The post-translational modifications (PTMs) occurring on microtubules have been implicated in the regulation of microtubule properties and functions. Acetylated K40 of α-tubulin, a hallmark of long-lived stable microtubules, is known to be negatively controlled by histone deacetylase 6 (HDAC6). However, the vital roles of HDAC6 in microtubule-related processes such as cell motility and cell division cannot be fully explained by the only known target site on tubulin. Here, we attempt to comprehensively map lysine acetylation sites on tubulin purified from mouse brain tissues. Furthermore, mass spectrometry-based quantitative comparison of acetylated peptides from wild-type vs HDAC6 knockout mice allowed us to identify six new deacetylation sites possibly mediated by HDAC6. Thus, adding new sites to the repertoire of HDAC6-mediated tubulin deacetylation events would further our understanding of the multi-faceted roles of HDAC6 in regulating microtubule stability and cellular functions.

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The compact CRISPR/Cas9 system, which can be delivered with their gRNA and a full-length promoter for expression by a single adeno-associated virus (AAV), is a promising platform for therapeutic applications. We previously identified a compact SauriCas9 that displays high activity and requires a simple NNGG PAM, but the specificity is moderate. Here, we identified three compact Cas9 orthologs, Staphylococcus lugdunensis Cas9 (SlugCas9), Staphylococcus lutrae Cas9 (SlutrCas9) and Staphylococcus haemolyticus Cas9 (ShaCas9), for mammalian genome editing. Of these three Cas9 orthologs, SlugCas9 recognizes a simple NNGG PAM and displays comparable activity to SaCas9. Importantly, we generated a SlugCas9-SaCas9 chimeric nuclease, which has both high specificity and high activity. We finally engineered SlugCas9 with mutations to generate a high-fidelity variant that maintains high specificity without compromising on-target editing efficiency. Our study offers important minimal Cas9 tools that are ideal for both basic research and clinical applications.

RNA helicase DDX21 plays vital roles in ribosomal RNA biogenesis, transcription, and the regulation of host innate immunity during virus infection. How DDX21 recognizes and unwinds RNA and how DDX21 interacts with virus remain poorly understood. Here, crystal structures of human DDX21 determined in three distinct states are reported, including the apo‐state, the AMPPNP plus single‐stranded RNA (ssRNA) bound pre‐hydrolysis state, and the ADP‐bound post‐hydrolysis state, revealing an open to closed conformational change upon RNA binding and unwinding. The core of the RNA unwinding machinery of DDX21 includes one wedge helix, one sensor motif V and the DEVD box, which links the binding pockets of ATP and ssRNA. The mutant D339H/E340G dramatically increases RNA binding activity. Moreover, Hill coefficient analysis reveals that DDX21 unwinds double‐stranded RNA (dsRNA) in a cooperative manner. Besides, the nonstructural (NS1) protein of influenza A inhibits the ATPase and unwinding activity of DDX21 via small RNAs, which cooperatively assemble with DDX21 and NS1. The structures illustrate the dynamic process of ATP hydrolysis and RNA unwinding for RNA helicases, and the RNA modulated interaction between NS1 and DDX21 generates a fresh perspective toward the virus–host interface. It would benefit in developing therapeutics to combat the influenza virus infection.

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There has been growing interest in applying tissue engineering to stem cell-based regeneration therapies. We have previously reported that zebrafish can faithfully regenerate complicated tissue structures through blastemal cell type conversions and tissue reorganization. To unveil the regenerative factors and engineering arts of blastemal regeneration, we conducted transcriptomal analyses at four time points corresponding to preamputation, re-epitheliation, blastemal formation, and respecification. By combining the hierarchical gene ontology term network, the DAVID annotation system, and Euclidean distance clustering, we identified four signaling pathways: foxi1-foxo1b-pou3f1, pax3a-mant3a-col11/col2, pou5f1-cdx4-kdrl, and isl1-wnt11 PCPsox9a. Results from immunohistochemical staining and promoter-driven transgenic fish suggest that these pathways, respectively, define wound epidermis reconstitution, cell type conversions, blastemal angiogenesis/vasculogenesis, and cartilage matrix-orientation. Foxi1 morpholinoknockdown caused expansions of Foxo1b-and Pax3a-expression in the basal layer-blastemal junction region. Moreover, foxi1 morphants displayed increased sox9a and hoxa2b transcripts in the embryonic pharyngeal arches. Thus, a Foxi1 signal switch is required to establish correct tissue patterns, including re-epitheliation and blastema formation. This study provides novel insight into a blastema regeneration strategy devised by epithelial cell transdifferentiation, blood vessel engineering, and cartilage matrix deposition. STEM CELLS 2015;33:806-818