Alternative exon skipping biases substrate preference of the deubiquitylase USP15 for mysterin/RNF213, the moyamoya disease susceptibility factor.

The deubiquitylating enzyme USP15 plays significant roles in multiple cellular pathways including TGF-ß signaling, RNA splicing, and innate immunity. Evolutionarily conserved skipping of exon 7 occurs during transcription of the mRNAs encoding USP15 and its paralogue USP4, yielding two major isoforms for each gene. Exon 7 of USP15 encodes a serine-rich stretch of 29 amino acid residues located in the inter-region linker that connects the N-terminal putative regulatory region and the C-terminal enzymatic region. Previous findings suggested that the variation in the linker region leads to functional differences between the isoforms of the two deubiquitylating enzymes, but to date no direct evidence regarding such functional divergence has been published. We found that the long isoform of USP15 predominantly recognizes and deubiquitylates mysterin, a large ubiquitin ligase associated with the onset of moyamoya disease. This observation represents the first experimental evidence that the conserved exon skipping alters the substrate specificity of this class of deubiquitylating enzymes. In addition, we found that the interactomes of the short and long isoforms of USP15 only partially overlapped. Thus, USP15, a key gene in multiple cellular processes, generates two functionally different isoforms via evolutionarily conserved exon skipping.

A Novel Fabrication Technique for Liquid-Tight Microchannels by Combination of a Paraffin Polymer and a Photo-Curable Silicone Elastomer.

The development and growth of microfluidics has been mainly based on various novel fabrication techniques for downsizing and integration of the micro/nano components. Especially, an effective fabrication technique of three-dimensional structures still continues to be strongly required in order to improve device performance, functionality, and device packing density because the conventional lamination-based technique for integrating several two-dimensional components is not enough to satisfy the requirement. Although three-dimensional printers have a high potential for becoming an effective tool to fabricate a three-dimensional microstructure, a leak caused by the roughness of a low-precision structure made by a 3D printer is a critical problem when the microfluidic device is composed of several parts. To build a liquid-tight microchannel on such a low-precision structure, we developed a novel assembly technique in which a paraffin polymer was used as a mold for a microchannel of photo-curable silicone elastomer on a rough surface. The shape and roughness of the molded microchannel was in good agreement with the master pattern. Additionally, the seal performance of the microchannel was demonstrated by an experiment of electrophoresis in the microchannel built on a substrate which has a huge roughness and a joint.