Small nucleolar RNAs (snoRNAs) and microRNAs are two classes of non-protein-coding RNAs with distinct functions in RNA modification or post-transcriptional gene silencing. In this study, we introduce novel insights to RNA-induced gene activity adjustments in human cells by identifying numerous snoRNA-derived molecules with miRNA-like function, including H/ACA box snoRNAs and C/D box snoRNAs. In particular, we demonstrate that several C/D box snoRNAs give rise to gene regulatory RNAs, named sno-miRNAs here. Our data are complementing the increasing number of studies in the field of small RNAs with regulatory functions. In massively deep sequencing of small RNA fractions we identified high copy numbers of sub-sequences from >30 snoRNAs with lengths of ≥18 nt. RNA secondary structure prediction indicated for a majority of candidates a location in predicted stem regions. Experimental analysis revealed efficient gene silencing for 11 box C/D sno-miRNAs, indicating cytoplasmic processing and recruitment to the RNA silencing machinery. Assays in four different human cell lines indicated variations in both the snoRNA levels and their processing to active sno-miRNAs. In addition we show that box D elements are predominantly flanking at least one of the sno-miRNA strands, while the box C element locates within the sequence of the sno-miRNA guide strand.

Astrin is a mitotic-spindle-associated protein expressed in most human cell lines and tissues. However, its functions in spindle organization and mitosis have not yet been determined. Sequence analysis revealed that astrin has an N-terminal globular domain and an extended coiled-coil domain. Recombinant astrin was purified and characterized by CD spectroscopy and electron microscopy. Astrin showed parallel dimers with head-stalk structures reminiscent of motor proteins, although no sequence similarities to known motor proteins were found. In physiological buffers, astrin dimers oligomerized via their globular head domains and formed aster-like structures. Silencing of astrin in HeLa cells by RNA interference resulted in growth arrest, with formation of multipolar and highly disordered spindles. Chromosomes did not congress to the spindle equator and remained dispersed. Cells depleted of astrin were normal during interphase but were unable to progress through mitosis and finally ended in apoptotic cell death. Possible functions of astrin in mitotic spindle organization are discussed.

Efficient transduction tools are a hallmark for both research and therapy development. Here, we introduce new insights into the generation of lentiviral vectors with improved performance by utilizing producer cells with increased production rates of extracellular vesicles through CD9 overexpression. Most human cells secrete small vesicles from their surface (microvesicles) or intraluminal endosome-derived membranes (exosomes). In particular, enhanced levels of the tetraspanin CD9 result in significantly increased numbers of extracellular vesicles with exosome-like features that were secreted from four different human cell lines. Intriguingly, exosomes and their biogenesis route display similarities to lentivirus and we examined the impact of CD9 expression on release and infectivity of recombinant lentiviral vectors. Although the titers of released viral particles were not increased upon production in high CD9 cells, we observed improved performance in terms of both speed and efficiency of lentiviral gene delivery into numerous human cell lines, including HEK293, HeLa, SH-SY5Y, as well as B and T lymphocytes. Here, we demonstrate that enhanced CD9 enables lentiviral transduction in the absence of any pseudotyping viral glycoprotein or fusogenic molecule. Our findings indicate an important role of CD9 for lentiviral vector and exosome biogenesis and point out a remarkable function of this tetraspanin in membrane fusion, viral infectivity, and exosome-mediated horizontal information transfer.

Research on cell-free vesicles revealed a multitude of characteristics, in particular of microvesicles and exosomes, that range from their potential as biomarkers to a function in horizontal transfer of genetic information from cell to cell and also include supportive functions in viral infection. Exosome-associated adeno-associated viruses (exo-AAVs) are of particular interest for the past couple of years, because they introduced a new source of highly potent recombinant AAVs with improved features, including accelerated transduction rates and more efficient immune escape. However, key factors like the mode of action, efficiency of production, or engineering of exo-AAVs remain elusive to a large extent. Here, we used the established system of CD9 overexpression to boost the exosome output of AAV producing HEK-AAV cells. The CD9-powered high-exosome environment was established during exo-AAV1 production, and we could demonstrate that the yield of exo-AAVs dramatically increased when compared to standard exo-AAVs. Furthermore, we report that exo-AAV-CD9GFP was more efficient in transduction of cells in the same titer ranges as standard exo-AAVs. Our results provide a technological approach for the generation of exo-AAVs with superior performance.

The enzyme choline kinase (CK), which catalyzes the phosphorylation of choline to phosphorylcholine in the presence of ATP, has an essential role in the biosynthesis of phosphatidylcholine, the major constituent of all mammalian cell membranes. CK is encoded by two separate genes expressing the three isoforms CKα1, CKα2 and CKβ that are active as homodimeric or heterodimeric species. Metabolic changes observed in various cancer cell lines and tumors have been associated with differential and marked up‐regulation of the CKα genes, and specific inhibition of CKα activity has been proposed as a potential anti‐cancer strategy. As a result, less attention has been given to CKβ and its interaction with CKα. With the aim of profiling the intracellular roles of CKα and CKβ, we used RNA interference (RNAi) as a molecular approach to down‐regulate the expression of CK in HeLa cells. Individual and simultaneous RNAi‐based silencing of the CK α and β isoforms was achieved using different combinations of knockdown strategies. Efficient knockdown was confirmed by immunodetection using our isoform‐specific antibodies and by quantitative real‐time PCR. Our analyses of the phenotypic consequences of CK depletion showed the expected lethal effect of CKα knockdown. However, CKβ‐ and CKα + CKβ‐silenced cells had no aberrant phenotype. Therefore, our results support the hypothesis that the balance of the α and β isoforms is critical for cancer cell survival. The suppression of the cancer cell killing effect of CKα silencing by simultaneous knockdown of both isoforms implies that a more effective CK‐based anti‐cancer strategy can be achieved by reducing cross‐reactivity with CKβ.

Here we demonstrate highly efficient RNA interference in ZFL, SJD and ZF4 cell lines derived from adult and embryonic zebrafish Danio rerio. Microinjection of siRNAs resulted in silencing in almost...