Identification of an intronic splicing regulatory element involved in auto‐regulation of alternative splicing of SCL33 pre‐mRNA

In Arabidopsis, pre‐mRNAs of serine/arginine‐rich (SR) proteins undergo extensive alternative splicing (AS). However, little is known about the cis‐elements and trans‐acting proteins involved in regulating AS. Using a splicing reporter (GFP–intron–GFP), consisting of the GFP coding sequence interrupted by an alternatively spliced intron of SCL33, we investigated whether cis‐elements within this intron are sufficient for AS, and which SR proteins are necessary for regulated AS. Expression of the splicing reporter in protoplasts faithfully produced all splice variants from the intron, suggesting that cis‐elements required for AS reside within the intron. To determine which SR proteins are responsible for AS, the splicing pattern of the GFP–intron–GFP reporter was investigated in protoplasts of three single and three double mutants of SR genes. These analyses revealed that SCL33 and a closely related paralog, SCL30a, are functionally redundant in generating specific splice variants from this intron. Furthermore, SCL33 protein bound to a conserved sequence in this intron, indicating auto‐regulation of AS. Mutations in four GAAG repeats within the conserved region impaired generation of the same splice variants that are affected in the scl33 scl30a double mutant. In conclusion, we have identified the first intronic cis‐element involved in AS of a plant SR gene, and elucidated a mechanism for auto‐regulation of AS of this intron.     

Genetic identification of multiple biological roles associated with the capsid protein of satellite panicum mosaic virus

Satellite panicum mosaic virus (SPMV), an 824-nucleotide, positive-sense, single-stranded RNA virus, depends on Panicum mosaic virus (PMV) for replication and spread in host plants. Compared with PMV infection alone, symptoms are intensified and develop faster on millet plants infected with SPMV and PMV. SPMV encodes a 157 amino acid capsid protein (CP) (17.5 kDa) to encapsidate SPMV RNA and form T = 1 satellite virions. The present study identifies additional biological activities of the SPMV CP, including the induction of severe chlorosis on proso millet plants (Panicum miliaceum cv. Sunup or Red Turghai). Initial deletion mutagenesis experiments mapped the chlorosis-inducing domain to amino acids 50 to 157 on the C-terminal portion of the SPMV CP. More defined analyses revealed that amino acids 124 to 135 comprised a critical domain associated with chlorosis induction and virion formation, whereas the extreme C-terminal residues 148 to 157 were not strictly essential for either role. The results also demonstrated that the absence of SPMV CP tended to stimulate the accumulation of defective RNAs. This suggests that the SPMV CP plays a significant role in maintaining the structural integrity of the full-length satellite virus RNA and harbors multiple functions associated with pathogenesis in SPMV-infected host plants.     

The capsid protein of satellite Panicum mosaic virus contributes to systemic invasion and interacts with its helper virus

Satellite panicum mosaic virus (SPMV) depends on its helper Panicum mosaic virus (PMV) for replication and spread in host plants. The SPMV RNA encodes a 17-kDa capsid protein (CP) that is essential for formation of its 16-nm virions. The results of this study indicate that in addition to the expression of the full-length SPMV CP from the 5'-proximal AUG start codon, SPMV RNA also expresses a 9.4-kDa C-terminal protein from the third in-frame start codon. Differences in solubility between the full-length protein and its C-terminal product were observed. Subcellular fractionation of infected plant tissues showed that SPMV CP accumulates in the cytosol, cell wall-, and membrane-enriched fractions. However, the 9.4-kDa protein exclusively cofractionated with cell wall- and membrane-enriched fractions. Earlier studies revealed that the 5'-untranslated region (5'-UTR) from nucleotides 63 to 104 was associated with systemic infection in a host-specific manner in millet plants. This study shows that nucleotide deletions and insertions in the 5'-UTR plus simultaneous truncation of the N-terminal part of the CP impaired SPMV spread in foxtail millet, but not in proso millet plants. In contrast, the expression of the full-length version of SPMV CP efficiently compensated the negative effect of the 5'-UTR deletions in foxtail millet. Finally, immunoprecipitation assays revealed the presence of a specific interaction between the capsid proteins of SPMV and its helper virus (PMV). Our findings show that the SPMV CP has several biological functions, including facilitating efficient satellite virus infection and movement in millet plants.     

The role of crude human saliva and purified salivary MUC5B and MUC7 mucins in the inhibition of Human Immunodeficiency Virus type 1 in an inhibition assay

Panicum mosaic virus (PMV) has a positive-sense, single-stranded RNA genome that serves as the mRNA for two 5'-proximal genes, p48 and p112. The p112 open reading frame (ORF) has a GDD-motif, a feature of virus RNA-dependent RNA polymerases. Replication assays in protoplasts showed that p48 and p112 are sufficient for replication of PMV and its satellite virus (SPMV). Differential centrifugation of extracts from PMV-infected plants showed that the p48 and p112 proteins are membrane-associated. The same fractions exhibited RNA polymerase activity in vitro on viral RNA templates, suggesting that p48 and p112 represent the viral replication proteins. Moreover, we identified a domain spanning amino acids 306 to 405 on the p48 and p112 PMV ORFs that is common to the Tombusviridae. Alanine scanning mutagenesis of the conserved domain (CD) revealed that several substitutions were lethal or severely debilitated PMV accumulation. Other substitutions did not affect RNA accumulation, yet they caused variable phenotypes suggestive of plant-dependent effects on systemic invasion and symptom induction. The mutants that were most debilitating to PMV replication were hydrophobic amino acids that we hypothesize are important for membrane localization and functional replicase activity.     

Defective interfering RNAs of a satellite virus

Panicum mosaic virus (PMV) is a recently molecularly characterized RNA virus with the unique feature of supporting the replication of two subviral RNAs in a few species of the family Gramineae. The subviral agents include a satellite RNA (satRNA) that is devoid of a coding region and the unrelated satellite panicum mosaic virus (SPMV) that encodes its own capsid protein. Here we report the association of this complex with a new entity in the RNA world, a defective-interfering RNA (DI) of a satellite virus. The specificity of interactions governing this four-component viral system is illustrated by the ability of the SPMV DIs to strongly interfere with the accumulation of the parental SPMV. The SPMV DIs do not interfere with PMV satRNA, but they do slightly enhance the rate of spread and titer of PMV. The SPMV-derived DIs provide an additional avenue by which to investigate fundamental biological questions, including the evolution and interactions of infectious RNAs.     

Identification of prognostic alternative splicing signatures in hepatitis B or/and C viruses related hepatocellular carcinoma

BackgroundAlternative splicing (AS) takes a crucial part in tumor process. We aim to analyze AS in Hepatitis B virus (HBV) or/and hepatitis C virus (HCV) related hepatocellular carcinoma (HCC).MethodsCox regression analysis was conducted to screen survival-associated AS events. The receiver operating characteristic curve used to evaluate the predictive accuracy. Splicing network was built to investigate the relationship between splicing factors and AS events.ResultsNinety-six survival-associated AS events were obtained by univariate Cox regression. Final prognostic model could significantly distinguish the prognosis. We identified RBFOX2 as the hub gene in splicing network based on differentially expressed splicing factors, and obtained MAP3K13_AT as the key AS event in survival-related splicing network.ConclusionOur results highlight the AS signatures in HCC patients with HBV or/and HCV infection. Meanwhile, AS events and splicing factors in different virus-infected HCC subgroups can provide novel perspectives as biomarkers and individualized therapeutic targets.     

Gene expression varies within and between enzootic and epizootic lineages of Batrachochytrium dendrobatidis (Bd) in the Americas

While much research focus is paid to hypervirulent fungal lineages during emerging infectious disease outbreaks, examining enzootic pathogen isolates can be equally fruitful in delineating infection dynamics and determining pathogenesis. The fungal pathogen of amphibians, Batrachochytrium dendrobatidis (Bd), exhibits markedly different patterns of disease in natural populations, where it has caused massive amphibian declines in some regions, yet persists enzootically in others. Here we compare in vitro gene expression profiles of a panel of Bd isolates representing both the enzootic Bd-Brazil lineage, and the more recently diverged, panzootic lineage, Bd-GPL. We document significantly different lineage-specific and intralineage gene expression patterns, with Bd-Brazil upregulating genes with aspartic-type peptidase activity, and Bd-GPL upregulating CBM18 chitin-binding genes, among others. We also find pronounced intralineage variation in membrane integrity and transmembrane transport ability within our Bd-GPL isolates. Finally, we highlight unexpectedly divergent expression profiles in sympatric panzootic isolates, underscoring microgeographic functional variation in a largely clonal lineage. This variation in gene expression likely plays an important role in the relative pathogenesis and host range of Bd-Brazil and Bd-GPL isolates. Together, our results demonstrate that functional genomics approaches can provide information relevant to studies of virulence evolution within the Bd clade.     

Daily temperature cycles promote alternative splicing of RNAs encoding SR45a,a splicing regulator in maize

Elevated temperatures enhance alternative RNA splicing in maize (Zea mays) with the potential to expand the repertoire of plant responses to heat stress. Alternative RNA splicing generates multiple RNA isoforms for many maize genes, and here we observed changes in the pattern of RNA isoforms with temperature changes. Increases in maximum daily temperature elevated the frequency of the major modes of alternative splices (AS), in particular retained introns and skipped exons. The genes most frequently targeted by increased AS with temperature encode factors involved in RNA processing and plant development. Genes encoding regulators of alternative RNA splicing were themselves among the principal AS targets in maize. Under controlled environmental conditions, daily changes in temperature comparable to field conditions altered the abundance of different RNA isoforms, including the RNAs encoding the splicing regulator SR45a, a member of the SR45 gene family. We established an “in protoplast” RNA splicing assay to show that during the afternoon on simulated hot summer days, SR45a RNA isoforms were produced with the potential to encode proteins efficient in splicing model substrates. With the RNA splicing assay, we also defined the exonic splicing enhancers that the splicing-efficient SR45a forms utilize to aid in the splicing of model substrates. Hence, with rising temperatures on hot summer days, SR45a RNA isoforms in maize are produced with the capability to encode proteins with greater RNA splicing potential.

RNA splicing patterns for SR45a, a major RNA splicing regulator in maize, change in response to maximum daily temperature and vary during the day in response to daily temperature cycles.     

Plasmodium chabaudi AS: Distinct CD4+CD25+Foxp3+ regulatory T cell responses during infection in DBA/2 and BALB/c mice

Malaria infections display variation patterns of clinical course and outcome. Although CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells play an essential role in immune homeostasis, the immune regulatory roles involved in malaria infection remains to be elucidated. Herein, we compared the disparity in Treg cells response during the course of blood stage Plasmodium chabaudi chabaudi AS (P. c chabaudi AS) infection in DBA/2 and BALB/c mice. BALB/c mice initiated a Th1/Th2 profile respond to P. c chabaudi AS infection, but DBA/2 mice failed to control P. c chabaudi AS infection and almost of them died post-peak parasitemia. At the peak parasitemia, we found that higher proportion of Treg cells with elevated Foxp3 expression in DBA/2 than in BALB/c mice. We used anti-CD25 mAb to deplete Treg cells and found that the survival time and rate were prolonged in DBA/2 mice treated with anti-CD25 mAb. Treatment with anti-CD25 mAb in vivo led to enhanced pro-inflammation responses and Foxp3 expression decline on Treg cells. In contrast, after DBA/2 was treatment with anti-IL-10R mAb, IL-10R blockade in vivo caused excessive pro-inflammation responses and Foxp3 expression loss on CD4⁺CD25⁺ T cells. Earlier death was found in all of DBA/2 mice with anti-IL-10R mAb. It suggested that IL-2 and IL-10 signal involved in maintaining Foxp3 expression on Treg cells. In all, the moderate suppressive activity of Treg cells may facilitate resistance to P. c chabaudi AS infection.     

Characterization of an alternative splicing by a NAGNAG splice acceptor site in the porcine KIT gene

The KIT gene has been shown to have multiple functions in hematopoiesis, melanogenesis, and gametogenesis. In addition, mutations of this gene cause pigmentation disorders in humans and mice and are responsible for coat color differences in pigs. While characterizing polymorphisms in the porcine KIT gene, we detected alternative splicing (AS) of the NAGNAG splice acceptor site at the boundary of intron 4 and exon 5. This AS event generated the E and I isoforms, characterized by insertion or deletion, respectively, of CAG at the borders of coding sequence. AS patterns measured in tissue samples from two randomly selected animals did not identified any tissue-specific outcomes. Analysis of AS patterns using three breeds demonstrated that Landrace and Large White pigs expressed both the E and I isoforms. In contrast, a subset of specimens from Korean Native Pigs (KNP) yielded a single I isoform. Alignment of the sequence from several species revealed that the region between the branch point sequence (BPS) and 3′ acceptor site is conserved. However, it is appeared that the selection of either the proximal or distal splice site varied between species. To test the breed specificity the NAGNAG splice acceptor site, we constructed two lineages of minigenes from KNP and Landrace pigs harboring breed-specific mutations. The minigene splicing assay demonstrated that both types of minigenes expressed both the E and I isoforms in two host cell lines, and no differences were detected in the AS pattern between the two breeds. We conclude that the AS at the NAGNAG splice acceptor site on intron 4/exon 5 in the porcine KIT gene is the result of noise selection at the splice site by the splicing machinery. Therefore, this AS event in the porcine KIT gene is unlikely to have any relationship with the coat color variations of Landrace and KNP breeds.