Abundant ϳ28-nucleotide RNAs that are thought to direct histone H3 lysine 9 (H3K9) methylation and promote the elimination of nearly 15 Mbp of DNA from the developing somatic genome are generated during Tetrahymena thermophila conjugation. To identify the protein(s) that generates these small RNAs, we studied three Dicer-related genes encoded within the Tetrahymena genome, two that contain both RNase III and RNA helicase motifs, Dicer 1 (DCR1) and DCR2, and a third that lacks the helicase domain, Dicer-like 1 (DCL1). DCL1 is expressed upon the initiation of conjugation, and the protein localizes to meiotic micronuclei when bidirectional germ line transcription occurs and small RNAs begin to accumulate. Cells in which we disrupted the DCL1 gene (⌬DCL1) grew normally and initiated conjugation as wild-type cells but arrested near the end of development and eventually died, unable to resume vegetative growth. These ⌬DCL1 cells failed to generate the abundant small RNAs but instead accumulated germ line-limited transcripts. Together, our findings demonstrate that these transcripts are the precursors of the small RNAs and that DCL1 performs RNA processing within the micronucleus. Postconjugation ⌬DCL1 cells die without eliminating the germ linelimited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicerrelated gene is required for programmed DNA rearrangements. Surprisingly, ⌬DCL1 cells were not deficient in overall H3K9 methylation, but this modification was not enriched on germ line-limited sequences as it is in wild-type cells, which clearly demonstrates that these small RNAs are essential for its targeting to specific loci.RNA interference (RNAi) describes an array of related mechanisms involved in diverse biological processes including defense against RNA viruses, specification of centromeric heterochromatin structure, and developmental control of gene expression (reviewed in reference 25). These mechanisms share the use of small RNAs to target specific effector protein complexes to homologous sequences via base-pairing interactions. The use of small, homologous RNAs as specificity factors imparts tremendous flexibility of targets on a single protein complex. These targeting RNAs are generated by RNase III enzymes, collectively called Dicer ribonucleases, that cleave longer, double-stranded RNA (dsRNA) into ϳ20-to 26-nucleotide (nt) species that are incorporated into the effector complexes (3, 24, 27, 30; reviewed in reference 6). The genomes of many eukaryotes encode multiple Dicer-related proteins, and the specific Dicer used to generate the small RNAs can determine the downstream pathway that they enter. For instance, in Arabidopsis thaliana, the Dicer-like 3 (Dcl3) gene product is required to produce endogenous short interfering RNAs (siRNAs), A. thaliana Dcl2 is necessary for accumulation of siRNAs in response to RNA virus infections, and A. thaliana Dcl1 is necessary to generate micro-RNAs (miRNAs) involved in the control of flower development (31,53). Similarly, th...

SummaryWe generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development.

Human pluripotent stem cells represent an accessible cell source for novel cell-based clinical research and therapies. With the realization of induced pluripotent stem cells (iPSCs), it is possible to produce almost any desired cell type from any patient's cells. Current developments in gene modification methods have opened the possibility for creating genetically corrected human iPSCs for certain genetic diseases that could be used later in autologous transplantation. Promising preclinical studies have demonstrated correction of disease-causing mutations in a number of hematological, neuronal and muscular disorders. This review aims to summarize these recent advances with a focus on iPSC generation techniques, as well as gene modification methods. We will then further discuss some of the main obstacles remaining to be overcome before successful application of human pluripotent stem cell-based therapy arrives in the clinic and what the future of stem cell research may look like.

Culture-independent studies of cystic fibrosis lung microbiota have provided few mechanistic insights into the polymicrobial basis of disease. Deciphering the specific contributions of individual taxa to CF pathogenesis requires comprehensive understanding of their ecophysiology at the site of infection. We hypothesize that only a subset of CF microbiota are translationally active and that these activities vary between subjects. Here, we apply bioorthogonal non-canonical amino acid tagging (BONCAT) to visualize and quantify bacterial translational activity in expectorated sputum. We report that the percentage of BONCAT-labeled (i.e. active) bacterial cells varies substantially between subjects (6-56%). We use fluorescence-activated cell sorting (FACS) and genomic sequencing to assign taxonomy to BONCAT-labeled cells. While many abundant taxa are indeed active, most bacterial species detected by conventional molecular profiling show a mixed population of both BONCAT-labeled and unlabeled cells, suggesting heterogeneous growth rates in sputum. Differentiating translationally active subpopulations adds to our evolving understanding of CF lung disease and may help guide antibiotic therapies targeting bacteria most likely to be susceptible.

Double-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of gene transcription and translation, including some with tandem DSRMs that act in small RNA biogenesis. We report the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1 and DRB2), two genes encoding nuclear proteins with tandem DSRMs in the ciliate Tetrahymena thermophila. Both proteins are expressed throughout growth and development but exhibit distinct peaks of expression, suggesting different biological roles. In support of this, we show that expression of DRB2 is essential for vegetative growth while DRB1 expression is not. During conjugation, Drb1p and Drb2p localize to distinct nuclear foci. Cells lacking all DRB1 copies are able to produce viable progeny, although at a reduced rate relative to wild-type cells. In contrast, cells lacking germ line DRB2 copies, which thus cannot express Drb2p zygotically, fail to produce progeny, arresting late into conjugation. This arrest phenotype is accompanied by a failure to organize the essential DNA rearrangement protein Pdd1p into DNA elimination bodies and execute DNA elimination and chromosome breakage. These results implicate zygotically expressed Drb2p in the maturation of these nuclear structures, which are necessary for reorganization of the somatic genome.Proteins containing a double-stranded RNA (dsRNA) binding motif (DSRM) participate in diverse biological pathways in a wide range of organisms. This motif was first identified in the developmentally essential gene Staufen of Drosophila melanogaster and has since been recognized to be encoded in the genomes in all three domains of living organisms, as well as in viruses (63; reviewed in references 20 and 67). DSRM proteins commonly act in developmental pathways (e.g., RNA localization by the Staufen family and developmental transcriptional regulation by the DIP1 family) (5,18,62,68) but also have ubiquitous roles in transcriptional and translational regulation (e.g., PKR family and PKR-associated proteins) (26,45,55,58). Proteins vital for RNA interference (RNAi) also contain DSRMs. These include members of the RNase III family (e.g., Dicer and Drosha family proteins) and their tandem DSRMcontaining partner proteins (e.g., RDE-4 of Caenorhabditis elegans, Pasha, R2D2, and Loqs in D. melanogaster, and their homologues in