Protective effects of Nogo-B deficiency in NAFLD mice and its multiomics analysis of gut microbiology and metabolism.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a prevalent chronic liver ailment that can lead to serious conditions such as cirrhosis and hepatocellular carcinoma. Hepatic Nogo-B regulates glucose and lipid metabolism, and its inhibition has been shown to be protective against metabolic syndrome. Increasing evidence suggests that imbalances in the gut microbiota (GM) and lipid metabolism disorders are significant contributors to NAFLD progression. Nevertheless, it is not yet known whether Nogo-B can affect NAFLD by influencing the gut microbiota and metabolites. Hence, the aim of the present study was to characterize this process and explore its possible underlying mechanisms. METHODS: A NAFLD model was constructed by administering a high-fat diet (HFD) to Nogo-B-/- and WT mice from the same litter, and body weight was measured weekly in each group. The glucose tolerance test (GTT) and insulin tolerance test (ITT) were performed to assess blood glucose levels. At the end of the 12-week period, samples of serum, liver, and intestinal contents were collected and used for serum biochemical marker and inflammatory factor detection; pathology evaluation; and gut microbiome and metabolomics analysis. Spearman's correlation analysis was performed to determine possible correlations between differential gut microbiota and differential serum metabolites between groups. RESULTS: Nogo-B deficiency attenuated the effects of the HFD, including weight gain, liver weight gain, impaired glucose tolerance, hepatic steatosis, elevated serum lipid biochemicals levels, and liver function. Nogo-B deficiency suppressed M1 polarization and promoted M2 polarization, thus inhibiting inflammatory responses. Furthermore, Nogo-B-/--HFD-fed mice presented increased gut microbiota richness and diversity, decreased Firmicutes/Bacteroidota (F/B) ratios, and altered serum metabolites compared with those of WT-HFD-fed mice. During analysis, several differential gut microbiota, including Lachnoclostridium, Harryflintia, Odoribacter, UCG-009, and unclassified_f_Butyricoccaceae, were screened between groups. These microbiota were found to be positively correlated with upregulated purine metabolism and bile acid metabolites in Nogo-B deficiency, while they were negatively correlated with downregulated corticosterone and tricarboxylic acid cyclic metabolites in Nogo-B deficiency. CONCLUSION: Nogo-B deficiency delayed NAFLD progression, as demonstrated by reduced hepatocellular lipid accumulation, attenuated inflammation and liver injury, and ameliorated gut microbiota dysbiosis and metabolic disorders. Importantly, Odoribacter was strongly positively correlated with ALB and taurodeoxycholic acid, suggesting that it played a considerable role in the influence of Nogo-B on the progression of NAFLD, a specific feature of NAFLD in Nogo-B-/- mice. The regulation of bile acid metabolism by the gut microbiota may be a potential target for Nogo-B deficiency to ameliorate NAFLD.

Amplifying protection against acute lung injury: Targeting both inflammasome and cGAS-STING pathway by Lonicerae Japonicae Flos-Forsythiae Fructus drug pair.

Objective: Acute lung injury (ALI) is characterized by inflammation and currently lacks an efficacious pharmacological intervention. The medicine combination of Lonicerae Japonicae Flos (LJF) and Forsythiae Fructus (FF) demonstrates combined properties in its anti-infective, anti-inflammatory, and therapeutic effects, particularly in alleviating respiratory symptoms. In previous studies, Chinese medicine has shown promising efficacy in lipopolysaccharides (LPS)-induced ALI. However, there have been no reports of LJF and FF pairing for lung injury. The aim of this study is to compare the efficacy of herb pair Lonicerae Japonicae Flos-Forsythiae Fructus (LF) with LJF or FF alone in the treatment of ALI, and to explore whether LJF and FF have a combined effect in the treatment of lung injury, along with the underlying mechanism involved. Methods: A total of 36 mice were divided into six groups (control, model, LJF, FF, LF, dexamethasone) based on the treatments they received after undergoing sham-operation/LPS tracheal instillation. H&E staining and pulmonary edema indexes were used to evaluate lung injury severity. Alveolar exudate cells (AECs) were counted based on cell count in bronchoalveolar lavage fluid (BALF), and neutrophil percentage in BALF was measured using flow cytometry. Myeloperoxidase (MPO) activity in BALF was measured using enzyme-linked immunosorbent assay (ELISA), while the production of IL-1ß, TNF-α, and IL-6 in the lung and secretion level of them in BALF were detected by quantitative polymerase chain reaction (qPCR) and ELISA. The effect of LJF, FF, and LF on the expression of Caspase-1 and IL-1ß proteins in bone marrow derived macrophages (BMDMs) supernatant was assessed using Western blot method under various inflammasome activation conditions. In addition, the concentration of IL-1ß and changes in lactatedehydrogenase (LDH) release levels in BMDMs supernatant after LJF, FF, and LF administration, respectively, were measured using ELISA. Furthermore, the effects of LJF, FF and LF on STING and IRF3 phosphorylation in BMDMs were detected by Western blot, and the mRNA changes of IFN-ß, TNF-α, IL-6 and CXCL10 in BMDMs were detected by qPCR. Results: LF significantly attenuated the damage to alveolar structures, pulmonary hemorrhage, and infiltration of inflammatory cells induced by LPS. This was evidenced by a decrease in lung index score and wet/dry weight ratio. Treatment with LF significantly reduced the total number of neutrophil infiltration by 75% as well as MPO activity by 88%. The efficacy of LF in reducing inflammatory factors IL-1ß, TNF-α, and IL-6 in the lungs surpasses that of LJF or FF, approaching the effectiveness of dexamethasone. In BMDMs, the co-administration of 0.2 mg/mL of LJF and FF demonstrated superior inhibitory effects on the expression of nigericin-stimulated Caspase-1 and IL-1ß, as well as the release levels of LDH, compared to individual treatments. Similarly, the combination of 0.5 mg/mL LJF and FF could better inhibit the phosphorylation levels of STING and IRF3 and the production of IFN-ß, TNF-α, IL-6, and CXCL10 in response to ISD stimulation. Conclusion: The combination of LJF and FF increases the therapeutic effect on LPS-induced ALI, which may be mechanistically related to the combined effect inhibition of cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and NOD-like receptor family protein 3 (NLRP3) inflammasomes pathways by LJF and FF. Our study provides new medicine candidates for the clinical treatment of ALI.

[Mechanism of total glucosides of White Paeony Capsules in ameliorating acute lung injury based on NLRP3 inflammasome].

This study deciphered the ameliorating effect and molecular mechanism of the total glucosides of White Paeony Capsules(TGP) in the treatment of mice model with acute lung injury(ALI) via NOD-like receptor thermal protein domain associated protein 3(NLRP3) signaling pathway of the inflammasome. The study established an inflammasome activation model of primed bone marrow-derived macrophages(BMDMs), and its molecular mechanism was investigated by Western blot(WB), immunofluorescence staining, enzyme-linked immunosorbent assay(ELISA), and flow cytometry. C57BL/6J mice were randomly divided into a blank control group, a TGP group, a model group(LPS group), LPS+low-and high-dose TGP groups, LPS+MCC950 group, and LPS+MCC950+TGP group, with eight mice per group. The ALI model was induced in mice. Finally, bronchoalveolar lavage fluid(BALF) and lung tissue were collected. Lung index and lung weight wet-to-dry ratio were determined for each group of mice. The pathological changes in lung tissue were observed through hematoxylin-eosin(HE) staining. The number of neutrophils in the BALF of each group was detected using flow cytometry. The levels of interleukin(IL)-1ß, IL-6, and tumor necrosis factor(TNF)-α in the BALF were determined by ELISA. The expressions of IL-1ß, IL-18, IL-6, and TNF-α in the lung tissue were determined by real-time quantitative PCR(RT-qPCR). This study demonstrated that TGP dramatically blocked the activation of the NLRP3 inflammasome by inhibiting the production of upstream mitochondrial reactive oxygen species(mtROS) and the subsequent oligomerization of apoptosis-associated specks(ASC). Additionally, in the ALI mice model, compared with the blank control group, the model group showed alveolar structure rupture, thic-kening of alveolar septa, and dramatically increased lung index, lung weight wet-to-dry ratio in lung tissue, neutrophil count, and inflammatory factor levels. Compared with the model group, the pathological morphology of lung tissue was significantly ameliorated in the TGP and MCC950 groups, and the lung index and lung weight wet-to-dry ratio were significantly reduced. Neutrophil counts were reduced, and levels of inflammatory factors were significantly downregulated. Notably, compared with the MCC950 group, there was no significant difference in effect in the MCC950+TGP group. Collectively, the study reveals that TGP may ameliorate ALI in mice by inhibiting the activation of NLRP3 inflammasome, providing a safe and effective drug candidate for the prevention or treatment of ALI/ARDS.

Total glucosides of paeony alleviates cGAS-STING-mediated diseases by blocking the STING-IRF3 interaction.

In the realm of autoimmune and inflammatory diseases, the cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) signaling pathway has been thoroughly investigated and established. Despite this, the clinical approval of drugs targeting the cGAS-STING pathway has been limited. The Total glucosides of paeony (TGP) is highly anti-inflammatory and is commonly used in the treatment of rheumatoid arthritis (RA), emerged as a subject of our study. We found that the TGP markedly reduced the activation of the cGAS-STING signaling pathway, triggered by various cGAS-STING agonists, in mouse bone marrow-derived macrophages (BMDMs) and Tohoku Hospital Pediatrics-1 (THP-1) cells. This inhibition was noted alongside the suppression of interferon regulatory factor 3 (IRF3) phosphorylation and the expression of interferon-beta (IFN-ß), C-X-C motif chemokine ligand 10 (CXCL10), and inflammatory mediators such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). The mechanism of action appeared to involve the TGP's attenuation of the STING-IRF3 interaction, without affecting STING oligomerization, thereby inhibiting the activation of downstream signaling pathways. In vivo, the TGP hindered the initiation of the cGAS-STING pathway by the STING agonist dimethylxanthenone-4-acetic acid (DMXAA) and exhibited promising therapeutic effects in a model of acute liver injury induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN). Our findings underscore the potential of the TGP as an effective inhibitor of the cGAS-STING pathway, offering a new treatment avenue for inflammatory and autoimmune diseases mediated by this pathway.

Flavonoid extracted from Epimedium attenuate cGAS-STING-mediated diseases by targeting the formation of functional STING signalosome.

Hyperactivation of the cyclic-GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signalling pathway has been shown to be associated with the development of a variety of inflammatory diseases, and the discovery of an inhibitor of the cGAS-STING signalling pathway holds great promise in the therapeutic interventions. Epimedium flavonoid (EF), a major active ingredient isolated from the medicinal plant Epimedium, has been reported to have good anti-inflammatory activity, but its exact mechanism of action remains unclear. In the present study, we found that EF in mouse bone marrow-derived macrophages (BMDMs), THP-1 (Tohoku Hospital Pediatrics-1) as well as in human peripheral blood mononuclear cells (hPBMC) inhibited the activation of the cGAS-STING signalling pathway, which subsequently led to a decrease in the expression of type I interferon (IFN-ß, CXCL10 and ISG15) and pro-inflammatory cytokines (IL-6 and TNF-α). Mechanistically, EF does not affect STING oligomerization, but inhibits the formation of functional STING signalosome by attenuating the interaction of interferon regulatory factor 3 (IRF3) with STING and TANK-binding kinase 1 (TBK1). Importantly, in vivo experiments, EF has shown promising therapeutic effects on inflammatory diseases mediated by the cGAS-STING pathway, which include the agonist model induced by DMXAA stimulation, the autoimmune inflammatory disease model induced by three prime repair exonuclease 1 (Trex1) deficiency, and the non-alcoholic steatohepatitis (NASH) model induced by a pathogenic amino acid and choline deficiency diet (MCD). To summarize, our study suggests that EF is a potent potential inhibitor component of the cGAS-STING signalling pathway for the treatment of inflammatory diseases mediated by the cGAS-STING signalling pathway.

The combination of Schisandrin C and Luteolin synergistically attenuates hepatitis B virus infection via repressing HBV replication and promoting cGAS-STING pathway activation in macrophages.

BACKGROUND: HBV infection can result in severe liver diseases and is one of the primary causes of liver cell carcinoma-related mortality. Liuwei Wuling tablet (LWWL) is a traditional Chinese medicine formula, with a protecting liver and decreasing enzyme activity, usually used to treat chronic hepatitis B with NAs in clinic. However, its main active ingredients and mechanism of action have not been fully investigated. Hence, we aimed to screen the active ingredient and effective ingredient combinations from Liuwei Wuling tablet to explore the anti-herpatitis B virus activity and mechanism. METHODS: Analysis and screening of effective antiviral components in LWWL by network pharmacology, luteolin (Lut) may be a compound with significant antiviral activity. The mechanism of antiviral action of Lut was also found by real-time PCR detection and western blotting. Meanwhile, we established a co-culture model to investigate the antiviral mechanism of Schisandrin C (SC), one of the main active components of Schisandra chinensis fructus (the sovereign drug of LWWL). Next, HBV-infected mice were established by tail vein injection of pAAV-HBV1.2 plasmid and administered continuously for 20 days. And their antiviral capacity was evaluated by checking serum levels of HBsAg, HBeAg, levels of HBV DNA, and liver levels of HBcAg. RESULTS: In this study, we conducted network pharmacology analysis on LWWL, and through in vitro experimental validation and data analysis, we found that luteolin (Lut) possessed obviously anti-HBV activity, inhibiting HBV replication by downregulating hepatocyte nuclear factor 4α (HNF4α) via the ERK pathway. Additionally, we established a co-culture system and proved that SC promoted activation of cGAS-STINIG pathway and IFN-ß production in THP-1 cells to inhibit HBV replication in HepG2.2.15 cells. Moreover, we found the combination of SC and Lut shows a greater effect in inhibiting HBV compared to SC or Lut alone in HBV-infected mice. CONCLUSION: Taken together, our study suggests that combination of SC and Lut may be potential candidate drug for the prevention and treatment of chronic hepatitis B.

Liuweiwuling Tablet relieves the inflammatory transformation of hepatocellular carcinoma by inhibiting the PI3K/AKT/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Liuweiwuling Tablet (LWWL) is a patented Chinese medicine approved by the Chinese National Medical Products Administration (NMPA). Clinically, it is used to treat a range of liver diseases that precede hepatocellular carcinoma (HCC), including hepatitis, liver fibrosis and cirrhosis. LWWL is hypothesized to inhibit the inflammatory transformation of HCC, which may have a positive impact on the prevention and treatment of HCC. However, its exact mechanism of action remains unknown. AIM OF THE STUDY: To investigate how LWWL is effective in the treatment of HCC and to validate the pathways involved in this process. MATERIALS AND METHODS: An in vivo model of HCC induced by diethylnitrosamine (DEN) was established to study the effect of LWWL on the development of HCC. The rat serum was analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transpeptidase (γ-GT). The rat liver tissues were stained with hematoxylin and eosin (HE) and Masson's trichrome for pathological analysis. Rat liver tissue was subjected to transcriptome sequencing. Expression of inflammatory and liver fibrosis-related factors in bone marrow-derived macrophages (BMDMs) and LX-2 cells was detected by QRT-PCR, ELISA and Western blot (WB). The expression of apoptosis and stemness genes in HepG2 and Huh7 cells was assessed through flow cytometry and QRT-PCR. Transcriptomics, network pharmacology, WB, and QRT-PCR were employed to validate the mechanisms associated with the amelioration of HCC development by LWWL. RESULTS: LWWL significantly reduced the severity of hepatitis and liver fibrosis, the expression of tumor stemness genes, and the incidence of HCC. In addition, LWWL inhibited the release of inflammatory substances and nuclear accumulation of P65 protein in BMDMs as well as the conversion of LX-2 cells to fibroblasts. LWWL inhibited the proliferation of HepG2 and Huh7 cells, including the initiation of apoptosis and the reduction of stemness gene expression. Importantly, LWWL regulates the PI3K/AKT/NF-κB pathway, which affects hepatic inflammation and cancer progression. CONCLUSION: LWWL inhibited the occurrence and development of HCC by modulating the severity of hepatitis and liver fibrosis, indicating the potential clinical relevance of LWWL in preventing and treating HCC.

Data mining-based analysis on medication rules of Chinese herbal medicine treating headache with blood stasis syndrome.

Objective: The incidence of headaches with blood stasis syndrome has increased. Herein, we used scientific, statistical methods to explore the medication rules of Chinese herbal medicines (CHMs) to treat headaches with blood stasis syndrome and provide a scientific and reliable theoretical basis for clinical treatment. Methods: First, we retrieved studies related to CHMs used to treat headaches with blood stasis syndrome from the VIP, CNKI, Wanfang, and PubMed databases. We used Excel 2013 to establish a database and SPSS Modeler 18.0 and SPSS 25.0 to conduct frequency, association rule, and cluster analyses. Results: Based on the screening criteria, we retrieved 126 CHM prescriptions for headaches with blood stasis syndrome involving 149 herbs. The top three high-frequency herbs were Chuanxiong Rhizoma (Chuanxiong), Angelica Sinensis Radix (Danggui), and Carthami Flos (Honghua). Blood-activating and stasis-eliminating herbs were the most frequently used herb efficacy categories. The liver meridian represented the most frequently used herb meridian tropism. The properties and taste of herbs were mainly warm and bitter, respectively. We obtained 21 association rules and five new clusters. The Chuanxiong Rhizoma (Chuanxiong) and Angelica Sinensis Radix (Danggui) herb pair had the strongest correlation. Conclusion: We analyzed published CHM prescriptions for headaches with blood stasis syndrome and eliminated factors that did not reach an agreement, such as herb dosage. We used different data mining and analysis methods to ensure that the method and process were scientific and the conclusion was reliable, comprising a valuable reference for selecting herbs for the clinical treatment of headaches with blood stasis syndrome. The Xuefu Zhuyu Decoction (XFZYD) was the primary CHM prescription for headaches with blood stasis syndrome. Xiaoyao San (XYS) and Buyang Huanwu Decoction (BYHWD) might also be clinical references for treatment selection. Meridian-inducing and insect herbs might be used according to syndromes.

Papaya CpERF9 acts as a transcriptional repressor of cell-wall-modifying genes CpPME1/2 and CpPG5 involved in fruit ripening.

KEY MESSAGE: CpERF9 controls papaya fruit ripening through transcriptional repression of cell-wall-modifying genes CpPME1/2 and CpPG5 by directly binding to their promoters. Papaya fruit ripening is an intricate and highly coordinated developmental process which is controlled by the action of ethylene and expression of numerous ethylene-responsive genes. Ethylene response factors (ERFs) representing the last regulators of ethylene-signaling pathway determine the specificities of ethylene response. However, knowledge concerning the transcriptional controlling mechanism of ERF-mediated papaya fruit ripening is limited. In the present work, a gene-encoding AP2/ERF protein with two ERF-associated amphiphilic repression (EAR) motifs, named CpERF9, was characterized from papaya fruit. CpERF9 was found to localize in nucleus, and possess transcriptional repression ability. CpERF9 expression steadily decreased during papaya fruit ripening, while several genes encoding pectin methylesterases (PMEs) and polygalacturonases (PGs), such as CpPME1/2 and CpPG5, were gradually increased, paralleling the decline of fruit firmness. Electrophoretic mobility shift assay (EMSA) demonstrated a specific binding of CpERF9 to promoters of CpPME1/2 and CpPG5, via the GCC-box motif. Transient expression of CpERF9 in tobacco repressed CpPME1/2 and CpPG5 promoter activities, which was depended on two EAR motifs of CpERF9 protein. Taken together, these findings suggest that papaya CpERF9 may act as a transcriptional repressor of several cell-wall modifying genes, such as CpPME1/2 and CpPG5, via directly binding to their promoters.

A conserved intronic U1 snRNP-binding sequence promotes trans-splicing in Drosophila.

Unlike typical cis-splicing, trans-splicing joins exons from two separate transcripts to produce chimeric mRNA and has been detected in most eukaryotes. Trans-splicing in trypanosomes and nematodes has been characterized as a spliced leader RNA-facilitated reaction; in contrast, its mechanism in higher eukaryotes remains unclear. Here we investigate mod(mdg4), a classic trans-spliced gene in Drosophila, and report that two critical RNA sequences in the middle of the last 5' intron, TSA and TSB, promote trans-splicing of mod(mdg4). In TSA, a 13-nucleotide (nt) core motif is conserved across Drosophila species and is essential and sufficient for trans-splicing, which binds U1 small nuclear RNP (snRNP) through strong base-pairing with U1 snRNA. In TSB, a conserved secondary structure acts as an enhancer. Deletions of TSA and TSB using the CRISPR/Cas9 system result in developmental defects in flies. Although it is not clear how the 5' intron finds the 3' introns, compensatory changes in U1 snRNA rescue trans-splicing of TSA mutants, demonstrating that U1 recruitment is critical to promote trans-splicing in vivo. Furthermore, TSA core-like motifs are found in many other trans-spliced Drosophila genes, including lola. These findings represent a novel mechanism of trans-splicing, in which RNA motifs in the 5' intron are sufficient to bring separate transcripts into close proximity to promote trans-splicing.

Conserved RNA cis-elements regulate alternative splicing of Lepidopteran doublesex.

Doublesex (dsx) is a downstream key regulator in insect sex determination pathway. In Drosophila, alternative splicing of Dm-dsx gene is sex-specifically regulated by transformer (tra), in which the functional TRA promotes female-specific Dm-dsx. However, the sex determination pathway in Lepidoptera is not well understood; here we focused on alternative splicing of doublesex (dsx) in two agricultural pests, Asian corn borer (Ostrinia furnacalis) and cotton bollworm (Helicoverpa armigera), as well as the silkworm (Bombyx mori). More than a dozen new alternative splicing isoforms of dsx were found in the Lepidopteran females, which exist in all tested developmental stages and differentiated tissues. Alignment of mRNA and protein sequences of doublesex revealed high conservation of this gene in Lepidoptera. Strength analysis of splice sites revealed a weak 5' splice site at intron 3 in Lepidopteran dsx, which was experimentally confirmed. Furthermore, we identified highly conserved RNA sequences in the Lepidopteran dsx, including RNA elements I (14 nt), II (11 nt), III (26 nt), IV (17 nt), 3E-1 (8 nt) and 3E-2 (8 nt). The RNA elements III and IV were previously found in exon 4 of B. mori dsx and bound with Bm-PSI, which suppressed the inclusion of exons 3 & 4 into the male-specific Bm-dsx. Then we identified and analyzed the homologous genes of Bm-psi in the two Lepidopteran pests, which expressed at similar levels and exhibited a unique isoform in the males and females from each Lepidoptera. Importantly, mutagenesis of Bm-dsx mini-genes and their expression in BmN cell line demonstrated that three RNA elements are involved in the female-specific alternative splicing of Bm-dsx. Mutations in the RNA cis-elements 3E-1 and 3E-2 resulted in decreased inclusion of exon 3 into the female-specific dsx mRNA, suggesting that these two elements would be exonic splicing enhancers that facilitate the recognition of the weak 5' splice site at intron 3 of Lepidopteran dsx. We propose that the 5' splice sites at intron 3 are weak, resulting in multiple alternative splicing events in intron 3 of female Lepidoptera dsx. Activation of the 5' splice site requires regulatory cis-elements in exons 3 for female-specific splicing of Lepidoptera dsx.

Splicing proofreading at 5' splice sites by ATPase Prp28p.

Fidelity and efficiency of pre-mRNA splicing are critical for generating functional mRNAs, but how such accuracy in 5' splice site (SS) selection is attained is not fully clear. Through a series of yeast genetic screens, we isolated alleles of prp28 that improve splicing of suboptimal 5'SS substrates, demonstrating that WT-Prp28p proofreads, and consequently rejects, poor 5'SS. Prp28p is thought to facilitate the disruption of 5'SS-U1 snRNA pairing to allow for 5'SS-U6 snRNA pairing in the catalytic spliceosome; unexpectedly, 5'SS proofreading by Prp28p is dependent on competition with the stability of the 5'SS:U6 duplex, but not the 5'SS:U1 duplex. E404K, the strongest prp28 allele containing a mutation located in the linker region between adenosine triphosphatase (ATPase) subdomains, exhibited lower RNA-binding activity and enhanced splicing of suboptimal substrates before first-step catalysis, suggesting that decreased Prp28p activity allows longer time for suboptimal 5'SS substrates to pair with U6 snRNA and thereby reduces splicing fidelity. Residue E404 is critical for providing high splicing activity, demonstrated here in both yeast and Drosophila cells. Thus, the subdomain linker in Prp28p plays important roles both in splicing efficiency across species and in proofreading of 5'SS.

Alternative splicing and trans-splicing events revealed by analysis of the Bombyx mori transcriptome.

Alternative splicing and trans-splicing events have not been systematically studied in the silkworm Bombyx mori. Here, the silkworm transcriptome was analyzed by RNA-seq. We identified 320 novel genes, modified 1140 gene models, and found thousands of alternative splicing and 58 trans-splicing events. Studies of three SR proteins show that both their alternative splicing patterns and mRNA products are conserved from insect to human, and one isoform of Srsf6 with a retained intron is expressed sex-specifically in silkworm gonads. Trans-splicing of mod(mdg4) in silkworm was experimentally confirmed. We identified integrations from a common 5'-gene with 46 newly identified alternative 3'-exons that are located on both DNA strands over a 500-kb region. Other trans-splicing events in B. mori were predicted by bioinformatic analysis, in which 12 events were confirmed by RT-PCR, six events were further validated by chimeric SNPs, and two events were confirmed by allele-specific RT-PCR in F(1) hybrids from distinct silkworm lines of JS and L10, indicating that trans-splicing is more widespread in insects than previously thought. Analysis of the B. mori transcriptome by RNA-seq provides valuable information of regulatory alternative splicing events. The conservation of splicing events across species and newly identified trans-splicing events suggest that B. mori is a good model for future studies.

[Mapping of non-lepis wing gene nlw in silkworm (Bombyx mori) using SSR and STS markers].

The non-lepis wing of silkworm (Bombyx mori) is controlled by the recessive gene, nlw. Owning to lack of crossing over in females, the reciprocal backcrossed F(1) (BC(1)) progenies were used for linkage analysis and mapping of nlw based on the SSR linkage map and STS markers using the wild type (+(nlw)/+(nlw)) silkworm strain P50 and U06 with scaleless wing (nlw/nlw). The nlw gene was linked to eight SSR markers and one STS marker. All the individuals with the wild type in the BC1F (Using F(1) as female to backcross to the recessive parent, that is (U06xP50)xU06) showed heterozygous profile of (U06xP50) F(1), and the ones with non-lepis wing in BC1F exhibited the homozygous profile of the strain U06. Using a reciprocal BC1M (Using F1 as male to backcross to the recessive parent, that is U06x(U06xP50))cross, we constructed a linkage map of 125.6 cM, and the distance between nlw and the nearest marker cash2p was 11.4 cM.

Gene expression profiling of resistant and susceptible Bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels.

We investigated variations in the gene expression of Bombyx mori following infection with a nucleopolyhedrovirus (BmNPV). Two B. mori strains, KN and 306, which are highly resistant and susceptible to BmNPV infection, respectively, were used in this study. The infection profiles of BmNPV in the B. mori KN and 306 larvae revealed that the virus invaded the midguts of both these strains. However, its proliferation was notably inhibited in the midgut of the resistant strain. By using the suppression subtractive hybridization method, two cDNA libraries were constructed in order to compare the BmNPV responsive gene expressions between the two silkworm lines. In total, 62 differentially expressed genes were obtained. Real-time qPCR analysis confirmed that eight genes were significantly up-regulated in the midgut of the KN strain following BmNPV infection. Our results imply that these up-regulated genes may be involved in the B. mori immune response against BmNPV infection.