Proteomics reveals the significance of vacuole Pi transporter in the adaptability of Brassica napus to Pi deprivation.

Vacuolar Pi transporters (VPTs) have recently been identified as important regulators of cellular Pi status in Arabidopsis thaliana and Oryza sativa. In the oil crop Brassica napus, BnA09PHT5;1a and BnC09PHT5;1a are two homologs of AtPHT5;1, the vacuolar Pi influx transporter in Arabidopsis. Here, we show that Pi deficiency induces the transcription of both homologs of PHT5;1a genes in B. napus leaves. Brassica PHT5;1a double mutants (DM) had smaller shoots and higher cellular Pi concentrations than wild-type (WT, Westar 10), suggesting the potential role of BnPHT5;1a in modulating cellular Pi status in B. napus. A proteomic analysis was performed to estimate the role of BnPHT5;1a in Pi fluctuation. Results show that Pi deprivation disturbs the abundance of proteins in the physiological processes involved in carbohydrate metabolism, response to stimulus and stress in B. napus, while disruption of BnPHT5;1a genes may exacerbate these processes. Besides, the processes of cell redox homeostasis, lipid metabolic and proton transmembrane transport are supposed to be unbalanced in BnPHT5;1a DM under the -Pi condition. Noteworthy, disruption of BnPHT5;1a genes severely alters the abundance of proteins related to ATP biosynthesis, and proton/inorganic cation transmembrane under normal Pi condition, which might contribute to B. napus growth limitations. Additionally, seven new protein markers of Pi homeostasis are identified in B. napus. Taken together, this study characterizes the important regulatory role of BnPHT5;1a genes as vacuolar Pi influx transporters in Pi homeostasis in B. napus.

The LIKE SEX FOUR 1-malate dehydrogenase complex functions as a scaffold to recruit ß-amylase to promote starch degradation.

In plant leaves, starch is composed of glucan polymers that accumulate in chloroplasts as the products of photosynthesis during the day; starch is mobilized at night to continuously provide sugars to sustain plant growth and development. Efficient starch degradation requires the involvement of several enzymes, including ß-amylase and glucan phosphatase. However, how these enzymes cooperate remains largely unclear. Here, we show that the glucan phosphatase LIKE SEX FOUR 1 (LSF1) interacts with plastid NAD-dependent malate dehydrogenase (MDH) to recruit ß-amylase (BAM1), thus reconstituting the BAM1-LSF1-MDH complex. The starch hydrolysis activity of BAM1 drastically increased in the presence of LSF1-MDH in vitro. We determined the structure of the BAM1-LSF1-MDH complex by a combination of cryo-electron microscopy, crosslinking mass spectrometry, and molecular docking. The starch-binding domain of the dual-specificity phosphatase and carbohydrate-binding module of LSF1 was docked in proximity to BAM1, thus facilitating BAM1 access to and hydrolysis of the polyglucans of starch, thus revealing the molecular mechanism by which the LSF1-MDH complex improves the starch degradation activity of BAM1. Moreover, LSF1 is phosphatase inactive, and the enzymatic activity of MDH was dispensable for starch degradation, suggesting nonenzymatic scaffold functions for LSF1-MDH in starch degradation. These findings provide important insights into the precise regulation of starch degradation.

Structural insights into DNA N6-adenine methylation by the MTA1 complex.

N6-methyldeoxyadenine (6mA) has recently been reported as a prevalent DNA modification in eukaryotes. The Tetrahymena thermophila MTA1 complex consisting of four subunits, namely MTA1, MTA9, p1, and p2, is the first identified eukaryotic 6mA methyltransferase (MTase) complex. Unlike the prokaryotic 6mA MTases which have been biochemically and structurally characterized, the operation mode of the MTA1 complex remains largely elusive. Here, we report the cryogenic electron microscopy structures of the quaternary MTA1 complex in S-adenosyl methionine (SAM)-bound (2.6 Å) and S-adenosyl homocysteine (SAH)-bound (2.8 Å) states. Using an AI-empowered integrative approach based on AlphaFold prediction and chemical cross-linking mass spectrometry, we further modeled a near-complete structure of the quaternary complex. Coupled with biochemical characterization, we revealed that MTA1 serves as the catalytic core, MTA1, MTA9, and p1 likely accommodate the substrate DNA, and p2 may facilitate the stabilization of MTA1. These results together offer insights into the molecular mechanism underpinning methylation by the MTA1 complex and the potential diversification of MTases for N6-adenine methylation.

Identification of vacuolar phosphate influx transporters in Brassica napus.

Recent progress has shown that vacuolar Pi transporters (VPTs) are important for cellular Pi homoeostasis in Arabidopsis thaliana and Oryza sativa under fluctuating external Pi supply, but the identity and involvement of VPTs in cellular Pi homoeostasis in Brassica napus is poorly understood. Here, we identified two vacuolar Pi influx transporters B. napus, BnA09PHT5;1b and BnCnPHT5;1b, and uncovered their necessity for cellular Pi homoeostasis through functional analysis. Both Brassica proteins are homologs of Arabidopsis AtPHT5;1 with a similar sequence, structure, tonoplast localization, and VPT activity. Brassica pht5;1b double mutants had smaller shoots and larger shoot cellular Pi concentrations than wild-type B. napus, which contrasts with a previous study of the Arabidopsis pht5;1 mutant, suggesting that PHT5;1-VPTs play different roles in cellular Pi homoeostasis in seedlings of B. napus and A. thaliana. Disruption of BnPHT5;1b genes also caused Pi toxicity in floral organs, reduced seed yield and impacted seed traits, consistent with the proposed role of AtPHT5;1 in floral Pi homoeostasis in Arabidopsis. Taken together, our studies identified two vacuolar Pi influx transporters in B. napus and revealed the distinct and conserved roles of BnPHT5;1bs in cellular Pi homoeostasis in this plant species.

Expression of sphingosine-1-phosphate receptor 2 is correlated with migration and invasion of human colon cancer cells: A preliminary clinical study.

Sphingosine-1-phosphate (S1P) is a bioactive phospholipid that serves as a potent mediator of cell proliferation, differentiation and apoptosis by binding to S1P receptors (S1PRs). S1P signalling is involved in the pathogenesis of numerous types of disease, including cancer. To the best of our knowledge, however, little is known about the expression patterns of S1PRs and their role in human colorectal cancer (CRC) cell migration and invasion. The aim of the present study was to investigate the role of S1P signalling in the metastasis of colon cancer cells and the expression of S1PRs in patients with CRC. The protein and mRNA expression levels of S1PRs and sphingosine kinases (SPHKs) in 55 patients with CRC were detected by western blotting (WB), immunohistochemical (IHC) analysis and reverse transcription-quantitative PCR. The levels of S1P in serum from patients and healthy individuals were quantified by ELISA. S1PRs antagonists JTE013, FTY720 and S1PR2-small interfering (si)RNA were used to determine the role of S1PR2 in human CRC LOVO and SW480 cell lines. Migration and invasion assays were performed for functional analysis. The levels of S1P in serum were significantly increased in patients with CRC compared with healthy individuals. The relative mRNA expression levels of S1PR2 were significantly downregulated in tumour compared with normal tissue, whereas S1PR1 and SPHK1 were upregulated. WB showed that 58% (32/55 cases) of patients presented downregulated S1PR2 protein expression. IHC analysis indicated that expression of S1PR2 was lower in tumour than in normal tissue in 65.5% (36/55 cases) of patients. Exogenous addition of S1P promoted migration and invasion in the different cell types. S1P stimulated the migration and invasion of SW480 cells. The inhibition of S1PR2 by JTE013 or S1PR2-siRNA significantly promoted the migration and invasion of SW480 cells, while FTY720 reversed these effects. The present study indicated that expression levels of S1PRs, particularly S1PR2, were associated with migration and invasion of CRC cells. The present findings revealed a novel mechanism by which S1P inhibited tumour cell migration and invasion via a S1PR2-dependent pathway, suggesting that S1PR2 may be a therapeutic target for treatment of colon cancer.

Transcriptomic profiling revealed key signaling pathways for cold tolerance and acclimation of two carp species.

BACKGROUND: Closely related species of the carp family (Cyprinidae) have evolved distinctive abilities to survive under cold stress, but molecular mechanisms underlying the generation of cold resistance remain largely unknown. In this study, we compared transcriptomic profiles of two carp species to identify key factors and pathways for cold tolerance and acclimation. RESULTS: Larvae of Songpu mirror carp and Barbless carp that were pretreated at 18 °C for 24 h significantly improved their survival rates under lethal cold temperature at 8 °C or 10 °C, indicating that two carp species possess the ability of cold acclimation. However, Songpu mirror carp exhibited stronger abilities of cold tolerance and acclimation than Barbless carp. Transcriptomic profiles of Songpu mirror carp and Barbless carp larvae at 28 °C and 18 °C were compared during cold acclimation through RNA-seq. Differentially expressed genes that are closely associated with the differences in cold acclimation between two carp species were identified through bioinformatics and Venn's diagram analysis. GO enrichment analysis of these genes indicated that cellular component assembly involved in morphogenesis, secondary alcohol metabolism and drug transport were the most up-regulated biological processes during cold acclimation of Songpu mirror carp. Conversely, positive regulation of macroautophagy, intracellular protein transport, and organonitrogen compound catabolism were the most down-regulated biological processes during cold acclimation of Barbless carp. KEGG enrichment analysis revealed that factors in the FoxO-related signaling pathways are mainly responsible for the development of differences in cold tolerance and acclimation between two carp species since altering the phosphorylation of key proteins in the FoxO-related signaling pathways with inhibitors or an activator significantly decreased the cold tolerance and acclimation of Songpu mirror carp. These data provided key clues for dissection of molecular mechanisms underlying the development of cold tolerance and acclimation in carps. CONCLUSIONS: These findings indicate that larvae of two carp species possess different abilities of cold tolerance and can build cold acclimation under mild low temperature. Multiple biological processes and FoxO-related signaling pathways are closely associated with the development of differences in cold tolerance and acclimation between two carp species.

Production of neoagarobiose from agar through a dual-enzyme and two-stage hydrolysis strategy.

The oligosaccharides from agar hydrolysis have special biological activities, and exhibit application prospects in cosmetic, food and pharmaceutical industry. In this study, two novel ß-agarases (AgaA and AgaB) were screened and characterized. It was found that the AgaA was an endo-type agarase which could efficiently hydrolyzed agar or agarose to form neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6), while the AgaB was an exo-type and bifunctional enzyme that showed activities towards both agarose and porphyran. Based on the properties of the two enzymes, we developed modular strategy for enzymatic production of neoagarobiose through a two-stage hydrolysis reaction. The cheap substrate agar was first liquefied by AgaA at high temperature to form neoagaroligosaccharides, which together with the sulfated polysaccharides were homogenized by AgaB to form neoagarobiose as the final product. High concentration of agar (10 g/L) was almost completely converted into neoagarobiose with high purity.

Efficient and risk-reduced genome editing using double nicks enhanced by bacterial recombination factors in multiple species.

Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.

Dynamic Phosphoproteome Profiling of Zebrafish Embryonic Fibroblasts during Cold Acclimation.

Temperature affects almost all aspects of the fish life. To cope with low temperature, fish have evolved the ability of cold acclimation for survival. However, intracellular signaling events underlying cold acclimation in fish remain largely unknown. Here, the formation of cold acclimation in zebrafish embryonic fibroblasts (ZF4) is monitored and the phosphorylation events during the process are investigated through a large-scale quantitative phosphoproteomic approach. In total, 11 474 phosphorylation sites are identified on 4066 proteins and quantified 5772 phosphosites on 2519 proteins. Serine, threonine, and tyrosine (Ser/Thr/Tyr) phosphorylation accounted for 85.5%, 13.3%, and 1.2% of total phosphosites, respectively. Among all phosphosites, 702 phosphosites on 510 proteins show differential regulation during cold acclimation of ZF4 cells. These phosphosites are divided into six clusters according to their dynamic changes during cold exposure. Kinase-substrate prediction reveals that mitogen-activated protein kinase (MAPK) among the kinase groups is predominantly responsible for phosphorylation of these phosphosites. The differentially regulated phosphoproteins are functionally associated with various cellular processes such as regulation of actin cytoskeleton and MAPK signaling pathway. These data enrich the database of protein phosphorylation sites in zebrafish and provide key clues for the elucidation of intracellular signaling networks during cold acclimation of fish.

The Characterization and Modification of a Novel Bifunctional and Robust Alginate Lyase Derived from Marinimicrobium sp. H1.

Alginase lyase is an important enzyme for the preparation of alginate oligosaccharides (AOS), that possess special biological activities and is widely used in various fields, such as medicine, food, and chemical industry. In this study, a novel bifunctional alginate lyase (AlgH) belonging to the PL7 family was screened and characterized. The AlgH exhibited the highest activity at 45 °C and pH 10.0, and was an alkaline enzyme that was stable at pH 6.0-10.0. The enzyme showed no significant dependence on metal ions, and exhibited unchanged activity at high concentration of NaCl. To determine the function of non-catalytic domains in the multi-domain enzyme, the recombinant AlgH-I containing only the catalysis domain and AlgH-II containing the catalysis domain and the carbohydrate binding module (CBM) domain were constructed and characterized. The results showed that the activity and thermostability of the reconstructed enzymes were significantly improved by deletion of the F5/8 type C domain. On the other hand, the substrate specificity and the mode of action of the reconstructed enzymes showed no change. Alginate could be completely degraded by the full-length and modified enzymes, and the main end-products were alginate disaccharide, trisaccharide, and tetrasaccharide. Due to the thermo and pH-stability, salt-tolerance, and bifunctionality, the modified alginate lyase was a robust enzyme which could be applied in industrial production of AOS.

GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2.

BACKGROUND & AIMS: Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. METHODS: B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. RESULTS: GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs. Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. CONCLUSIONS: GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.

Molecular characterization of the giant freshwater prawn (Macrobrachium rosenbergii) beta-actin gene promoter.

Constitutive promoters are important tools for gene function studies and transgenesis. The Beta-actin (actb1) gene promoter has been isolated from many species but remains to be cloned from the giant freshwater prawn (Macrobrachium rosenbergii). In this study, we cloned and characterized the Mractb1 gene promoter. Two alternative promoters were identified for the Mractb1 gene, which direct the generation of two transcripts with different 5' untranslated regions. Three CpG islands were predicted in the upstream sequence, which are intimately related to transcription initiation and promoter activity. In addition to the CCAAT-box and the CArG-box, molecular dissection of the flanking sequence revealed the existence of one negative and two positive elements in the upstream region and the first intron. Finally, the Mractb1 promoter demonstrated comparative activity to the carp (Cyprinus carpio) actb1 promoter. Our investigations provide a valuable genetic tool for gene function studies and shed light on the regulation of the Mractb1 gene.

AQP4­knockout alleviates the lipopolysaccharide­induced inflammatory response in astrocytes via SPHK1/MAPK/AKT signaling.

To date, aquaporin­4 (AQP4) has been considered as a critical contributor to neuroinflammation, but little is known about the underlying mechanism. Previous studies have shown that a critical enzyme involved in the sphingomyelin cycle, sphingosine kinase 1 (SPHK1), is implicated in inflammatory processes and contributes to chronic neuroinflammation. The present study investigated the role of AQP4 in proinflammatory cytokine release from astrocytes, with an emphasis on the SPHK1/mitogen­activated protein kinase (MAPK)/protein kinase B (AKT) pathway. Using primary cultures isolated from AQP4+/+ and AQP4­/­ embryos, the production of tumor necrosis factor­α (TNF­α)/interleukin­6 (IL­6) from astrocytes challenged by lipopolysaccharide (LPS) was compared. The results showed increased secretion of TNF­α/IL­6 in the two groups following LPS treatment, but a significantly lower level was observed in the AQP4­/­ group compared with that in the AQP4+/+ group. Although upregulation of SPHK1 was detected in the two genotypes, only a mild increase in SPHK1 was found in the AQP4­/­ genotype. The phosphorylation of MAPK/AKT was also confirmed to be attenuated in the AQP4­/­ group, suggesting decreased MAPK/AKT signaling over time in AQP4­/­ astrocytes. Overall, the study findings demonstrated that AQP4 deficiency alleviates proinflammatory cytokine release from astrocytes, in association with the SPHK1/MAPK/AKT pathway. This data improves our understanding of AQP4 in neuroinflammatory events, highlighting a novel profile of SPHK1 as a potential target for the treatment of CNS inflammation.

LRH-1 senses signaling from phosphatidylcholine to regulate the expansion growth of digestive organs via synergy with Wnt/ß-catenin signaling in zebrafish.

Liver receptor homolog-1 (LRH-1) is an orphan nuclear receptor that is critical for the growth and proliferation of cancer cells and other biological processes, including lipid transportation and metabolism, sexual determination and steroidogenesis. However, because homozygous lrh-1-/- mice die in utero, the regulatory mechanisms involved in embryonic development mediated by this receptor are poorly understood. In the present study, we performed transcription activator-like effector nuclease (TALEN)-mediated loss-of-function assays, taking advantage of zebrafish external fertilization, to investigate the function of lrh-1. The digestive organs were affected by lrh-1 depletion as a result of cell-cycle arrest (at the checkpoint of G1 to S phase), but not cell apoptosis. Biochemical analysis revealed that LRH-1 augments the transcriptional activity of ß-catenin 1 and 2 via physical interactions. Screening the specific ligand(s) sensed by LRH-1 during organogenesis revealed that phosphatidylcholine (PC), a potential ligand, is the upstream target of LRH-1 during endoderm development. These data provide evidence for the crosstalk between the PC/LRH-1 and Wnt/ß-catenin signaling pathways during the expansion growth of endoderm organs.

Transcriptional events co-regulated by hypoxia and cold stresses in Zebrafish larvae.

BACKGROUND: Hypoxia and temperature stress are two major adverse environmental conditions often encountered by fishes. The interaction between hypoxia and temperature stresses has been well documented and oxygen is considered to be the limiting factor for the thermal tolerance of fish. Although both high and low temperature stresses can impair the cardiovascular function and the cross-resistance between hypoxia and heat stress has been found, it is not clear whether hypoxia acclimation can protect fish from cold injury. RESULTS: Pre-acclimation of 96-hpf zebrafish larvae to mild hypoxia (5% O2) significantly improved their resistance to lethal hypoxia (2.5% O2) and increased the survival rate of zebrafish larvae after lethal cold (10°C) exposure. However, pre-acclimation of 96-hpf larvae to cold (18°C) decreased their tolerance to lethal hypoxia although their ability to endure lethal cold increased. RNA-seq analysis identified 132 up-regulated and 41 down-regulated genes upon mild hypoxia exposure. Gene ontology enrichment analyses revealed that genes up-regulated by hypoxia are primarily involved in oxygen transport, oxidation-reduction process, hemoglobin biosynthetic process, erythrocyte development and cellular iron ion homeostasis. Hypoxia-inhibited genes are enriched in inorganic anion transport, sodium ion transport, very long-chain fatty acid biosynthetic process and cytidine deamination. A comparison with the dataset of cold-regulated gene expression identified 23 genes co-induced by hypoxia and cold and these genes are mainly associated with oxidation-reduction process, oxygen transport, hemopoiesis, hemoglobin biosynthetic process and cellular iron ion homeostasis. The alleviation of lipid peroxidation damage by both cold- and hypoxia-acclimation upon lethal cold stress suggests the association of these genes with cold resistance. Furthermore, the alternative promoter of hmbsb gene specifically activated by hypoxia and cold was identified and confirmed. CONCLUSIONS: Acclimation responses to mild hypoxia and cold stress were found in zebrafish larvae and pre-acclimation to hypoxia significantly improved the tolerance of larvae to lethal cold stress. RNA-seq and bioinformatics analyses revealed the biological processes associated with hypoxia acclimation. Transcriptional events co-induced by hypoxia and cold may represent the molecular basis underlying the protection of hypoxia-acclimation against cold injury.

Transcriptomic characterization of cold acclimation in larval zebrafish.

BACKGROUND: Temperature is one of key environmental parameters that affect the whole life of fishes and an increasing number of studies have been directed towards understanding the mechanisms of cold acclimation in fish. However, the adaptation of larvae to cold stress and the cold-specific transcriptional alterations in fish larvae remain largely unknown. In this study, we characterized the development of cold-tolerance in zebrafish larvae and investigated the transcriptional profiles under cold stress using RNA-seq. RESULTS: Pre-exposure of 96 hpf zebrafish larvae to cold stress (16°C) for 24 h significantly increased their survival rates under severe cold stress (12°C). RNA-seq generated 272 million raw reads from six sequencing libraries and about 92% of the processed reads were mapped to the reference genome of zebrafish. Differential expression analysis identified 1,431 up- and 399 down-regulated genes. Gene ontology enrichment analysis of cold-induced genes revealed that RNA splicing, ribosome biogenesis and protein catabolic process were the most highly overrepresented biological processes. Spliceosome, proteasome, eukaryotic ribosome biogenesis and RNA transport were the most highly enriched pathways for genes up-regulated by cold stress. Moreover, alternative splicing of 197 genes and promoter switching of 64 genes were found to be regulated by cold stress. A shorter isoform of stk16 that lacks 67 amino acids at the N-terminus was specifically generated by skipping the second exon in cold-treated larvae. Alternative promoter usage was detected for per3 gene under cold stress, which leading to a highly up-regulated transcript encoding a truncated protein lacking the C-terminal domains. CONCLUSIONS: These findings indicate that zebrafish larvae possess the ability to build cold-tolerance under mild low temperature and transcriptional and post-transcriptional regulations are extensively involved in this acclimation process.