A small RNA system ensures accurate homologous pairing and unpaired silencing of meiotic chromosomes.

During meiosis, chromosomes with homologous partners undergo synaptonemal complex (SC)-mediated pairing, while the remaining unpaired chromosomes are heterochromatinized through unpaired silencing. Mechanisms underlying homolog recognition during SC formation are still unclear. Here, we show that the Caenorhabditis elegans Argonaute proteins, CSR-1 and its paralog CSR-2, interacting with 22G-RNAs, are required for synaptonemal complex formation with accurate homology. CSR-1 in nuclei and meiotic cohesin, constituting the SC lateral elements, were associated with nonsimple DNA repeats, including minisatellites and transposons, and weakly associated with coding genes. CSR-1-associated CeRep55 minisatellites were expressing 22G-RNAs and long noncoding (lnc) RNAs that colocalized with synaptonemal complexes on paired chromosomes and with cohesin regions of unpaired chromosomes. CeRep55 multilocus deletions reduced the efficiencies of homologous pairing and unpaired silencing, which were supported by the csr-1 activity. Moreover, CSR-1 and CSR-2 were required for proper heterochromatinization of unpaired chromosomes. These findings suggest that CSR-1 and CSR-2 play crucial roles in homology recognition, achieving accurate SC formation between chromosome pairs and condensing unpaired chromosomes by targeting repeat-derived lncRNAs.

Versatile strategy using vaccinia virus-capping enzyme to synthesize functional 5' cap-modified mRNAs.

The potential of synthetic mRNA as a genetic carrier has increased its application in scientific fields. Because the 5' cap regulates the stability and translational activity of mRNAs, there are concerted efforts to search for and synthesize chemically-modified 5' caps that improve the functionality of mRNA. Here, we report an easy and efficient method to synthesize functional mRNAs by modifying multiple 5' cap analogs using a vaccinia virus-capping enzyme. We show that this enzyme can introduce a variety of GTP analogs to the 5' end of RNA to generate 5' cap-modified mRNAs that exhibit different translation levels. Notably, some of these modified mRNAs improve translation efficiency and can be conjugated to chemical structures, further increasing their functionality. Our versatile method to generate 5' cap-modified mRNAs will provide useful tools for RNA therapeutics and biological research.

Monitoring and visualizing microRNA dynamics during live cell differentiation using microRNA-responsive non-viral reporter vectors.

MicroRNA (miRNA) activity differs with cell type, suggesting it can be used as a cell marker. In this study, we developed novel miRNA-responsive non-viral reporter vectors to continuously monitor and visualize miRNA dynamics during differentiation and to efficiently purify target living cells. Each vector codes miRNA-responsive and reference reporter genes in a single mRNA. These two genes are independent modules but transcribed by a single promoter, which enables us to distinguish miRNA-mediated post-transcriptional repression from transcriptional repression. We generated stable, miRNA-responsive vector-containing human induced pluripotent stem cells (hiPSCs) using the piggyBac transposon or episomal vectors. We could continuously monitor the differentiation status of living hiPSCs by detecting the activity of hiPSC-specific miRNA (miR-302a*). In addition, we could selectively sort hiPSC-derived cardiomyocytes using cardiomyocyte-specific miRNA (miR-208a or miR-1)-reporter vectors. Our miRNA reporter system provides a simple way to quantitatively and continuously monitor and visualize changes in the cellular state and should facilitate a broad range of studies that depend on cellular changes including drug discovery and cell-fate conversion.

Directing and Boosting of Cell Migration by the Entropic Force Gradient in Polymer Solution.

Noncontact manipulation of nano/micromaterials presents a great challenge in fields ranging from biotechnology to nanotechnology. In this study we developed a new strategy for the manipulation of molecules and cells based on diffusiophoresis driven by a concentration gradient of a polymer solute. By using laser focusing in a microfluidic device, we created a sharp concentration gradient of poly(ethylene glycol) (PEG) in a solution of this polymer. Because diffusiophoresis essentially depends on solute gradients alone, PEG solute contrast resulted in trapping of DNA and eukaryotic cells with little material dependence. Furthermore, quantitative analysis revealed that the motility of migrating cells was enhanced with the PEG concentration, consistent with a theoretical model of boosted cell migration. Our results support that a solute contrast of polymer can exert an interfacial force gradient that physically propels objects and may have application for the manipulation of soft materials.

Expression of constitutive androstane receptor splice variants in rat liver and lung and their functional properties.

The mammalian constitutive androstane receptor (CAR) is a transcription factor that participates in controlling the expression of xenobiotic metabolizing and transporting genes in response to xenobiotics in an organ-specific manner. In addition to the wild-type CAR (CAR WT) mRNA, mRNAs for five splice variants (SVs) could be detected in the liver of 7-week-old male Wistar rats by RT-PCR using primer pairs covering a full-length mRNA derived from 9 exons; insertion of 18 bp at the 5'-end of intron 8 with or without deletion of 3 bp from the 5'-end of exon 7 (CAR SV1 or SV2), deletion of 4 bp from the 5'-end of exon 8 (CAR SV3), insertion of 195 bp intron 7 (CAR SV4), and insertion of 91 bp intron 6 (CAR SV5). In contrast, only CAR SV5 was detected in lung. Due to the introduction of novel stop codons, all the SVs were considered to code for premature proteins. The liver homogenate gave two protein bands in the vicinity of 37 kDa on Western blotting. They were attributable to CAR WT and SV-complex, respectively, based on their putative molecular weights in descending order. Upon cotransfection with the reporter plasmid, only the cells transfected with the CAR SV4-expression plasmid showed enhanced luciferase activity similar to the WT-transfected cells, for which the further splicing of the remaining intron 7 seemed to be responsible. The transactivation-defective SVs downregulated CAR WT-induced luciferase activity to some extent in the cotransfection experiments.