Yuchao Feng

APASdb: a database describing alternative poly(A) sites and selection of heterogeneous cleavage sites downstream of poly(A) signals

Increasing amounts of genes have been shown to utilize alternative polyadenylation (APA) 3′-processing sites depending on the cell and tissue type and/or physiological and pathological conditions at the time of processing, and the construction of genome-wide database regarding APA is urgently needed for better understanding poly(A) site selection and APA-directed gene expression regulation for a given biology. Here we present a web-accessible database, named APASdb (http://mosas.sysu.edu.cn/utr), which can visualize the precise map and usage quantification of different APA isoforms for all genes. The datasets are deeply profiled by the sequencing alternative polyadenylation sites (SAPAS) method capable of high-throughput sequencing 3′-ends of polyadenylated transcripts. Thus, APASdb details all the heterogeneous cleavage sites downstream of poly(A) signals, and maintains near complete coverage for APA sites, much better than the previous databases using conventional methods. Furthermore, APASdb provides the quantification of a given APA variant among transcripts with different APA sites by computing their corresponding normalized-reads, making our database more useful. In addition, APASdb supports URL-based retrieval, browsing and display of exon-intron structure, poly(A) signals, poly(A) sites location and usage reads, and 3′-untranslated regions (3′-UTRs). Currently, APASdb involves APA in various biological processes and diseases in human, mouse and zebrafish.

Characterizing the evolution and functions of the M-superfamily conotoxins

Conotoxins from cone snails are valuable in physiology research and therapeutic applications. Evolutionary mechanisms of conotoxins have been investigated in several superfamilies, but there is no phylogenetic analysis on M-superfamily conotoxins. In this study, we characterized identical sequences, gene structure, novel cysteine frameworks, functions and evolutionary mechanisms of M-superfamily conotoxins. Identical M-superfamily conotoxins can be found in different Conus species from the analysis of novel 467 M-superfamily conotoxin sequences and other published M-superfamily conotoxins sequences. M-superfamily conotoxin genes consist of two introns and three exons from the results of genome walking. Eighteen cysteine frameworks were identified from the M-superfamily conotoxins, and 10 of the 18 may be generated from framework III. An analysis between diet types and phylogeny of the M-superfamily conotoxins indicate that M-superfamily conotoxins might not evolve in a concerted manner but were subject to birth-and-death evolution. Codon usage analysis shows that position-specific codon conservation is not restricted to cysteines, but also to other conserved residues. By analysing primary structures and physiological functions of M-superfamily conotoxins, we proposed a hypothesis that insertions and deletions, especially insertions in the third cysteine loop, are involved in the creation of new functions and structures of the M-superfamily conotoxins.

The role of alternative polyadenylation in the antiviral innate immune response

Alternative polyadenylation (APA) is an important regulatory mechanism of gene functions in many biological processes. However, the extent of 3′ UTR variation and the function of APA during the innate antiviral immune response are unclear. Here, we show genome-wide poly(A) sites switch and average 3′ UTR length shortens gradually in response to vesicular stomatitis virus (VSV) infection in macrophages. Genes with APA and mRNA abundance change are enriched in immune-related categories such as the Toll-like receptor, RIG-I-like receptor, JAK-STAT and apoptosis-related signalling pathways. The expression of 3′ processing factors is down-regulated upon VSV infection. When the core 3′ processing factors are knocked down, viral replication is affected. Thus, our study reports the annotation of genes with APA in antiviral immunity and highlights the roles of 3′ processing factors on 3′ UTR variation upon viral infection.

Soluble expression and sodium channel activity of lt16a, a novel framework XVI conotoxin from the M-superfamily.

A peptide toxin, lt16a, from the venom of the worm-hunting Conus litteratus, shares the typical signal peptide sequences of M-superfamily conotoxins, which usually contain six cysteine residues that are arranged in a CC-C-C-CC pattern. Interestingly, lt16a comprises 21 amino acid residues in its mature region and has a cysteine framework XVI, which is arranged in a C-C-CC pattern. The coding region of lt16a was cloned into the pTRX vector and the fusion protein was overexpressed in Escherichia coli. After cleaving the fusion protein and purifying the protein lt16a using chromatography, the mass of lt16a was found by mass spectrometry to be consistent with the expected mass of 2357.7 Da. Whole-cell patch clamp experiments demonstrated that lt16a could inhibit both the TTX-sensitive and TTX-resistant sodium currents in adult rat dorsal root ganglion neurons. The inhibition of lt16a on TTX-resistant sodium currents was stronger than on TTX-sensitive sodium currents. To our knowledge, this is the first report of a framework XVI conotoxin that can inhibit voltage-gated sodium channel currents in mammalian sensory neurons. This report helps facilitates an understanding of the sequence diversity of conotoxins.

Small Nuclear Ribonucleoprotein Polypeptide A–Mediated Alternative Polyadenylation of STAT5B during Th1 Cell Differentiation

T cells are activated and differentiated into Th cells depending on the rapid and accurate changes in the cell transcriptome. In addition to changes in mRNA expression, the sequences of many transcripts are altered by alternative splicing and alternative polyadenylation (APA). We profiled the APA sites of human CD4+ T cell subsets with high-throughput sequencing and found that Th1 cells harbored more genes with shorter tandem 3′ untranslated regions (UTRs) than did naive T cells. We observed that STAT5B, a key regulator of Th1 differentiation, possessed three major APA sites and preferred shorter 3′ UTRs in Th1 cells. In addition, small nuclear ribonucleoprotein polypeptide A (SNRPA) was found to bind directly to STAT5B 3′ UTR and facilitate its APA switching. We also found that p65 activation triggered by TCR signaling could promote SNRPA transcription and 3′ UTR shortening of STAT5B. Thus we propose that the APA switching of STAT5B induced by TCR activation is mediated by SNRPA.

Dynamic Regulation of Tandem 3' Untranslated Regions in Zebrafish Spleen Cells during Immune Response.

Alternative polyadenylation (APA) has been found to be involved in tumorigenesis, development, and cell differentiation, as well as in the activation of several subsets of immune cells in vitro. Whether APA takes place in immune responses in vivo is largely unknown. We profiled the variation in tandem 3′ untranslated regions (UTRs) in pathogen-challenged zebrafish and identified hundreds of APA genes with ∼10% being immune response genes. The detected immune response APA genes were enriched in TLR signaling, apoptosis, and JAK-STAT signaling pathways. A greater number of microRNA target sites and AU-rich elements were found in the extended 3′ UTRs than in the common 3′ UTRs of these APA genes. Further analysis suggested that microRNA and AU-rich element–mediated posttranscriptional regulation plays an important role in modulating the expression of APA genes. These results indicate that APA is extensively involved in immune responses in vivo, and it may be a potential new paradigm for immune regulation.

Chapter 12 – Primitive Adaptive Immune System of Amphioxus

In the chapter, we discuss the evidence about the origins of the vertebrate adaptive immune system. Amphioxus is very likely to have lymphocyte-like cells and related transcription factors and to have an antecedent MHC-linked genomic region. More importantly, a novel DNA transposon discovered in amphioxus has proven to be the long-sought RAG transposon, which is supposed to give rise to the later RAG1 and RAG2 genes that mediate the Ig/TCR somatic recombination.