Discovery of Novel MicroRNAs in Rat Kidney Using Next Generation Sequencing and Microarray Validation

MicroRNAs (miRNAs) are small non-coding RNAs that regulate a variety of biological processes. The latest version of the miRBase database (Release 18) includes 1,157 mouse and 680 rat mature miRNAs. Only one new rat mature miRNA was added to the rat miRNA database from version 16 to version 18 of miRBase, suggesting that many rat miRNAs remain to be discovered. Given the importance of rat as a model organism, discovery of the completed set of rat miRNAs is necessary for understanding rat miRNA regulation. In this study, next generation sequencing (NGS), microarray analysis and bioinformatics technologies were applied to discover novel miRNAs in rat kidneys. MiRanalyzer was utilized to analyze the sequences of the small RNAs generated from NGS analysis of rat kidney samples. Hundreds of novel miRNA candidates were examined according to the mappings of their reads to the rat genome, presence of sequences that can form a miRNA hairpin structure around the mapped locations, Dicer cleavage patterns, and the levels of their expression determined by both NGS and microarray analyses. Nine novel rat hairpin precursor miRNAs (pre-miRNA) were discovered with high confidence. Five of the novel pre-miRNAs are also reported in other species while four of them are rat specific. In summary, 9 novel pre-miRNAs (14 novel mature miRNAs) were identified via combination of NGS, microarray and bioinformatics high-throughput technologies

Conscious uncoupling between FANCI and FANCD2 in DNA repair

The Fanconi anemia (FA)-BRCA pathway mediates repair of DNA interstrand crosslinks. The FA core complex, a multi-subunit ubiquitin ligase, participates in the detection of DNA lesions and monoubiquitinates two downstream FA proteins, FANCD2 and FANCI (or the ID complex). However, the regulation of the FA core complex itself is poorly understood. Here we show that the FA core complex proteins are recruited to sites of DNA damage and form nuclear foci in S and G2 phases of the cell cycle. ATR kinase activity, an intact FA core complex and FANCM-FAAP24 were crucial for this recruitment. Surprisingly, FANCI, but not its partner FANCD2, was needed for efficient FA core complex foci formation. Monoubiquitination or ATR-dependent phosphorylation of FANCI were not required for the FA core complex recruitment, but FANCI deubiquitination by USP1 was. Additionally, BRCA1 was required for efficient FA core complex foci formation. These findings indicate that FANCI functions upstream of FA core complex recruitment independently of FANCD2, and alter the current view of the FA-BRCA pathway

Expression of a B-cell-restricted isoform of CD45 is associated with maturity in rat serosal and connective-tissue mast cells

A number of technical limitations make differential staining with dyes such as Alcian blue and Safranin O unsatisfactory as the primary method for assessing the maturity of connective tissue mast cells in rodents. We demonstrated for the first time that in the steady state, most serosal mast cells in normal rats express a high-molecular-weight isoform of CD45 that reacts with monoclonal antibody OX33 (OX33+) and has been reported previously only on B cells. However, a small proportion did not express this isoform (OX33−) and this subset predominated during the early stages of mast cell regeneration in the peritoneal cavity. Furthermore, there was a progressive increase in the proportion of donor-derived OX33+ cells after intraperitoneal adoptive transfer of purified OX33− mast cells to CD45 congenic recipients. These results indicate that serosal mast cells up-regulate expression of the OX33-reactive CD45 isoform(s) as they mature. Interestingly, acquisition of OX33-reactive CD45 did not correlate temporally with sulphation of glycosaminglycan in the mast cell granules. Expression of this isoform was used also to assess the maturity of connective tissue mast cells during mastocytosis in synovium associated with T-cell-mediated experimental polyarthritis. Together, our results demonstrate that OX33-reactive CD45 is a marker that can be used to assess the maturity of serosal and connective tissue mast cells during normal homeostasis and during pathological processes. The significance of differential expression of CD45 isoforms may be to regulate the sensitivity of maturing mast cells to the actions of growth factors and activating stimuli

Endogenous antigen tunes the responsiveness of naive B cells but not T cells

In humans up to 75% of newly generated B cells and about 30% of mature B cells exhibit some degree of autoreactivity(1). Yet, how B cells establish and maintain tolerance in the face of autoantigen exposure during and after development is not certain. Studies of model BCR transgenic systems have highlighted the critical role played by functional unresponsiveness or ‘anergy’(2,3). Unlike T cells, evidence suggests that receptor editing and anergy, rather than deletion, account for much of B cell tolerance(4,5). However, it remains unclear whether the mature diverse B cell repertoire of mice contains anergic autoreactive B cells, and if so, whether antigen was encountered during or after their development. By taking advantage of a reporter mouse in which B cell antigen receptor (BCR) signaling rapidly and robustly induces GFP expression under the control of the Nur77 regulatory region, antigen-dependent and – independent BCR signaling events in vivo during B cell maturation were visualized. Here we show that B cells encounter antigen during development in the spleen, and that this antigen exposure in turn tunes the responsiveness of BCR signaling in B cells at least partly by down-modulating expression of surface IgM but not IgD BCRs, and by modifying basal calcium levels. By contrast, no analogous process occurs in naive mature T cells. Our data demonstrate not only that autoreactive B cells persist in the mature repertoire, but that functional unresponsiveness or ‘anergy’ exists in the mature B cell repertoire along a continuum, a fact that has long been suspected, but never yet shown. These results have important implications for understanding how tolerance in T and B cells is differently imposed, and how these processes might go awry in disease