Summary By analyzing gene expression data in gliobastoma in combination with matched microRNA profiles, we have uncovered a post-transcriptional regulation layer of surprising magnitude, comprising over 248,000 microRNA (miR)-mediated interactions. These include ~7,000 genes whose transcripts act as miR ‘sponges’ and 148 genes that act through alternative, non-sponge interactions. Biochemical analyses in cell lines confirmed that this network regulates established drivers of tumor initiation and subtype, including PTEN, PDGFRA, RB1, VEGFA, STAT3, and RUNX1, suggesting that these interactions mediate crosstalk between canonical oncogenic pathways. RNA silencing of 13 microRNA-mediated PTEN regulators, whose locus deletions are predictive of PTEN expression variability, was sufficient to downregulate PTEN in a 3′ UTR-dependent manner and to increase tumor-cell growth rates. Thus, this miR-mediated network provides a mechanistic, experimentally validated rationale for the loss of PTEN expression in a large number of glioma samples with an intact PTEN locus.

In contrast to microRNAs and Piwi-associated RNAs, short interfering RNAs (siRNAs) are seemingly dispensable for host-directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA-generating enzyme Dicer-2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology 1,2 . Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes (hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors (Dicer-2, Hen1 (known as CG12367) and Argonaute 2) with a canonical microRNA factor (Loquacious) to generate ~21-nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila.Artificial, long-inverted repeat transcripts are efficiently processed by a Dicer-2 (Dcr-2)/ Argonaute 2 (AGO2)-driven RNA interference (RNAi) pathway in transgenic Drosophila 1 , 3 . We hypothesized that this might reflect the existence of an endogenous pathway that accepts long, inverted repeat transcripts. To test this idea, we searched for inverted repeats using EINVERTED 4 and selected putative hairpins containing mapped small RNA reads (see Methods). Out of 8,132 candidate regions, most consisted of the terminal inverted repeats of individual transposable elements or long terminal repeats of tandem inverted transposable elements. The remaining loci corresponded to inverted tandem duplications of messenger RNA-or transfer RNA-encoding genes, a microRNA (miRNA) gene (mir-997), a novel tandem pair of short hairpins (chou39-1 and chou39-2, Supplementary Fig. 1), and a handful of singlegene annotations and unannotated regions.We analysed the size distribution of cloned RNAs from all of the non-transposable-element EINVERTED hits. Although these mostly exhibited a broad length distribution across the ~18-

Drosophila utilizes two small-RNA systems to restrict transposon activity in the germline (mostly via piRNAs) and in the soma (mostly via siRNAs).

Summary microRNAs (miRNAs) are ~22 nucleotide regulatory RNAs derived from hairpins generated either by Drosha cleavage (canonical substrates) or by splicing and debranching of short introns (mirtrons). The 5′ end of the highly conserved Drosophila mirtron-like locus mir-1017 is coincident with the splice donor, but a substantial “tail” separates its hairpin from the 3′ splice acceptor. Genetic and biochemical studies define a biogenesis pathway involving splicing, lariat debranching, and RNA exosome-mediated “trimming”, followed by conventional dicing and loading into AGO1 to yield a miRNA that can repress seed-matched targets. Analysis of cloned small RNAs yielded six additional candidate 3′ tailed mirtrons in D. melanogaster. Altogether, these data reveal an unexpected role for the exosome in the biogenesis of miRNAs from hybrid mirtron substrates.

microRNAs (miRNAs) are an abundant class of ~22 nucleotide (nt) regulatory RNAs that are pervasive in higher eukaryotic genomes. In order to fully understand their prominence in genomes, it is necessary to elucidate the molecular mechanisms that can diversify miRNA activities. In this review, we describe some of the many strategies that allow novel miRNA functions to emerge, with particular emphasis on how miRNA genes evolve in animals. These mechanisms include changes in their sequence, processing, or expression pattern; acquisition of miRNA* functionality or antisense processing; and de novo gene birth. The facility and versatility of miRNAs to evolve and change likely underlies how they have become dominant constituents of higher genomes.

Mirtrons are intronic hairpin substrates of the dicing machinery that generate functional microRNAs. In this study, we describe experimental assays that defined the essential requirements for entry of introns into the mirtron pathway. These data informed a bioinformatic screen that effectively identified functional mirtrons from the Drosophila melanogaster transcriptome. These included 17 known and six confident novel mirtrons among the top 51 candidates, and additional candidates had limited read evidence in available small RNA data. Our computational model also proved effective on Caenorhabditis elegans, for which the identification of 14 cloned mirtrons among the top 22 candidates more than tripled the number of validated mirtrons in this species. A few low-scoring introns generated mirtron-like read patterns from atypical RNA structures, but their paucity suggests that relatively few such loci were not captured by our model. Unexpectedly, we uncovered examples of clustered mirtrons in both fly and worm genomes, including a <8-kb region in C. elegans harboring eight distinct mirtrons. Altogether, we demonstrate that discovery of functional mirtrons, unlike canonical miRNAs, is amenable to computational methods independent of evolutionary constraint.[Supplemental material is available for this article. Small RNA data have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). A full list of accession numbers can be found in Supplemental Table S1.]Canonical microRNAs (miRNAs) are ;22-nucleotide (nt) regulatory RNAs derived from inverted repeat transcripts whose biogenesis involves a defined series of processing events (Kim et al. 2009). Primary-miRNA (pri-miRNA) transcripts are first cleaved by the nuclear RNase III enzyme Drosha (also known as RNASEN) to yield pre-miRNA hairpins. Following their cytoplasmic export via exportin 5, pre-miRNAs are cleaved on their terminal loop side by a Dicer-class RNase III enzyme to release miRNA/miRNA* duplexes. One side of the duplex, designated the mature miRNA, is preferentially transferred into an Argonaute protein and guides it to regulate target transcripts. Its partner miRNA* strand is inferred to be preferentially degraded on account of its lower steady-state accumulation, although miRNA* species may still be transferred into Argonaute proteins and have regulatory activities. Since RNase III enzymes typically cleave substrates leaving signature 2-nt 39 overhangs, an appropriate geometry of cloned small RNA duplex ends provides evidence for their transit via a Drosha-Dicer pathway (Ambros et al. 2003;Friedlander et al. 2008;Berezikov et al. 2010;Chiang et al. 2010).Since thousands of miRNAs are now known (Griffiths-Jones et al. 2008), one might presume that sufficient information exists to segregate bona fide miRNA genes from bulk genomic hairpins. Although bioinformatic strategies can enrich for genuine miRNA genes, the number of plausible pri-miRNA hairpins in a typical animal genome exceeds the number of confirmed miRNA hairpins by se...

We introduce a method for simultaneous prediction of microRNA–target interactions and their mediated competitive endogenous RNA (ceRNA) interactions. Using high-throughput validation assays in breast cancer cell lines, we show that our integrative approach significantly improves on microRNA–target prediction accuracy as assessed by both mRNA and protein level measurements. Our biochemical assays support nearly 500 microRNA–target interactions with evidence for regulation in breast cancer tumors. Moreover, these assays constitute the most extensive validation platform for computationally inferred networks of microRNA–target interactions in breast cancer tumors, providing a useful benchmark to ascertain future improvements.

SignificanceRAS mutant cancers represent a large unmet clinical need. Kras mutant genetically engineered mouse models (GEMMs) of cancer recapitulate disease characteristics and are relied upon preclinically to validate targets and test therapies. Our integrative analysis of GEMM tumors revealed significantly evolved genetic heterogeneity, a common feature of human tumors that undermines therapeutic responses. Moreover, interspecies comparative analyses showed the extent of gene-level fidelity between altered oncogenes and tumor suppressors. The genomic diversity represents an unrecognized opportunity to identify therapeutically susceptible genomic subsets preclinically. Moreover, this more-thorough understanding of the unappreciated complexity in these model systems ultimately allows for better interpretation and translatability of preclinical GEMM data for the benefit of cancer patients.