Project description:PolyA, Position, Profiling (3P-seq) for mouse tissues and cell lines There is no biological replicates. For six mouse tissues, 3P-seq libraries were constructed in wild-type and miR-22 knockout mouse.
Project description:The recent reports of two circular RNAs (circRNAs) with strong potential to act as microRNA (miRNA) sponges suggest that circRNAs might play important roles in regulating gene expression. However, the global properties of circRNAs are not well understood. We developed a computational pipeline to identify circRNAs and quantify their relative abundance from RNA-seq data. Applying this pipeline to a large set of non-poly(A)-selected RNA-seq data from the ENCODE project, we annotated 7,112 human circRNAs that were estimated to comprise at least 10% of the transcripts accumulating from their loci. Most circRNAs are expressed in only a few cell types and at low abundance, but they are no more cell-type–specific than are mRNAs with similar overall expression levels. Although most circRNAs overlap protein-coding sequences, ribosome profiling provides no evidence for their translation. We also annotated 635 mouse circRNAs, and although 20% of them are orthologous to human circRNAs, the sequence conservation of these circRNA orthologs is no higher than that of their flanking linear exons. The previously proposed miR-7 sponge, CDR1as, is one of only two circRNAs with more miRNA sites than expected by chance, with the next best miRNA-sponge candidate deriving from a primate-specific zinc-finger gene, ZNF91. These results provide a new framework for future investigation of this intriguing topological isoform while raising doubts regarding a biological function of most circRNAs. Examination of 9 samples in 1 cell type Note: The ENCODE data we used are under GEO SuperSeries GSE26284 (all samples labeled "_cell_total"). But they were not used in the processing of the U2OS data.
Project description:Thousands of large intervening non-coding RNAs (lincRNAs) have been identified in mammals. To better understand the evolution and functions of these enigmatic RNAs, we used chromatin marks, poly(A)-site mapping and RNA-Seq data, to identify more than 550 distinct lincRNAs in zebrafish. Although these shared many characteristics with mammalian lincRNAs, only 29 had detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Other lincRNAs had conserved genomic locations without detectable sequence conservation. Antisense reagents targeting conserved regions of two zebrafish lincRNAs caused developmental defects. Reagents targeting splice sites caused the same defects and were rescued by adding either the mature lincRNA or its human or mouse ortholog. Our study provides a roadmap for identification and analysis of lincRNAs in model organisms and shows that lincRNAs play crucial biological roles during embryonic development with functionality conserved despite limited sequence conservation. H3K4me3, H3K36me3 chromatin maps, 3P-Seq and RNA-Seq were used to identify lincRNAs in the zebrafish genome
Project description:Human KLK8/neuropsin, a kallikrein-related serine peptidase, is mostly expressed in skin and hippocampus, where it regulates memory formation by synaptic remodeling. Recombinantly expressed KLK8 revealed a good accordance two substrate profiling methods: positional scanning with fluorogenic tetrapeptides and a novel version of the proteomic PICS approach. Besides a strong preference for Arg in the P1 position, the enzyme favors Thr in P4, basic P3 residues, aliphatic P2, small P1′ and aliphatic P2′ residues. Enzyme kinetic studies with synthesized substrates showed stimulatory and inhibitory effects of Ca2+ and Zn2+, respectively, which are important for physiological functions. Two crystal structures of KLK8 with the active site inhibitor leupeptin explain the subsite specificity and display Ca2+ bound to the 75-loop, which is unique among the KLKs. The mutants D70K and H99A confirmed the antagonistic role of the cation binding sites, for which a comparison with the murine apo-KLK8 structure clarified further mechanistic details. Molecular docking and dynamics calculations provided insights in substrate binding and the dual regulation by Ca2+ and Zn2+, involving an allosteric surface loop network.
Project description:The conversion of male germ cell chromatin to a nucleoprotamine structure is fundamental to the life cycle yet the underlying molecular details remain obscure. Here we show that an essential step is the genome-wide incorporation of TH2B, a histone H2B variant of hitherto unknown function. Using mouse models in which TH2B is depleted or C-terminally modified we show that TH2B directs the final transformation of dissociating nucleosomes into protamine-packed structures. Depletion of TH2B induces compensatory mechanisms that permit histone removal by up-regulating H2B and programming nucleosome instability through targeted histone modifications, including lysine crotonylation and arginine methylation. Furthermore, after fertilization, TH2B re-assembles onto the male genome during protamine-to-histone exchange. Thus, TH2B is a unique histone variant, which plays a key role in the histone-to-protamine packing of the male genome and guides genome-wide chromatin transitions that both precede and follow transmission of the male genome to the egg. Examination of TH2B binding on chromatin in meiotic (spermatocytes) and post-meiotic (round spermatids) male germ cells from adult Th2b+/tag mice (TH2B C-terminally fused to three consecutive affinity tags: His, Flag and Ha). Examination of TH2B (wild type) binding on chromatin in male germ cells from adult wt mice.
Project description:In enteric bacteria, the transcription factor ?E maintains membrane homeostasis by inducing expression of proteins involved in membrane repair and of two small, regulatory RNAs (sRNAs) that downregulate synthesis of abundant membrane porins. Here, we describe the discovery of a third ?E-dependent sRNA, MicL, transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308 nt primary transcript that is processed to an 80 nt form. Both forms possess features typical of Hfq-binding sRNAs, but surprisingly only target a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that ?E activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new ?E regulatory loop that opposes membrane stress. Together MicA, RybB and MicL allow ?E to repress the expression of all abundant outer membrane proteins in response to stress. 12 samples mRNA-seq data, 2 samples ribosome profiling data. For mRNA-seq data, samples were gathered at the indicated time (in min) after induction of either vector (WT), long (MicL), and short (MicL-S) forms of MicL.