of data, we present an automated workflow to identify polyadenylation sites by integrating NGS data cleaning, processing, mapping, normalizing, and clustering. In this pipeline, a series of Perl scripts are seamlessly integrated to iteratively map the single- or paired-end sequences to the reference genome. After mapping, the poly(A) tags (PATs) at the same genome coordinate are grouped into one cleavage site, and the internal priming artifacts removed. Then the ambiguous region is introduced to parse the genome annotation for cleavage site clustering. Finally, cleavage sites within a close range of 24 nucleotides and from different samples can be clustered into poly(A) clusters. This procedure could be used to identify thousands of reliable poly(A) clusters from millions of NGS sequences in different tissues or treatments.

The NCBI manages the SRA (Sequence Read Archive) database to store RNA-Seq data generated from different NGS technologies. With ever increasing finished and ongoing genome and transcriptome sequencing projects, the data in SRA expand rapidly and present a treasure for mining useful information to facilitate our understanding of biological issues like mRNA 3'-end formation and alternative polyadenylation. We developed a bioinformatics pipeline that can process raw SRA sequence data and obtain high quality poly(A) sites and poly(A) cluster sites with detailed expression information. This pipeline is designed to be generic and can be utilized for polyadenylation studies in any eukaryotic species.