Gao, X. et al. (2017) International Worm Meeting "Transcriptome analysis of C. elegans smn-1 mutant."

Xu, J., Gan, W., Ma, L., Hu, S., Gao, X.

[International Worm Meeting, 2017]

Spinal muscular atrophy (SMA) is caused by mutations in the highly conserved gene survival motor neuron 1 (SMN1). Although many efforts have been made to elucidate the molecular functions of SMN1, mechanisms underlying the pathogenesis of SMA are not well understood. SMN1 plays important roles in several biological processes including the assembly of U snRNPs and pre-mRNA splicing. The nematode Caenorhabditis elegans carries a single ortholog of SMN1, smn-1, and has been used as a model for studying the functions of SMN. To understand how SMN-1 affects mRNA splicing and expression, we determined the transcriptomes of wild type, smn-1(ok355), uaf-1(n4588) and smn-1(ok355); uaf-1(n4588) mutants on day 2 post L1, when the deleterious phenotypes of smn-1 mutants are less obvious. We found that mutations in smn-1 or uaf-1 cause widespread alternations of gene expression. KEGG analysis suggests that the differentially expressed genes (DEGs) in smn-1 mutants are enriched in neuronal ligand-receptor interactions, phagosomes and cancer-related signaling pathways, implying that these pathways might be involved in smn-1 functions. We are verifying these changes using RT-PCR experiments. In addition, the defective expression of some genes in smn-1(ok355) mutant is reversed by uaf-1(n4588), which is consistent with our previous findings that uaf-1(n4588) mutation could partially suppress the lifespan and locomotion defects of smn-1(ok355) mutant. Our study might provide new insights into the functions of SMN-1 and SMA pathology.

Gao X et al. (2008) Genomics "Proteome-wide prediction of PKA phosphorylation sites in eukaryotic kingdom."

Ren J, Yao X, Xue Y, Gao X, Jin C

[Genomics, 2008]

Protein phosphorylation is one of the most essential post-translational modifications (PTMs), and orchestrates a variety of cellular functions and processes. Besides experimental studies, numerous computational predictors implemented in various algorithms have been developed for phosphorylation sites prediction. However, large-scale predictions of kinase-specific phosphorylation sites have not successfully pursued and remained to be a great challenge. In this work, we raised a "kiss farewell" model and conducted a high-throughput prediction of cAMP-dependent kinase (PKA) phosphorylation sites. Since a protein kinase (PK) should at least "kiss" its substrates and then run away, we proposed a PKA-binding protein to be a potential PKA substrate if at least one PKA site was predicted. To improve the prediction specificity, we reduced false positive rate (FPR) less than 1% when the cut-off value was set as 4. Successfully, we predicted 1387, 630, 568 and 912 potential PKA sites from 410, 217, 173 and 260 PKA-interacting proteins in Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster and Homo sapiens, respectively. Most of these potential phosphorylation sites remained to be experimentally verified. In addition, we detected two sites in one of PKA regulatory subunits to be conserved in eukaryotes as potentially ancient regulatory signals. Our prediction results provide an excellent resource for delineating PKA-mediated signaling pathways and their system integration underlying cellular dynamics and plasticity.

Gao X et al. (2024) J Appl Toxicol "Mulberrin extends lifespan in Caenorhabditis elegans through detoxification function."

Wu C, Yu J, Fang M, Huang C, Yan Y, Shi H, Fan S, Gao X, Han Y, Zhang L, Liu Y

[J Appl Toxicol, 2024]

Mulberrin, a naturally occurring flavone found in mulberry and Romulus Mori, exhibits diverse biological functions. Here, we showed that mulberrin extended both the lifespan and healthspan in C. elegans. Moreover, mulberrin increased the worms' resistance to toxicants and activated the expression of detoxification genes. The longevity-promoting effect of mulberrin was attenuated in nuclear hormone receptor (NHR) homologous nhr-8 and daf-12 mutants, indicating that the lifespan extending effects of mulberrin in C. elegans may depend on nuclear hormone receptors NHR-8/DAF-12. Further analyses revealed the potential associations between the longevity effects of mulberrin and the insulin/insulin-like growth factor signaling (IIS) and adenosine 5'-monophosphate-activated protein kinase (AMPK) pathways. Together, our findings suggest that mulberrin may prolong lifespan and healthspan by activating detoxification functions mediated by nuclear receptors.

Gao X et al. (2016) RNA Biol "Compartmentalization of functions and predicted miRNA regulation among contiguous regions of the Nematode Intestine."

Jasmer DP, Mitreva M, Ly A, Magrini V, Tyagi R, Gao X

[RNA Biol, 2016]

The intestine of parasitic nematodes has proven an important target for therapies aimed at prevention and treatment of diseases caused by these pathogens in humans, animals and plants. We have developed a unique research model with the intestine of Ascaris suum, the large round worm of swine and humans, that will enhance biological research on this tissue. To expand utility of this model, we quantitatively compared expression of 15,382 coding RNAs and 277 noncoding, micro RNAs (miRNAs) among three contiguous regions of the adult A. suum intestine. Differentially expressed transcripts were identified among regions, with the largest number expressed at significantly higher levels in the anterior region, identifying this region as the most functionally unique compared to middle and posterior regions. We further identified 64 exon splice variants (from 47 genes) that are differentially expressed among these regions. A total of 2,063 intestinal mRNA transcripts were predicted to be targeted by intestinal miRNA, and negative correlation coefficients for miRNA:mRNA abundances predicted 22 likely influential miRNAs and 503 likely associated miRNA:mRNA pairs. A. suum intestinal miRNAs were identified that are conserved with intestinal miRNAs from C. elegans (10 mature sequences and 13 seed sequences conserved), and prospective intestinal miRNAs from the murine gastrointestinal nematode, Heligmosomoides polygyrus (5 mature and 11 seeds). Most of the conserved intestinal miRNAs were also high abundance miRNAs. The data provide the most comprehensive compilation of constitutively and differentially expressed genes along the length of the intestine for any nematode species. The information will guide prospective development of many hypotheses on nematode intestinal functions encoded by mRNAs, miRNAs and interactions between these RNA populations.

Gao X et al. (2009) Int J Parasitol "Molecular cloning and DNA binding characterization of DAF-16 orthologs from Ancylostoma hookworms."

Gao X, Hawdon JM, Frank D

[Int J Parasitol, 2009]

Infective hookworm L3s encounter a host-specific signal during infection that re-initiates a suspended developmental pathway, resulting in development to the adult stage. This resumption of development in the host is analogous to recovery of developmentally arrested Caenorhabditis elegans dauer larvae in response to favorable environmental signals. Dauer recovery in C. elegans dauers and hookworm L3s is mediated by insulin-like signaling (ILS). A key output of ILS in C. elegans is the forkhead transcription factor DAF-16, which controls the expression of genes required for maintenance of the dauer stage. The similarity between recovery pathways of L3s and dauers suggests that DAF-16 functions similarly in hookworm L3 activation. To test this, orthologs of Ce-DAF-16 were isolated from the hookworms Ancylostoma caninum and Ancylostoma ceylanicum. The protein sequences of hookworm DAF-16 DNA binding domains were identical, and shared 94% identity with the b and c isoforms of Ce-DAF-16. Ac-DAF-16 expressed in HEK293 kidney cells bound strongly to the conserved DAF family binding element (DBE), but not to a random DNA sequence. Ac-DAF-16 was able to drive transcription of a reporter gene located downstream of six copies of the DBE in NIH3T3 cells under starved conditions. Addition of serum caused a decrease in reporter gene expression, indicating that DAF-16 is negatively regulated by growth factor stimulation. These data confirm the presence of DAF-16 orthologs in hookworms, and demonstrate that Ac-DAF-16 binds to and drives transcription from a conserved DAF-16 family DNA binding element.

Gao, X. et al. (2013) International Worm Meeting "Altered-function mutations of the U2AF large subunit are protective modifiers of the C. elegans SMN mutant defects."

Gao, X., Ma, L.

[International Worm Meeting, 2013]

Spinal muscular atrophy (SMA) is caused by loss-of-function mutations in the highly conserved and ubiquitously expressed protein SMN. Mutations in SMN can lead to defective RNA splicing. However, little is known about the in vivo interactions of SMN with RNA splicing factor genes. To understand how SMN affects RNA splicing, we examined the splicing of the endogenous splicing reporter gene tos-1 in the C. elegans smn-1(ok355D) (SMN null) mutants. We found that smn-1(ok355D) caused increased tos-1 intron 1 retention. By analyzing tos-1 transgenes carrying different substituted 3' splice sites, we found that smn-1 is required for efficient splicing only at weak 3' splice sites. Surprisingly, altered-function mutations of the U2AF large subunit gene uaf-1 could rescue the lifespan and locomotion defects of smn-1(ok355D) mutants. Our study suggests that in C. elegans the splicing at strong 3' splice sites is likely not dependent on SMN-1 and identifies altered-function mutations of the U2AF large subunit gene as novel rescuing modifiers of SMN mutant defects.

Gao X et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Identification of Hookworm DAF-16/FOXO Response Elements and Direct Gene Targets"

Mitreva M, Gao X, Wang Z, Hawdon J, Martin J, Abubucker S, Zhang X

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

The infective stage of the parasitic nematode hookworm is developmentally arrested in the environment and needs to infect a specific host to complete its life cycle. The canine hookworm (Ancylostoma caninum) is an excellent model for investigating the human hookworm infections. The forkhead transcription factor of A. caninum, Ac-DAF-16, which has a characteristic forkhead /""winged helix"" DNA binding domain (DBD), has been implicated in the resumption of hookworm development in the host. However, the precise roles of Ac-DAF-16 in hookworm parasitism and its downstream effectors are unknown. In the present study, we combined molecular techniques and bioinformatics to identify a group of Ac-DAF-16 binding sites and target genes. The DNA binding domain of Ac-DAF-16 was used to select genomic fragments by in vitro genomic selection. Twenty four bound genomic fragments were analyzed for the presence of the DAF-16 family binding element (DBE) and possible alternative Ac-DAF-16 bind motifs. The 22 genes linked to these genomic fragments were identified using bioinformatics tools and defined as candidate direct gene targets of Ac-DAF-16. Their developmental stage-specific expression patterns were examined. Our results show that Ac-DAF-16 is involved in diverse biological processes throughout hookworm development. Further investigation of these target genes will provide insights into the molecular basis by which Ac-DAF-16 regulates its downstream gene network in hookworm infection.

Gao X et al. (2019) Front Genet "Defective Expression of Mitochondrial, Vacuolar H+-ATPase and Histone Genes in a C. elegans Model of SMA."

Zhou C, Xue D, Gao X, Ma L, Ma YC, Xu J, Chen H, Pan W

[Front Genet, 2019]

Spinal muscular atrophy (SMA) is a severe motor neuron degenerative disease caused by loss-of-function mutations in the survival motor neuron gene <i>SMN1</i>. It is widely posited that defective gene expression underlies SMA. However, the identities of these affected genes remain to be elucidated. By analyzing the transcriptome of a <i>Caenorhabditis elegans</i> SMA model at the pre-symptomatic stage, we found that the expression of numerous nuclear encoded mitochondrial genes and vacuolar H⁺-ATPase genes was significantly down-regulated, while that of histone genes was significantly up-regulated. We previously showed that the <i>uaf-1</i> gene, encoding key splicing factor U2AF large subunit, could affect the behavior and lifespan of <i>smn-1</i> mutants. Here, we found that <i>smn-1</i> and <i>uaf-1</i> interact to affect the recognition of 3' and 5' splice sites in a gene-specific manner. Altogether, our results suggest a functional interaction between <i>smn-1</i> and <i>uaf-1</i> in affecting RNA splicing and a potential effect of <i>smn-1</i> on the expression of mitochondrial and histone genes.

Gao X et al. (2014) RNA Biol "The survival motor neuron gene smn-1 interacts with the U2AF large subunit gene uaf-1 to regulate Caenorhabditis elegans lifespan and motor functions."

Ma L, Li M, Teng Y, Huang L, Ma YC, Zhang Z, Luo J, Gao X

[RNA Biol, 2014]

Spinal muscular atrophy (SMA), the most frequent human congenital motor neuron degenerative disease, is caused by loss-of-function mutations in the highly conserved survival motor neuron gene SMN1. Mutations in SMN could affect several molecular processes, among which aberrant pre-mRNA splicing caused by defective snRNP biogenesis is hypothesized as a major cause of SMA. To date little is known about the interactions of SMN with other splicing factor genes and how SMN affects splicing in vivo. The nematode Caenorhabditis elegans carries a single ortholog of SMN, smn-1, and has been used as a model for studying the molecular functions of SMN. We analyzed RNA splicing of reporter genes in an smn-1 deletion mutant and found that smn-1 is required for efficient splicing at weak 3' splice sites. Genetic studies indicate that the defective lifespan and motor functions of the smn-1 deletion mutants could be significantly improved by mutations of the splicing factor U2AF large subunit gene uaf-1. In smn-1 mutants we detected a reduced expression of U1 and U5 snRNAs and an increased expression of U2, U4 and U6 snRNAs. Our study verifies an essential role of smn-1 for RNA splicing in vivo, identifies the uaf-1 gene as a potential genetic modifier of smn-1 mutants, and suggests that SMN-1 has multifaceted effects on the expression of spliceosomal snRNAs.

Gao X et al. (2023) Int J Biol Macromol "Protective effects of pumpkin polysaccharide hydrolysates on oxidative stress injury and its potential mechanism - Antioxidant mechanism of pumpkin polysaccharide hydrolysates."

Jiang W, Gao X, Li X, Yang Z, Huang L, Li J, Wang S, Zhang Y, Zuo S

[Int J Biol Macromol, 2023]

Pumpkin polysaccharides (PPe) exhibit multiple bioactive properties, including the ability to reduce blood sugar and lipids. Our prior investigation discovered that hydrolysates (PPe-s) derived from PPe demonstrated stronger antioxidant capabilities than PPe. The objective of the current study was to explore the potential mechanism of PPe-s, utilizing Caenorhabditis elegans and MIN6 cells as models. The results of this investigation revealed that PPe-s exhibited strong scavenging ability towards ABTS⁺ and OH&#xb7;in vitro. Additionally, PPe-s extended the lifespan of C. elegans under hydrogen peroxide stress (p&#x202f;&lt;&#x202f;0.05) by upregulating the mRNA expression of daf-16, sod-1, sod-3, and skn-1 (all &gt;1.43-fold, p&#x202f;&lt;&#x202f;0.05). Furthermore, PPe-s enhanced the proliferation activity of MIN6 cells, induced by alloxan, increased insulin secretion and cAMP levels, and excreted intracellular excessive Ca²⁺ in a concentration-dependent manner. Our study demonstrated that PPe-s upregulated the expression levels of antioxidative-related genes and augmented the antioxidant defense system.