Alex S. Flynt

Biological principles of microRNA-mediated regulation: shared themes amid diversity

Regulation of gene activity by microRNAs is critical to myriad aspects of eukaryotic development and physiology. Amidst an extensive regulatory web that is predicted to involve thousands of transcripts, emergent themes are now beginning to illustrate how microRNAs have been incorporated into diverse settings. These include potent inhibition of individual key targets, fine-tuning of target activity, the coordinated regulation of target batteries, and the reversibility of some aspects of microRNA-mediated repression. Such themes may reflect some of the inherent advantages of exploiting microRNA control in biological circuits, and provide insight into the consequences of microRNA dysfunction in disease.

Zebrafish miR-214 modulates Hedgehog signaling to specify muscle cell fate

Numerous microRNAs (miRNAs) have been discovered in the genomes of higher eukaryotes, and functional studies indicate that they are important during development. However, little is known concerning the function of individual miRNAs. We approached this problem in zebrafish by combining identification of miRNA expression, functional analyses and experimental validation of potential targets. We show that miR-214 is expressed during early segmentation stages in somites and that varying its expression alters the expression of genes regulated by Hedgehog signaling. Inhibition of miR-214 results in a reduction or loss of slow-muscle cell types. We show that su(fu) mRNA, encoding a negative regulator of Hedgehog signaling, is targeted by miR-214. Through regulation of su(fu), miR-214 enables precise specification of muscle cell types by sharpening cellular responses to Hedgehog.

SIRNA THERAPEUTICS: BIG POTENTIAL FROM SMALL RNAS

RNA interference (RNAi) is now an umbrella term referring to post-transcriptional gene silencing mediated by either degradation or translation arrest of target RNA. This process is initiated by double-stranded RNA with sequence homology driving specificity. The discovery that 21–23 nucleotide RNA duplexes (small-interfering RNAs, siRNAs) mediate RNAi in mammalian cells opened the door to the therapeutic use of siRNAs. While much work remains to optimize delivery and maintain specificity, the therapeutic advantages of siRNAs for treatment of viral infection, dominant disorders, cancer, and neurological disorders show great promise.

Dicing of viral replication intermediates during silencing of latent Drosophila viruses

Previous studies revealed roles for RNA interference (RNAi) in the immediate cellular response to viral infection in plants, nematodes and flies. However, little is known about how RNAi combats viruses during persistent or latent infections. Our analysis of small RNAs cloned from Drosophila cells latently infected with Flock House Virus (FHV) failed to reveal signatures of bulk degradation of the viral genome. Instead, this + strand virus specifically generated Dicer-2-dependent, 21-nucleotide siRNAs that derived in equal proportion from + and − strands. Curiously, luciferase reporters that are fully complementary to abundant viral siRNAs were poorly repressed. Moreover, although the viral siRNAs that were incorporated into an effector complex associated with Argonaute2, bulk FHV siRNAs in latently infected cells were not loaded into any Argonaute protein. Together, these data suggest that direct dicing of viral replication intermediates plays an important role in maintaining the latent viral state. In addition, the denial of bulk viral siRNAs from effector complexes suggests that criteria beyond the structural competency of RNA duplexes influence the assembly of functional silencing complexes.

Evolutionary flux of canonical microRNAs and mirtrons in Drosophila.

Next-generation sequencing technologies generate vast catalogs of short RNA sequences from which to mine microRNAs. However, such data must be vetted to appropriately categorize microRNA precursors and interpret their evolution. A recent study annotated hundreds of microRNAs in three Drosophila species on the basis of singleton reads of heterogeneous length1. Our multi-million read datasets indicated that most of these were not substrates of RNAse III cleavage, and comprised many mRNA degradation fragments. We instead identified a distinct and smaller set of novel microRNAs supported by confident cloning signatures, including a high proportion of evolutionarily nascent mirtrons. Our data support a much lower rate in the emergence of lineage-specific microRNAs than previously inferred1, with a net flux of ~1 microRNA/million years of Drosophilid evolution.

DJ-1 Binds Androgen Receptor Directly and Mediates Its Activity in Hormonally Treated Prostate Cancer Cells

The oncogene DJ-1 has been associated with multiple cancers, including prostate cancer, where it can be stabilized by androgens and antiandrogens. However, little data exist on the expression pattern and function of DJ-1 in prostate cancer. To address the function of DJ-1 in prostate, a yeast two-hybrid screen was done to identify novel DJ-1 binding proteins. The androgen receptor (AR) was identified and confirmed as a DJ-1 binding partner. This is the first evidence that DJ-1 directly interacts with AR. We also show that modulation of DJ-1 expression regulated AR transcriptional activity. Importantly, both the subcellular localization of DJ-1 and the interaction with AR are regulated by androgens and antiandrogens. Additionally, immunohistochemical staining on two human prostate cancer tissue arrays was done providing the first large-scale expression analysis of DJ-1 in prostate. DJ-1 expression did not change with Gleason pattern but increased after androgen deprivation therapy, indicating that it may be involved in the development of androgen independence. These data provide a novel mechanism where DJ-1-mediated regulation of AR may promote the progression of prostate cancer to androgen independence.

miR-8 microRNAs regulate the response to osmotic stress in zebrafish embryos

MicroRNAs (miRNAs) are highly conserved small RNAs that act as translational regulators of gene expression, exerting their influence by selectively targeting mRNAs bearing complementary sequence elements. These RNAs function in diverse aspects of animal development and physiology. Because of an ability to act as rapid responders at the level of translation, miRNAs may also influence stress response. In this study, we show that the miR-8 family of miRNAs regulates osmoregulation in zebrafish embryos. Ionocytes, which are a specialized cell type scattered throughout the epidermis, are responsible for pH and ion homeostasis during early development before gill formation. The highly conserved miR-8 family is expressed in ionocytes and enables precise control of ion transport by modulating the expression of Nherf1, which is a regulator of apical trafficking of transmembrane ion transporters. Ultimately, disruption of miR-8 family member function leads to an inability to respond to osmotic stress and blocks the ability to properly traffic and/or cluster transmembrane glycoproteins at the apical surface of ionocytes.

MicroRNA-Related Cofilin Abnormality in Alzheimer's Disease

Rod-like structures composed of actin and the actin-binding protein cofilin are found in Alzheimers disease (AD) patients. However, the mechanisms underlying formation of these structures and their pathological consequences are still largely unknown. We found that microRNAs 103 and 107 repress translation of cofilin, and that reduced levels of miR-103 or miR-107 are associated with elevated cofilin protein levels and formation of rod-like structures in a transgenic mouse model of AD. These results suggest that microRNAs may play an important role in cytoskeletal pathology in AD.

MiRNA expression analysis during normal zebrafish development and following inhibition of the Hedgehog and Notch signaling pathways.

microRNAs (miRNAs) are small (∼22 nucleotide) non‐coding RNAs that regulate gene expression at the post‐transcriptional level, typically by inhibiting translation. The genes encoding these small RNAs are estimated to comprise approximately 2–3% of animal genomes yet potentially regulate a majority of protein‐coding genes including those involved in cell specification and development. A key remaining question is to identify target mRNAs regulated by microRNAs. As a means to identify potential targets, we designed a sensitive microarray to analyze global miRNA expression patterns at twelve developmental stages in zebrafish. Further, we conducted arrays on zebrafish embryos treated with small molecule inhibitors of the Hedgehog and Notch signaling pathways to enable identification of differentially expressed miRNAs that target genes controlling key developmental pathways during early embryogenesis. Developmental Dynamics 236:2172–2180, 2007.

Drosophila piwi Mutants Exhibit Germline Stem Cell Tumors that Are Sustained by Elevated Dpp Signaling

Drosophila Piwi is the founding member of a gonadal clade of Argonaute proteins that serve as silencing effectors for ∼26-32 nt Piwi-interacting RNAs (piRNAs) [1], and piwi mutants exhibit dramatically rudimentary ovaries [2]. It was proposed that somatic Piwi maintains germline stem cells (GSCs) by promoting Dpp signaling, presumably via cap cells that form the somatic niche for GSCs [3-5]. However, we unexpectedly observed that piwi mutants exhibit high-frequency GSC-like tumors that persist throughout adult life. Multiple readouts demonstrated hyperactive Dpp signaling in piwi mutants, including the failure to express the germline differentiation factor bag-of-marbles (bam), and restoration of bam expression relieved piwi GSC-like tumors. Tissue-specific rescue and knockdown experiments indicate that Piwi is not required in cap cells, the source of niche Dpp, but instead is required in gonadal intermingled cells (ICs, the progenitor cells of escort cells). Adult-specific knockdown of dpp in escort cells substantially rescued piwi tumors, demonstrating that they are driven by excess Dpp signaling. However, the temporal requirement for piwi to restrict GSC numbers was much earlier, during the wandering third-instar larval stage. Indeed, piwi mutant larval gonads exhibited defective morphology and loss of Bam. Our data indicate that loss of Piwi causes defects in ICs and escort cells, leading to ectopic Dpp signaling and consequent blockage of GSC differentiation.