东方蜜蜂微孢子虫基因的可变剪接及可变腺苷酸化解析

本研究旨在基于已获得的第三代纳米孔全长转录组数据对东方蜜蜂微孢子虫Nosema ceranae基因的可变剪接(alternative splicing,AS)和可变多聚腺苷酸化(alternative polyadenylation,APA)进行分析.通过Astalavista软件鉴定东方蜜蜂微孢子虫基因的AS事件类型...     

Evolution of exon-intron structure and alternative splicing

Despite significant advances in high-throughput DNA sequencing, many important species remain understudied at the genome level. In this study we addressed a question of what can be predicted about the genome-wide characteristics of less studied species, based on the genomic data from completely sequenced species. Using NCBI databases we performed a comparative genome-wide analysis of such characteristics as alternative splicing, number of genes, gene products and exons in 36 completely sequenced model species. We created statistical regression models to fit these data and applied them to loblolly pine (Pinus taeda L.), an example of an important species whose genome has not been completely sequenced yet. Using these models, the genome-wide characteristics, such as total number of genes and exons, can be roughly predicted based on parameters estimated from available limited genomic data, e.g. exon length and exon/gene ratio.     

RNA structure and the mechanisms of alternative splicing

Alternative splicing is a widespread means of increasing protein diversity and regulating gene expression in eukaryotes. Much progress has been made in understanding the proteins involved in regulating alternative splicing, the sequences they bind to, and how these interactions lead to changes in splicing patterns. However, several recent studies have identified other players involved in regulating alternative splicing. A major theme emerging from these studies is that RNA secondary structures play an under appreciated role in the regulation of alternative splicing. This review provides an overview of the basic aspects of splicing regulation and highlights recent progress in understanding the role of RNA secondary structure in this process.     

染色质结构与mRNA可变剪接

mRNA的可变剪接(alternative splicing)是一种由一个mRNA前体(pre-mRNA)通过不同的剪接方式产生多个mRNA变异体(variants)的RNA加工过程。在过去很长一段时间里,人们认为mRNA剪接过程是独立于转录过程的一个转录后RNA加工过程。然而,越来越多的实验证明mRNA剪接在很大程度上是与转录偶联发生的。因此,剪接调控会受到与转录相关因素的调控。本文将对染色质与mRNA剪接调控的相关性和染色质结构调控可变剪接的分子机制进行阐述。     

Genomic structure and alternative splicing of chicken angiopoietin-2

Angiopoietin-1 (Ang-1) prevents endothelial cell apoptosis and promotes blood vessel stability, while angiopoietin-2 (Ang-2), a natural antagonist of Ang-1, disrupts blood vessel structure and induces apoptosis. We have sequenced the chicken angiopoietin-2 gene that spans about 46 kb of DNA and is split in 9 exons by 8 introns. Alternative splicing of the gene gives rise to three different species of Ang-2 mRNAs: Ang-2A, Ang-2B, and Ang-2C. The three mRNA isoforms, also present in humans, codify for proteins with an identical fibrinogen-like C-terminal domain and a different coiled-coil N-terminal domain. Ang-2A and particularly Ang-2C are expressed in immature testis and regressed adult testis undergoing vascular remodeling, while their expression is barely detectable in quiescent adult testis. Conversely, Ang-2B is only detectable in adult testis. The new isoforms with truncated amino-terminal domains may modulate the Tie2 receptor during vascular angiogenesis and regression.     

Human and rhesus APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H demonstrate a conserved capacity to restrict Vif-deficient HIV-1

Successful intracellular pathogens must evade or neutralize the innate immune defenses of their host cells and render the cellular environment permissive for replication. For example, to replicate efficiently in CD4(+) T lymphocytes, human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral infectivity factor (Vif) that promotes pathogenesis by triggering the degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3 proteins have been implicated in HIV-1 restriction, but the relevant repertoire remains ambiguous. Here we present the first comprehensive analysis of the complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction. In addition to APOBEC3G, we find that three other human APOBEC3 proteins, APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors. These four proteins are expressed in CD4(+) T lymphocytes, are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, and they are all neutralized by the simian immunodeficiency virus Vif protein. On the other hand, neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant impact on HIV-1 replication. These data strongly implicate a combination of four APOBEC3 proteins--APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H--in HIV-1 restriction.     

Identification of a Cullin5-ElonginB-ElonginC E3 complex in degradation of feline immunodeficiency virus Vif-mediated feline APOBEC3 proteins

Various feline APOBEC3 (fA3) proteins exhibit broad antiviral activities against a wide range of viruses, such as feline immunodeficiency virus (FIV), feline foamy virus (FFV), and feline leukemia virus (FeLV), as well as those of other species. This activity can be counteracted by the FIV Vif protein, but the mechanism by which FIV Vif suppresses fA3s is unknown. In the present study, we demonstrated that FIV Vif could act via a proteasome-dependent pathway to overcome fA3s. FIV Vif interacted with feline cellular proteins Cullin5 (Cul5), ElonginB, and ElonginC to form an E3 complex to induce degradation of fA3s. Both the dominant-negative Cul5 mutant and a C-terminal hydrophilic replacement ElonginC mutant potently disrupted the FIV Vif activity against fA3s. Furthermore, we identified a BC-box motif in FIV Vif that was essential for the recruitment of E3 ubiquitin ligase and also required for FIV Vif-mediated degradation of fA3s. Moreover, despite the lack of either a Cul5-box or a HCCH zinc-binding motif, FIV Vif specifically selected Cul5. Therefore, FIV Vif may interact with Cul5 via a novel mechanism. These finding imply that SOCS proteins may possess distinct mechanisms to bind Cul5 during formation of the Elongin-Cullin-SOCS box complex.     

APOBEC3A, APOBEC3B, and APOBEC3H haplotype 2 restrict human T-lymphotropic virus type 1

The human APOBEC3 family consists of seven cytidine deaminases (A3A to A3H), some of which display potent antiretroviral activity against HIV-1 and other retroviruses. Studies that analyzed the effect of A3G on human T-lymphotropic virus type 1 (HTLV-1) infectivity resulted in conflicting findings, and our knowledge of HTLV-1 restriction by other A3 proteins remains limited. Since HTLV-1, much like HIV, targets CD4(+) T cells, we hypothesized that A3 proteins other than A3G restrict HTLV-1. All seven human A3 proteins were tested in HTLV-1 reporter and HIV-1 infectivity assays. We show that A3A, A3B, and A3H haplotype 2 (A3H hapII) acted as potent inhibitors of HTLV-1. Wild-type HIV-1, in contrast, was restricted by A3B and A3H hapII, but not by A3A. Catalytic site mutants of A3A, A3B, and A3H hapII showed that A3A and A3B restriction of HTLV-1 required deaminase activity. However, A3H hapII acted in a deaminase-independent manner when restricting HTLV-1, while requiring deaminase activity for HIV-1 restriction. We also analyzed A3 editing of HTLV-1 in five T-cell lines obtained from HTLV-1-infected patients. These cell lines contained extensively edited HTLV-1 sequences with G-to-A mutations in dinucleotide contexts suggestive of APOBEC3 mutagenesis. Comparison of the A3-induced mutations from reporter cells and the patient-derived cell lines indicate that A3G but also other A3 members, possibly A3A and A3B, affect HTLV-1 in vivo. Taken together, our data indicate that HTLV-1 is a likely target for multiple A3 proteins.     

Species‐specific differences in the ability of feline lentiviral Vif to degrade feline APOBEC3 proteins

How host–virus co‐evolutionary relationships manifest is one of the most intriguing issues in virology. To address this topic, the mammal–lentivirus relationship can be considered as an interplay of cellular and viral proteins, particularly apolipoprotein B mRNA editing enzyme catalytic polypeptide‐like 3 (APOBEC3) and viral infectivity factor (Vif). APOBEC3s enzymatically restrict lentivirus replication, whereas Vif antagonizes the host anti‐viral action mediated by APOBEC3. In this study, the focus was on the interplay between feline APOBEC3 proteins and two feline immunodeficiency viruses in cats and pumas. To our knowledge, this study provides the first evidence of non‐primate lentiviral Vif being incapable of counteracting a natural host's anti‐viral activity mediated via APOBEC3 protein.     

Gene structure, multiple alternative splicing, and expression in gonads of zebrafish Dmrt1

Many basic cellular processes are shared across vast phylogenetic distances, whereas sex-determining mechanisms are highly variable between phyla although the existence of two sexes is nearly universal in the animal kingdom. The only molecular similarity in sex determination found so far between phyla is among the fly doublesex, worm mab-3, and vertebrate Dmrt1/DMY, which contain a zinc-finger-like DNA-binding motif, DM domain. Here we report that three isoforms of the zebrafish Dmrt1 were generated in gonads by multiple alternative splicing, which encoded predicted proteins with 267, 246, and 132 amino acids, respectively. By cDNA cloning and genomic structure analysis, we found that there were seven exons of Dmrt1, which were alternatively spliced to generate the Dmrt1 isoforms. Northern blotting analysis revealed that expression of zebrafish Dmrt1 was higher in testis than ovary. Real time fluorescent quantitative RT-PCR indicated that expression of isoform a of Dmrt1 was dominantly higher than those of Dmrt1 b and c. Furthermore, in situ hybridization to gonads sections showed that Dmrt1 was expressed in developing germ cells of both testis and ovary, suggesting that the Dmrt1 gene is not only associated with testis development, but also, may be important in ovary differentiation of zebrafish.