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In eukaryotic cells, both alternative splicing and alternative polyadenylation (APA) play essential roles in the gene regulation network. U1 small ribonucleoprotein particle (U1 snRNP) is a major component of spliceosome, and U1 snRNP complex can suppress proximal APA sites through crosstalking with 3′ end processing factors. However, here we show that both knockdown and overexpression of SNRPA, SNRPC, SNRNP70, and SNRPD2, the U1 snRNP proteins, promote the usage of proximal APA sites at the transcriptome level. SNRNP70 can drive the phase transition of PABPN1 from droplet to aggregate, which may reduce the repressive effects of PABPN1 on the proximal APA sites. Additionally, SNRNP70 can also promote the proximal APA sites by recruiting CPSF6, suggesting that the function of CPSF6 on APA is related with other RNA-binding proteins and cell context-dependent. Consequently, these results reveal that, on the contrary to U1 snRNP complex, the free proteins of U1 snRNP complex can promote proximal APA sites through the interaction with 3′ end processing machinery. [ABSTRACT FROM AUTHOR]

Fruit flesh cell vacuoles play a pivotal role in fruit growth and quality formation. In the present study, intact vacuoles were carefully released and collected from protoplasts isolated from flesh cells at five sampling times along fig fruit development. Label-free quantification and vacuole proteomic analysis identified 1,251 proteins, 1,137 of which were recruited as differentially abundant proteins (DAPs) by fold change ≥ 1.5, P  < 0.05. DAPs were assigned to 10 functional categories; among them, 238, 186, 109, 93 and 90 were annotated as metabolism, transport proteins, membrane fusion or vesicle trafficking, protein fate and stress response proteins, respectively. Decreased numbers of DAPs were uncovered along fruit development. The overall changing pattern of DAPs revealed two major proteome landscape conversions in fig flesh cell vacuoles: the first occurred when fruit developed from late-stage I to mid-stage II, and the second occurred when the fruit started ripening. Metabolic proteins related to glycosidase, lipid and extracellular proteins contributing to carbohydrate storage and vacuole expansion, and protein-degrading proteins determining vacuolar lytic function were revealed. Key tonoplast proteins contributing to vacuole expansion, cell growth and fruit quality formation were also identified. The revealed comprehensive changes in the vacuole proteome during flesh development were compared with our previously published vacuole proteome of grape berry. The information expands our knowledge of the vacuolar proteome and the protein basis of vacuole functional evolution during fruit development and quality formation. [ABSTRACT FROM AUTHOR]

The discovery of ProtoRAG in amphioxus indicated that vertebrate RAG recombinases originated from an ancient transposon. However, the sequences of ProtoRAG terminal inverted repeats (TIRs) were obviously dissimilar to the consensus sequence of mouse 12/23RSS and recombination mediated by ProtoRAG or RAG made them incompatible with each other. Thus, it is difficult to determine whether or how 12/23RSS persisted in the vertebrate RAG system that evolved from the TIRs of ancient RAG transposons. Here, we found that the activity of ProtoRAG is highly dependent on its asymmetric 5′TIR and 3′TIR, which are composed of conserved TR1 and TR5 elements and a partially conserved TRsp element of 27/31 bp to separate them. Similar to the requirements for the recombination signal sequences (RSSs) of RAG recombinase, the first CAC in TR1, the three dinucleotides in TR5 and the specific length of the partially conserved TRsp are important for the efficient recombination activity of ProtoRAG. In addition, the homologous sequences flanking the signal sequences facilitate ProtoRAG- but not RAG-mediated recombination. In addition to the diverged TIRs, two differentiated functional domains in BbRAG1L were defined to coordinate with the divergence between TIRs and RSSs. One of these is the CTT* domain, which facilitates the specific TIR recognition of the BbRAGL complex, and the other is NBD*, which is responsible for DNA binding and the protein stabilization of the BbRAGL complex. Thus, our findings reveal that the functional requirement for ProtoRAG TIRs is similar to that for RSS in RAG-mediated recombination, which not only supports the common origin of ProtoRAG TIRs and RSSs from the asymmetric TIRs of ancient RAG transposons, but also reveals the development of RAG and RAG-like machineries during chordate evolution. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) encounter various types of stress during industrial processes and gastrointestinal transit. Catalase (CAT) and bile salt hydrolase (BSH) can protect bacteria from oxidative stress or damage caused by bile salts by decomposing hydrogen peroxide (HO) or deconjugating the bile salts, respectively. Lactobacillus casei is a valuable probiotic strain and is often deficient in both CAT and BSH. In order to improve the resistance of L. casei to both oxidative and bile salts stress, the catalase gene katA from L. sakei and the bile salt hydrolase gene bsh1 from L. plantarum were coexpressed in L. casei HX01. The enzyme activities of CAT and BSH were 2.41 μmol HO/min/10 colony-forming units (CFU) and 2.11 μmol glycine/min/ml in the recombinant L. casei CB, respectively. After incubation with 8 mM HO, survival ratio of L. casei CB was 40-fold higher than that of L. casei CK. Treatment of L. casei CB with various concentrations of sodium glycodeoxycholate (GDCA) showed that ~10 CFU/ml cells survived after incubation with 0.5% GDCA, whereas almost all the L. casei CK cells were killed when treaded with 0.4% GDCA. These results indicate that the coexpression of CAT and BSH confers high-level resistance to both oxidative and bile salts stress conditions in L. casei HX01. [ABSTRACT FROM AUTHOR]

Deep-sea hydrothermal vent chimneys harbor a high diversity of largely unknown microorganisms. Although the phylogenetic diversity of these microorganisms has been described previously, the adaptation and metabolic potential of the microbial communities is only beginning to be revealed. A pyrosequencing approach was used to directly obtain sequences from a fosmid library constructed from a black smoker chimney 4143-1 in the Mothra hydrothermal vent field at the Juan de Fuca Ridge. A total of 308 034 reads with an average sequence length of 227 bp were generated. Comparative genomic analyses of metagenomes from a variety of environments by two-way clustering of samples and functional gene categories demonstrated that the 4143-1 metagenome clustered most closely with that from a carbonate chimney from Lost City. Both are highly enriched in genes for mismatch repair and homologous recombination, suggesting that the microbial communities have evolved extensive DNA repair systems to cope with the extreme conditions that have potential deleterious effects on the genomes. As previously reported for the Lost City microbiome, the metagenome of chimney 4143-1 exhibited a high proportion of transposases, implying that horizontal gene transfer may be a common occurrence in the deep-sea vent chimney biosphere. In addition, genes for chemotaxis and flagellar assembly were highly enriched in the chimney metagenomes, reflecting the adaptation of the organisms to the highly dynamic conditions present within the chimney walls. Reconstruction of the metabolic pathways revealed that the microbial community in the wall of chimney 4143-1 was mainly fueled by sulfur oxidation, putatively coupled to nitrate reduction to perform inorganic carbon fixation through the Calvin-Benson-Bassham cycle. On the basis of the genomic organization of the key genes of the carbon fixation and sulfur oxidation pathways contained in the large genomic fragments, both obligate and facultative autotrophs appear to be present and contribute to biomass production. [ABSTRACT FROM AUTHOR]

TCR signaling is mediated by intracellular signaling molecules and nuclear transcription factors, which are tightly regulated by interaction with regulatory proteins such as Grb2 and SLAP. We reported recently that TCR stimulation induces the expression of cytokine‐induced SH2 protein (CIS). The expression of CIS promotes TCR‐mediated activation. We have now found specific interactions between CIS and activated protein kinase C (PKC) α, β and ϑ in TCR‐stimulated T cells. CIS was shown by in vitro kinase assay to associate with activated PKC. In CIS‐expressing T cells isolated from CIS‐transgenic mice, the amount of activated PKC associated with CIS was found to increase following TCR stimulation. By immunohistochemical analysis, CIS was also found to co‐localize with PKCϑ at the plasma membrane of activated T cells. In addition to the interaction and intracellular co‐localization of the CIS and PKC, an increase in the activation of AP‐1 and NF‐κB was noted in CIS‐expressing T cells, after stimulation by either anti‐CD3/CD28 or phorbol myristate acetate + ionomycin. These results suggest that CIS regulates PKC activation, and that this may be important for the activation of both the AP‐1 and NF‐κB pathways in TCR signaling. [ABSTRACT FROM PUBLISHER]

In this study we have compared the interaction of human MHC class I molecules with IgG heavy chain (HC) binding protein (BiP), calnexin, calreticulin, tapasin and TAP in β 2 -microglobulin (β 2 m)- or TAP-deficient cells, as well as in wild-type B-LCL cells. Distinct differences between the association of HC and these endoplasmic reticulum (ER) proteins were found in the three cell lines. In the absence of β 2 m (Daudi cells), HC associated with both BiP and calnexin. A prominent portion of HC was complexed simultaneously to both chaperones, as indicated by co-precipitation with either anti-calnexin or anti-class I antisera. In the presence of β 2 m, but absence of TAP (T2 cells), HC could be co-precipitated with calnexin, whereas no detectable interaction with BiP could be demonstrated. This suggests that calnexin interacts with HC at a later stage than BiP. In B-LCL cells, HC–β 2 m associated with calreticulin and tapasin, whereas no interaction with calnexin and BiP was observed. In the absence of β 2 m, HC were rapidly degraded in the ER, while the ER retained HC were stabilized in the presence of β 2 m, even in the absence of TAP. The dissociation of class I molecules from TAP in B-LCL cells correlated with the kinetics of appearance of class I molecules on the cell surface, suggesting that TAP retains peptide-free class I molecules in the ER. Taken together, our results suggest the model that BiP and calnexin sequentially control the folding of MHC class I, before MHC class I molecules associate with the loading complex. [ABSTRACT FROM PUBLISHER]

The assembly of MHC class I molecules is regulated by a multi-protein complex in the endoplasmic reticules (ER) termed the loading complex. Tapasin is suggested to be one of the molecules forming this complex on the basis of its interaction with both the transporter associated with antigen processing (TAP) and MHC class I molecules. To address whether TAP is indispensable for the processing of the assembly of tapasin-associated MHC class I molecules, we studied the association of MHC class I molecules with tapasin, the assembly of tapasin-associated MHC class I with peptides and the peptide-mediated dissociation of MHC class I from tapasin in TAP-mutant T2 cells. In the absence of TAP, MHC class I heavy chain and β2-microglobulin dimers were found to be properly associated with tapasin. The stable MHC class I dimer was required for its association with tapasin in the ER. In the absence of TAP, tapasin retained MHC class I molecules much longer in the ER than in the presence of TAP. This low off-rate of MHC class I from tapasin was due to the absence of high-affinity peptides in the ER of TAP-mutant cells but not to the absence of TAP per se. The introduction of peptides into permeabilized microsomes of TAP-mutant cells led to effective loading of the peptides onto tapasin-associated MHC class I and to the subsequent dissociation of MHC class I from tapasin. These results demonstrate that regulation of the assembly of tapasin-associated MHC class I is independent of the interaction of tapasin with TAP, but is dependent upon the peptides transported by TAP. [ABSTRACT FROM PUBLISHER]

Lancelet (amphioxus) represents the most basally divergent extant chordate (cephalochordates) that diverged from the other two chordate lineages (urochordates and vertebrates) more than half a billion years ago. As it occupies a key position in evolution, it is considered as one of the best proxies for understanding the chordate ancestral state. Thus, the construction of a database with multiple lancelet genomes and gene annotation data, including protein domains, is urgently needed to investigate the loss and gain of domains in orthologues among species, especially ancient domain types (non-vertebrate-specific domains) and novel domain combination, which is helpful for providing new insight into the chordate ancestral state and vertebrate evolution. Here, we present an integrated genome database for lancelet, LanceletDB, which provides reference haploid genome sequence and annotation data for lancelet (Branchiostoma belcheri), including gene models and annotation, protein domain types, gene expression pattern in embryogenesis, different expression sequence tag sets and alternative polyadenylation (APA) sites profiled by the sequencing APA sites method. Especially, LanceletDB allows comparison of domain types and combination in orthologues among type species so as to decode the ancient domain types and novel domain combination during evolution. We also integrated the released diploid lancelet genome annotation data (Branchiostoma floridae) to expand LanceletDB and extend its usefulness. These data are available through the search and analysis page, basic local alignment search tool page and genome browser to provide an integrated display. [ABSTRACT FROM AUTHOR]