Promiscuous splicing-derived hairpins are dominant substrates of tailing-mediated defense of miRNA biogenesis in mammals.

Canonical microRNA (miRNA) hairpins are processed by the RNase III enzymes Drosha and Dicer into ∼22 nt RNAs loaded into an Argonaute (Ago) effector. In addition, splicing generates numerous intronic hairpins that bypass Drosha (mirtrons) to yield mature miRNAs. Here, we identify hundreds of previously unannotated, splicing-derived hairpins in intermediate-length (∼50-100 nt) but not small (20-30 nt) RNA data.

miR-486 is essential for muscle function and suppresses a dystrophic transcriptome.

miR-486 is a muscle-enriched microRNA, or "myomiR," that has reduced expression correlated with Duchenne muscular dystrophy (DMD). To determine the function of miR-486 in normal and dystrophin-deficient muscles and elucidate miR-486 target transcripts in skeletal muscle, we characterized knockout mice ( KO). KO mice developed disrupted myofiber architecture, decreased myofiber size, decreased locomotor activity, increased cardiac fibrosis, and metabolic defects were exacerbated in KO: (DKO) mice.

Integrator Recruits Protein Phosphatase 2A to Prevent Pause Release and Facilitate Transcription Termination.

Efficient release of promoter-proximally paused RNA Pol II into productive elongation is essential for gene expression. Recently, we reported that the Integrator complex can bind paused RNA Pol II and drive premature transcription termination, potently attenuating the activity of target genes. Premature termination requires RNA cleavage by the endonuclease subunit of Integrator, but the roles of other Integrator subunits in gene regulation have yet to be elucidated.

Mechanism and Function of Antiviral RNA Interference in Mice.

Distinct mammalian RNA viruses trigger Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNA) and encode unrelated proteins to suppress vsiRNA biogenesis. However, the mechanism and function of the mammalian RNA interference (RNAi) response are poorly understood. Here, we characterized antiviral RNAi in a mouse model of infection with Nodamura virus (NoV), a mosquito-transmissible positive-strand RNA virus encoding a known double-stranded RNA (dsRNA)-binding viral suppressor of RNAi (VSR), the B2 protein.

Dual Strategies for Argonaute2-Mediated Biogenesis of Erythroid miRNAs Underlie Conserved Requirements for Slicing in Mammals.

While Slicer activity of Argonaute is central to RNAi, conserved roles of slicing in endogenous regulatory biology are less clear, especially in mammals. Biogenesis of erythroid Dicer-independent mir-451 involves Ago2 catalysis, but mir-451-KO mice do not phenocopy Ago2 catalytic-dead (Ago2-CD) mice, suggesting other needs for slicing. Here, we reveal mir-486 as another dominant erythroid miRNA with atypical biogenesis.

Selective Suppression of the Splicing-Mediated MicroRNA Pathway by the Terminal Uridyltransferase Tailor.

Several terminal uridyltransferases (TUTases) are known to modulate small RNA biogenesis and/or function via diverse mechanisms. Here, we demonstrate that Drosophila splicing-derived pre-miRNAs (mirtrons) are efficiently modified by the previously uncharacterized TUTase, Tailor. Tailor is necessary and sufficient for mirtron hairpin uridylation, and this modification inhibits mirtron biogenesis.

Multiple In Vivo Biological Processes Are Mediated by Functionally Redundant Activities of Drosophila mir-279 and mir-996.

While most miRNA knockouts exhibit only subtle defects, a handful of miRNAs are profoundly required for development or physiology. A particularly compelling locus is Drosophila mir-279, which was reported as essential to restrict the emergence of CO2-sensing neurons, to maintain circadian rhythm, and to regulate ovarian border cells. The mir-996 locus is located near mir-279 and bears a similar seed, but they otherwise have distinct, conserved, non-seed sequences, suggesting their evolutionary maintenance for separate functions.

Age- and pregnancy-associated DNA methylation changes in mammary epithelial cells.

Postnatal mammary gland development and differentiation occur during puberty and pregnancy. To explore the role of DNA methylation in these processes, we determined the genome-wide DNA methylation and gene expression profiles of CD24(+)CD61(+)CD29(hi), CD24(+)CD61(+)CD29(lo), and CD24(+)CD61(-)CD29(lo) cell populations that were previously associated with distinct biological properties at different ages and reproductive stages. We found that pregnancy had the most significant effects on CD24(+)CD61(+)CD29(hi) and CD24(+)CD61(+)CD29(lo) cells, inducing distinct epigenetic states that were maintained through life.

Alteration of miRNA activity via context-specific modifications of Argonaute proteins.

miRNAs are enclosed within Argonaute (Ago) proteins, the downstream effectors of small RNA-mediated gene silencing. Because miRNAs mediate extensive networks of post-transcriptional control, cells have evolved multiple strategies to control their activity with precision. A growing theme of recent years is how post-translational modifications of Ago proteins, such as prolyl hydroxylation, phosphorylation, ubiquitination, and poly-ADP-ribosylation, alter miRNA activity at global or specific levels.

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila.

Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is 'sliced' by Ago2, then 3'-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors.

The receptor tyrosine kinase ErbB3 maintains the balance between luminal and basal breast epithelium.

ErbB3 harbors weak kinase activity, but strongly activates downstream phosphatidylinositol 3-kinase/Akt signaling through heterodimerization with and activation by other ErbB receptor tyrosine kinases. We report here that ErbB3 loss in the luminal mammary epithelium of mice impaired Akt and MAPK signaling and reduced luminal cell proliferation and survival. ERBB3 mRNA expression levels were highest in luminal mammary populations and lowest in basal cell/stem cell populations.

Dicer recognizes the 5' end of RNA for efficient and accurate processing.

A hallmark of RNA silencing is a class of approximately 22-nucleotide RNAs that are processed from double-stranded RNA precursors by Dicer. Accurate processing by Dicer is crucial for the functionality of microRNAs (miRNAs). The current model posits that Dicer selects cleavage sites by measuring a set distance from the 3' overhang of the double-stranded RNA terminus.

Overcoming hypoxia-induced apoptotic resistance through combinatorial inhibition of GSK-3β and CDK1.

Tumor hypoxia is an inherent impediment to cancer treatment that is both clinically significant and problematic. In this study, we conducted a cell-based screen to identify small molecules that could reverse the apoptotic resistance of hypoxic cancer cells. Among the compounds, we identified were a structurally related group that sensitized hypoxic cancer cells to apoptosis by inhibiting the kinases GSK-3β and cyclin-dependent kinase (CDK) 1.