MOCAT is a highly configurable, modular pipeline for fast, standardized processing of single or paired-end sequencing data generated by the Illumina platform. The pipeline uses state-of-the-art programs to quality control, map, and assemble reads from metagenomic samples sequenced at a depth of several billion base pairs, and predict protein-coding genes on assembled metagenomes. Mapping against reference databases allows for read extraction or removal, as well as abundance calculations. Relevant statistics for each processing step can be summarized into multi-sheet Excel documents and queryable SQL databases. MOCAT runs on UNIX machines and integrates seamlessly with the SGE and PBS queuing systems, commonly used to process large datasets. The open source code and modular architecture allow users to modify or exchange the programs that are utilized in the various processing steps. Individual processing steps and parameters were benchmarked and tested on artificial, real, and simulated metagenomes resulting in an improvement of selected quality metrics. MOCAT can be freely downloaded at http://www.bork.embl.de/mocat/.
Mutations that enhance the response to double-stranded RNA (dsRNA) have revealed components of the RNA interference (RNAi) pathway or related small RNA pathways. To explore these small RNA pathways, we screened for Caenorhabditis elegans mutants displaying an enhanced response to exogenous dsRNAs. Here we describe the isolation of mutations in two adjacent, divergently transcribed open reading frames (eri-6 and eri-7) that fail to complement. eri-6 and eri-7 produce separate pre-messenger RNAs (pre-mRNAs)are trans-spliced to form a functional mRNA, eri-6/7. Trans-splicing of eri-6/7 is mediated by a direct repeat that flanks the eri-6 gene. Adenosine to inosine editing within untranslated regions (UTRs) of eri-6 and eri-7 pre-mRNAs reveals a double-stranded pre-mRNA intermediate, forming in the nucleus before splicing occurs. The ERI-6/7 protein is a superfamily I helicase that both negatively regulates the exogenous RNAi pathway and functions in an endogenous RNAi pathway.RNAi pathways act throughout phylogeny as both an experimental gene-silencing tool and a regulator of endogenous gene expression 1,2 . The endonuclease Dicer 3 and various Argonaute proteins act in specialized roles in most silencing pathways 4 . C. elegans Dicer interacts with negative regulators of exogenous RNAi 5 , such as the RNA-dependent RNA polymerase (RdRP) ) and the exonuclease ERI-1 ( ref. 7 ). These proteins are required for the production or stability of a subset of endogenous short interfering RNA (siRNAs) suggesting a competition with the exogenous RNAi pathway for shared, rate-limiting factors 5,8 .RdRPs amplify siRNAs on mRNA templates in nematodes 9-11 , fungi and plants 1 . The feedforward nature of RNAi and the still unexplained resistance of neurons to RNAi 12-14 suggest that there are undiscovered negative regulators of RNAi. To identify such regulators, we conducted a genetic screen for mutations that confer an enhanced response to dsRNA. Here we describe the identification and characterization of adjacent genes, eri-6 and eri-7, which are assembled by a dsRNA-mediated trans-splicing mechanism to regulate RNAi negatively. The dsRNA-dependent production of an RNAi factor suggests that there may be an autoregulatory feedback mechanism in RNAi. Characterization of eri mutantsTo identify negative regulators of C. elegans RNAi, we genetically screened for mutants having an enhanced RNAi (Eri) phenotype. These mutants, unlike wild type, exhibited an enhanced
The transcription factor sigma(F), which is activated in a cell-specific manner during sporulation in B. subtilis, is initially held in an inactive complex by the anti-sigma factor SpoIIAB. The anti-anti-sigma factor SpoIIAA reacts with SpoIIAB.sigma(F) to induce the release of free sigma(F) and free SpoIIAB. We now report that free SpoIIAB is subject to proteolysis and that it is protected from degradation by sigma(F) in the SpoIIAB.sigma(F) complex and by SpoIIAA in an alternative complex. Proteolysis requires residues located near the extreme C terminus of SpoIIAB and is dependent upon the ClpCP protease. The reaction of SpoIIAA with SpoIIAB.sigma(F) and the resulting degradation of newly released SpoIIAB could set up a self-reinforcing cycle that locks on the activation of sigma(F).
We synthesized a series of poly(disulfide)s by ring-opening polymerization and demonstrated that the copolymerization of monomer 1 containing diethylenetriamine moieties and monomer 2 containing guanidyl ligands could generate an efficient delivery platform for different forms of CRISPR-Cas9-based genome editors, including plasmid, mRNA, and protein. The excellent delivery performance of designed poly(disulfide)s stems from their delicate molecular structures to interact with genome-editing biomacromolecules, unique delivery pathways to mediate the cellular uptake of CRISPR-Cas9 cargoes, and strong ability to escape the endosome. The degradation of poly(disulfide)s by intracellular glutathione not only promotes the timely release of CRISPR-Cas9 machineries into the cytosol but also minimizes the cytotoxicity that nondegradable polymeric carriers often encounter. These merits collectively account for the excellent ability of poly(disulfide)s to mediate different forms of CRISPR-Cas9 for their efficient genome-editing activities in vitro and in vivo.
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