MicroRNA profiles of fibroblasts derived from in vivo fertilized and fat-1 transgenic cattle.

Fat-1 transgenic cattle have high levels of ω-3 fatty acids, which regulate several genes in fatty acid metabolism. In the current study, fibroblasts derived from in vivo fertilized (Ferti) and fat-1 transgenic (TG) Luxi cattle (Bos taurus), a local breed in China, were cultured and their miRNA expression was characterized. Expression of 352 known miRNAs differed in cells from Ferti and TG cattle: 83 miRNAs were found to be specifically expressed in cells from Ferti cattle while 23 miRNAs were found to be specifically expressed in cells from TG cattle. Novel differences in miRNA expression were also found in cells from Ferti and TG cattle. The identity of seven differentially expressed miRNAs was verified using quantitative real-time PCR, and target genes were identified computationally. GO and KEGG analysis revealed that these miRNAs were involved in seven major biological pathways, including metabolism, MAPK signaling, calcium signaling, purine metabolism, ubiquitin mediated proteolysis, pyrimidine metabolism, and the cell cycle. Overexpression of one of these miRNAs, miR-21-5p, was found to suppress expression of fibroblast growth factor 10 (FGF10) and adipose triglyceride lipase (ATGL) in fibroblasts from TG cattle and 3T3-L1 pre-adipocytes. Conversely, knockdown of miR-21-5p stimulated expression. Together, these results suggest that miRNAs potentially play a role in expression of lipogenic and lipolytic genes as well as in synthesis of ω-3 fatty acids facilitated by fat-1.

MicroRNA Profiles in Spontaneous Decidualized Menstrual Endometrium and Early Pregnancy Decidua with Successfully Implanted Embryos.

To comparatively analyze the human microRNA (miRNA) profiles between spontaneous decidualized menstrual endometrium and early pregnancy decidua by an in-depth sequencing of miRNAs. The specific miRNAs expressed at conception might be involved in pregnancy establishment and expression of let-7f-5p and let-7g-5p was experimentally up-regulated or inhibited to assess the effect on the expression of IGF2BP-1 and IGF2R in vitro, respectively. Samples of endometria and deciduas were obtained from 25 women who suffered from tubal or male factor subfertility and from 35 early pregnant women who underwent pregnancy termination at 6-8 weeks gestation were irrespectively collected and comparatively analyzed by miRNA sequencing and differential expression of known and novel miRNAs was analyzed using bioinformatics. The 2042 miRNA expression was analyzed in the study and the differential expression of six miRNAs was validated by qRT-PCR. The expression of four miRNAs in decidua samples was down-regulated (miR-34c, miR-92a, miR-181a-5p, and miR-191), whereas the expression of miR-10a-5p and let-7f-5p was significantly up-regulated. The expression of IGF2BP-1 and IGF2R declined and increased with overexpression and inhibition of let-7f-5p and let-7g-5p, respectively. Changes in the expression of particular miRNAs might play a role in the physiology of decidualization following successful embryo implantation, ultimately resulting in continuous decidualization.

MicroRNAome in decidua: a new approach to assess the maintenance of pregnancy.

OBJECTIVE: To comparatively analyze the human microRNAomes between normal pregnant and miscarriage deciduas by an in-depth sequencing of microRNA (miRNA); and to specifically examine miRNA-199b-5p and serum/glucocorticoid regulated kinase 1 (SGK1) in vivo and in vitro for their possible roles in pregnancy maintenance. DESIGN: Samples of deciduas from 6-8-week spontaneous miscarriages and normal pregnant women were irrespectively collected and comparatively analyzed by miRNA sequencing. The miR-199b-5p and SGK1 expressions were validated in vivo and in vitro. SETTING: University research and clinical institutes. PATIENT(S): In this experimental study, samples of deciduas were obtained from October 2011 to April 2012 from 29 women with spontaneous miscarriages and 35 normal pregnant women (control group) who underwent pregnancy termination at 6-8 weeks at our university gynecology unit. INTERVENTION(S): Endometrial biopsies, cell transfection, and production of an miR-199b-5p transgenic mouse model. MAIN OUTCOME MEASURE(S): In-depth sequencing of the miRNAome on human deciduas was performed for statistically significant differences in miRNA expression. Expression levels of SGK1 were detected by quantitative polymerase chain reaction and immunoblotting (Western blot) in vitro while miR-199b-5p is overexpressed or knockdown in miR-199b-5p transgenic mice. RESULT(S): Expression of the 1,921 known miRNAs was analyzed in the study. In aborted deciduas, 0.57% of the miRNAs were expressed abundantly (>10,000 transcripts per million) and represented 86.38% of all the miRNA reads. Six miRNAs were down-regulated (let-7a-5p, let-7f-5p, let-7g-5p, let-7e-5p, let-7d-5p, and miR-98), whereas miR-199b-5p was significantly up-regulated. Overexpression or knockdown of miR-199b-5p in HEK293T and Ishikawa cells decreased or increased SGK1 expression. Furthermore, overexpression of miR-199b-5p in human endometrial stromal cells or in transgenic mouse decreased SGK1 expression at the mRNA and protein levels, respectively. CONCLUSION(S): Among the miRNAomes, the abundant expression of the let-7 members was decreased in aborted samples, whereas miR-199b-5p expression was consistently increased. A significant inverse correlation was found between miR-199b-5p and SGK1 in vivo and in vitro.

Structure-function analysis of Plasmodium RNA triphosphatase and description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphatases and CYTH proteins.

RNA triphosphatase catalyzes the first step in mRNA capping. The RNA triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA triphosphatase as an anti-infective target. Fungal and protozoan RNA triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic beta-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name "triphosphate tunnel metalloenzyme," is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 5'-end processing.

Mechanism of nonhomologous end-joining in mycobacteria: a low-fidelity repair system driven by Ku, ligase D and ligase C.

DNA double-strand breaks (DSBs) can be repaired either via homologous recombination (HR) or nonhomologous end-joining (NHEJ). Both pathways are operative in eukaryotes, but bacteria had been thought to rely on HR alone. Here we provide direct evidence that mycobacteria have a robust NHEJ pathway that requires Ku and a specialized polyfunctional ATP-dependent DNA ligase (LigD). NHEJ of blunt-end and complementary 5'-overhang DSBs is highly mutagenic ( approximately 50% error rate). Analysis of the recombination junctions ensuing from individual NHEJ events highlighted the participation of several DNA end-remodeling activities, including template-dependent fill-in of 5' overhangs, nontemplated addition of single nucleotides at blunt ends, and nucleolytic resection. LigD itself has the template-dependent and template-independent polymerase functions in vitro that compose the molecular signatures of NHEJ in vivo. Another ATP-dependent DNA ligase (LigC) provides a backup mechanism for LigD-independent error-prone repair of blunt-end DSBs. We speculate that NHEJ allows mycobacteria to evade genotoxic host defense.

Biochemical and genetic analysis of the four DNA ligases of mycobacteria.

Mycobacterium tuberculosis encodes an NAD(+)-dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD(+) and ATP, respectively, whereas LigC and LigD, which have ATP-specific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA-adenylate intermediate. All four of the mycobacterial ligases are monomeric enzymes. LigA has a low K(m) for NAD(+) (1 microm) and is sensitive to a recently described pyridochromanone inhibitor of NAD(+)-dependent ligases. LigA is able to sustain growth of Saccharomyces cerevisiae in lieu of the essential yeast ligase Cdc9, but LigB, LigC, and LigD are not. LigB is distinguished by its relatively high K(m) for ATP (0.34 mm) and its dependence on a distinctive N-terminal domain for nick joining. None of the three ATP-dependent ligases are essential for mycobacterial growth. M. tuberculosis ligDDelta cells are defective in nonhomologous DNA end joining.

Structure-function analysis of Trypanosoma brucei RNA triphosphatase and evidence for a two-metal mechanism.

Trypanosoma brucei RNA triphosphatase TbCet1 is a 252-amino acid polypeptide that catalyzes the first step in mRNA cap formation. By performing an alanine scan of TbCet1, we identified six amino acids that are essential for triphosphatase activity (Glu-52, Arg-127, Glu-168, Arg-186, Glu-216, and Glu-218). These results consolidate the proposal that protozoan, fungal, and Chlorella virus RNA triphosphatases belong to a single family of metal-dependent NTP phosphohydrolases with a unique tunnel active site composed of eight beta strands. Limited proteolysis of TbCet1 suggests that the hydrophilic N terminus is surface-exposed, whereas the catalytic core domain is tightly folded with the exception of a protease-sensitive loop (76WKGRRARKT84) between two of the putative tunnel strands. The catalytic domain of TbCet1 is extraordinarily thermostable. It remains active after heating for 2 h at 75 degrees C. Analysis by zonal velocity sedimentation indicates that TbCet1 is a monomeric enzyme, unlike fungal RNA triphosphatases, which are homodimers. We show that tripolyphosphate is a potent competitive inhibitor of TbCet1 (Ki 1.4 microm) that binds more avidly to the active site than the ATP substrate (Km 25 microm). We present evidence of synergistic activation of the TbCet1 triphosphatase by manganese and magnesium, consistent with a two-metal mechanism of catalysis. Our findings provide new insight to the similarities (in active site tertiary structure and catalytic mechanism) and differences (in quaternary structure and thermal stability) among the different branches of the tunnel enzyme family.

Mapping the active site of vaccinia virus RNA triphosphatase.

The RNA triphosphatase component of vaccinia virus mRNA capping enzyme (the product of the viral D1 gene) belongs to a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, Chlorella virus, and baculoviruses. The family is defined by two glutamate-containing motifs (A and C) that form the metal-binding site. Most of the family members resemble the fungal and Chlorella virus enzymes, which have a complex active site located within the hydrophilic interior of a topologically closed eight-stranded beta barrel (the so-called "triphosphate tunnel"). Here we queried whether vaccinia virus capping enzyme is a member of the tunnel subfamily, via mutational mapping of amino acids required for vaccinia triphosphatase activity. We identified four new essential side chains in vaccinia D1 via alanine scanning and illuminated structure-activity relationships by conservative substitutions. Our results, together with previous mutational data, highlight a constellation of six acidic and three basic amino acids that likely compose the vaccinia triphosphatase active site (Glu37, Glu39, Arg77, Lys107, Glu126, Asp159, Lys161, Glu192, and Glu194). These nine essential residues are conserved in all vertebrate and invertebrate poxvirus RNA capping enzymes. We discerned no pattern of clustering of the catalytic residues of the poxvirus triphosphatase that would suggest structural similarity to the tunnel proteins (exclusive of motifs A and C). We infer that the poxvirus triphosphatases are a distinct lineage within the metal-dependent RNA triphosphatase family. Their unique active site, which is completely different from that of the host cell's capping enzyme, recommends the poxvirus RNA triphosphatase as a molecular target for antipoxviral drug discovery.

Chlorella virus RNA triphosphatase. Mutational analysis and mechanism of inhibition by tripolyphosphate.

Chlorella virus RNA triphosphatase (cvRtp1) is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi, protozoa, poxviruses, and baculoviruses. The primary structure of cvRtp1 is more similar to that of the yeast RNA triphosphatase Cet1 than it is to the RNA triphosphatases of other DNA viruses. To evaluate the higher order structural similarities between cvRtp1 and the fungal enzymes, we performed an alanine scan of individual residues of cvRtp1 that were predicted, on the basis of the crystal structure of Cet1, to be located at or near the active site. Twelve residues (Glu(24), Glu(26), Asp(64), Arg(76), Lys(90), Glu(112), Arg(127), Lys(129), Arg(131), Asp(142), Glu(163), and Glu(165)) were deemed essential for catalysis by cvRtp1, insofar as their replacement by alanine reduced phosphohydrolase activity to <5% of the wild-type value. Structure-activity relationships were elucidated by introducing conservative substitutions at the essential positions. The mutational results suggest that the active site of cvRtp1 is likely to adopt a tunnel fold like that of Cet1 and that a similar constellation of side chains within the tunnel is responsible for metal binding and reaction chemistry. Nonetheless, there are several discordant mutational effects in cvRtp1 versus Cet1, which suggest that different members of the phosphohydrolase family vary in their reliance on certain residues within the active site tunnel. We found that tripolyphosphate and pyrophosphate were potent competitive inhibitors of cvRtp1 (K(i) = 0.6 microm tripolyphosphate and 2.4 microm pyrophosphate, respectively), whereas phosphate had little effect. cvRtp1 displayed a weak intrinsic tripolyphosphatase activity (3% of its ATPase activity) but was unable to hydrolyze pyrophosphate.