Project description:RNA-sequencing (RNA-Seq) protocols and bioinformatic pipelines are designed to streamline downstream analyses on sequences believed to be the most important. Here, we have challenged this dogma by preserving ribosomal RNA (rRNA) in our samples and by lowering the minimal RNA size window of our small RNA-Seq analyses to 8 nt
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Although long thought to act cell autonomously, mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. We have previously shown that mutant KRAS induces EGFR-ligand trafficking to exosomes and drastically alters exosomal protein contents, leading to activities that contribute to neoplastic growth. We have performed small library RNAseq analysis on cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status to determine whether mutant KRAS regulates the composition of secreted small RNAs. Exosomal small RNA profiles were distinct from cellular profiles, with principle component analysis showing clusters of mutant KRAS cell-derived exosomes distinct from wild type KRAS cell-derived exosomes. Secreted RNA species encompassed several different classes of small RNAs, including ribosomal and tRNA fragments, as well as mature miRNA sequences. miR-10b, was selectively increased in wild type KRAS-derived exosomes, whereas miR-100 was selectively increased in mutant KRAS-derived exosomes. Ceramide inhibition resulted in accumulation of miR-100 in mutant KRAS cells, suggesting KRAS-dependent miRNA export. In Transwell cell culture experiments, mutant, but not wild type, KRAS donor cells conferred miR-100-mediated target repression in wild type KRAS recipient cells miRNAseq deep sequencing for both cell and exosome mirnas of Dks-8, DLD-1, and DKO-1 cell lines. The DKs-8 line contains a wild type KRAS alleles, the DLD-1 line contains both wild type and mutant (G13D) KRAS alleles, and the DKO-1 line contains only a mutant KRAS allele.
Project description:We established a BCG-infected THP-1 cell model in vitro, obtained the total RNA of cell culture supernatant exosomes and total RNA of the infected group and the uninfected group, and performed miRNA sequencing analysis to find exosomes related to tuberculosis infection The body-derived miRNA is verified by cell lines and primary cells, and the diagnostic value of small sample clinical samples and preliminary functional research and exploration are explored for candidate molecules.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Although long thought to act cell autonomously, mutant KRAS colorectal cancer (CRC) cells release protein-laden exosomes that can alter the tumor microenvironment. We have previously shown that mutant KRAS induces EGFR-ligand trafficking to exosomes and drastically alters exosomal protein contents, leading to activities that contribute to neoplastic growth. We have performed small library RNAseq analysis on cells and matched exosomes from isogenic CRC cell lines differing only in KRAS status to determine whether mutant KRAS regulates the composition of secreted small RNAs. Exosomal small RNA profiles were distinct from cellular profiles, with principle component analysis showing clusters of mutant KRAS cell-derived exosomes distinct from wild type KRAS cell-derived exosomes. Secreted RNA species encompassed several different classes of small RNAs, including ribosomal and tRNA fragments, as well as mature miRNA sequences. miR-10b, was selectively increased in wild type KRAS-derived exosomes, whereas miR-100 was selectively increased in mutant KRAS-derived exosomes. Ceramide inhibition resulted in accumulation of miR-100 in mutant KRAS cells, suggesting KRAS-dependent miRNA export. In Transwell cell culture experiments, mutant, but not wild type, KRAS donor cells conferred miR-100-mediated target repression in wild type KRAS recipient cells
Project description:As the evolution of miRNA genes has been found to be one of the important factors in formation of the modern type of man, we performed a comparative analysis of the evolution of miRNA genes in two archaic hominines, Homo sapiens neanderthalensis and Homo sapiens denisova, and elucidated the expression of their target mRNAs in bain.A comparative analysis of the genomes of primates, including species in the genus Homo, identified a group of miRNA genes having fixed substitutions with important implications for the evolution of Homo sapiens neanderthalensis and Homo sapiens denisova. The mRNAs targeted by miRNAs with mutations specific for Homo sapiens denisova exhibited enhanced expression during postnatal brain development in modern humans. By contrast, the expression of mRNAs targeted by miRNAs bearing variations specific for Homo sapiens neanderthalensis was shown to be enhanced in prenatal brain development.Our results highlight the importance of changes in miRNA gene sequences in the course of Homo sapiens denisova and Homo sapiens neanderthalensis evolution. The genetic alterations of miRNAs regulating the spatiotemporal expression of multiple genes in the prenatal and postnatal brain may contribute to the progressive evolution of brain function, which is consistent with the observations of fine technical and typological properties of tools and decorative items reported from archaeological Denisovan sites. The data also suggest that differential spatial-temporal regulation of gene products promoted by the subspecies-specific mutations in the miRNA genes might have occurred in the brains of Homo sapiens denisova and Homo sapiens neanderthalensis, potentially contributing to the cultural differences between these two archaic hominines.