Irene Bozzoni

Concerted microRNA control of Hedgehog signalling in cerebellar neuronal progenitor and tumour cells

MicroRNAs (miRNA) are crucial post‐transcriptional regulators of gene expression and control cell differentiation and proliferation. However, little is known about their targeting of specific developmental pathways. Hedgehog (Hh) signalling controls cerebellar granule cell progenitor development and a subversion of this pathway leads to neoplastic transformation into medulloblastoma (MB). Using a miRNA high‐throughput profile screening, we identify here a downregulated miRNA signature in human MBs with high Hh signalling. Specifically, we identify miR‐125b and miR‐326 as suppressors of the pathway activator Smoothened together with miR‐324‐5p, which also targets the downstream transcription factor Gli1. Downregulation of these miRNAs allows high levels of Hh‐dependent gene expression leading to tumour cell proliferation. Interestingly, the downregulation of miR‐324‐5p is genetically determined by MB‐associated deletion of chromosome 17p. We also report that whereas miRNA expression is downregulated in cerebellar neuronal progenitors, it increases alongside differentiation, thereby allowing cell maturation and growth inhibition. These findings identify a novel regulatory circuitry of the Hh signalling and suggest that misregulation of specific miRNAs, leading to its aberrant activation, sustain cancer development.

Release of U18 snoRNA from its host intron requires interaction of Nop1p with the Rnt1p endonuclease

An external stem, essential for the release of small nucleolar RNAs (snoRNAs) from their pre‐mRNAs, flanks the majority of yeast intron‐encoded snoRNAs. Even if this stem is not a canonical Rnt1p substrate, several experiments have indicated that the Rnt1p endonuclease is required for snoRNA processing. To identify the factors necessary for processing of intron‐encoded snoRNAs, we have raised in vitro extracts able to reproduce such activity. We found that snoRNP factors are associated with the snoRNA‐ coding region throughout all the processing steps, and that mutants unable to assemble snoRNPs have a processing‐deficient phenotype. Specific depletion of Nop1p completely prevents U18 snoRNA synthesis, but does not affect processing of a dicistronic snoRNA‐coding unit that has a canonical Rnt1p site. Correct cleavage of intron‐encoded U18 and snR38 snoRNAs can be reproduced in vitro by incubating together purified Nop1p and Rnt1p. Pull‐down experiments showed that the two proteins interact physically. These data indicate that cleavage of U18, snR38 and possibly other intron‐encoded snoRNAs is a regulated process, since the stem is cleaved by the Rnt1p endonuclease only when snoRNP assembly has occurred.

Exon 45 Skipping Through U1-snRNA Antisense Molecules Recovers the Dys-nNOS Pathway and Muscle Differentiation in Human DMD Myoblasts

Exon skipping has been demonstrated to be a successful strategy for the gene therapy of Duchenne muscular dystrophy (DMD): the rational being to convert severe Duchenne forms into milder Becker ones. Here, we show the selection of U1 snRNA-antisense constructs able to confer effective rescue of dystrophin synthesis in a Δ44 Duchenne genetic background, through skipping of exon 45; moreover, we demonstrate that the resulting dystrophin is able to recover timing of myogenic marker expression, to relocalize neuronal nitric oxide synthase (nNOS) and to rescue expression of miRNAs previously shown to be sensitive to the Dystrophin-nNOS-HDAC2 pathway. Becker mutations display different phenotypes, likely depending on whether the shorter protein is able to reconstitute the wide range of wild-type functions. Among them, efficient assembly of the dystrophin-associated protein complex (DAPC) and nNOS localization are important. Comparing different Becker deletions we demonstrate the correlation between the ability of the mutant dystrophin to relocalize nNOS and the expression levels of two miRNAs, miR-1 and miR29c, known to be involved in muscle homeostasis and to be controlled by the Dys-nNOS-HDAC2 pathway.