MOV10 and FMRP regulate AGO2 association with microRNA recognition elements.

The fragile X mental retardation protein FMRP regulates translation of its bound mRNAs through incompletely defined mechanisms. FMRP has been linked to the microRNA pathway, and we show here that it associates with the RNA helicase MOV10, also associated with the microRNA pathway. FMRP associates with MOV10 directly and in an RNA-dependent manner and facilitates MOV10's association with RNAs in brain and cells, suggesting a cooperative interaction. We identified the RNAs recognized by MOV10 using RNA immunoprecipitation and iCLIP. Examination of the fate of MOV10 on RNAs revealed a dual function for MOV10 in regulating translation: it facilitates microRNA-mediated translation of some RNAs, but it also increases expression of other RNAs by preventing AGO2 function. The latter subset was also bound by FMRP in close proximity to the MOV10 binding site, suggesting that FMRP prevents MOV10-mediated microRNA suppression. We have identified a mechanism for FMRP-mediated translational regulation through its association with MOV10.

Particle display: a quantitative screening method for generating high-affinity aptamers.

We report an aptamer discovery technology that reproducibly yields higher affinity aptamers in fewer rounds compared to conventional selection. Our method (termed particle display) transforms libraries of solution-phase aptamers into "aptamer particles", each displaying many copies of a single sequence on its surface. We then use fluorescence-activated cell sorting (FACS) to individually measure the relative affinities of >10(8) aptamer particles and sort them in a high-throughput manner. Through mathematical analysis, we identified experimental parameters that enable optimal screening, and demonstrate enrichment performance that exceeds the theoretical maximum achievable with conventional selection by many orders of magnitude. We used particle display to obtain high-affinity DNA aptamers for four different protein targets in three rounds, including proteins for which previous DNA aptamer selection efforts have been unsuccessful. We believe particle display offers an extraordinarily efficient mechanism for generating high-quality aptamers in a rapid and economic manner, towards accelerated exploration of the human proteome.

Novel primate miRNAs coevolved with ancient target genes in germinal zone-specific expression patterns.

Major nonprimate-primate differences in cortico-genesis include the dimensions, precursor lineages, and developmental timing of the germinal zones (GZs). microRNAs (miRNAs) of laser-dissected GZ compartments and cortical plate (CP) from embryonic E80 macaque visual cortex were deep sequenced. The CP and the GZ including ventricular zone (VZ) and outer and inner subcompartments of the outer subventricular zone (OSVZ) in area 17 displayed unique miRNA profiles. miRNAs present in primate, but absent in rodent, contributed disproportionately to the differential expression between GZ subregions. Prominent among the validated targets of these miRNAs were cell-cycle and neurogenesis regulators. Coevolution between the emergent miRNAs and their targets suggested that novel miRNAs became integrated into ancient gene circuitry to exert additional control over proliferation. We conclude that multiple cell-cycle regulatory events contribute to the emergence of primate-specific cortical features, including the OSVZ, generated enlarged supragranular layers, largely responsible for the increased primate cortex computational abilities.

Efficient selection of biomineralizing DNA aptamers using deep sequencing and population clustering.

DNA-based information systems drive the combinatorial optimization processes of natural evolution, including the evolution of biominerals. Advances in high-throughput DNA sequencing expand the power of DNA as a potential information platform for combinatorial engineering, but many applications remain to be developed due in part to the challenge of handling large amounts of sequence data. Here we employ high-throughput sequencing and a recently developed clustering method (AutoSOME) to identify single-stranded DNA sequence families that bind specifically to ZnO semiconductor mineral surfaces. These sequences were enriched from a diverse DNA library after a single round of screening, whereas previous screening approaches typically require 5-15 rounds of enrichment for effective sequence identification. The consensus sequence of the largest cluster was poly d(T)30. This consensus sequence exhibited clear aptamer behavior and was shown to promote the synthesis of crystalline ZnO from aqueous solution at near-neutral pH. This activity is significant, as the crystalline form of this wide-bandgap semiconductor is not typically amenable to solution synthesis in this pH range. High-resolution TEM revealed that this DNA synthesis route yields ZnO nanoparticles with an amorphous-crystalline core-shell structure, suggesting that the mechanism of mineralization involves nanoscale coacervation around the DNA template. We thus demonstrate that our new method, termed Single round Enrichment of Ligands by deep Sequencing (SEL-Seq), can facilitate biomimetic synthesis of technological nanomaterials by accelerating combinatorial selection of biomolecular-mineral interactions. Moreover, by enabling direct characterization of sequence family demographics, we anticipate that SEL-Seq will enhance aptamer discovery in applications employing additional rounds of screening.

Origin of the PSEN1 E280A mutation causing early-onset Alzheimer's disease.

BACKGROUND: A mutation in presenilin 1 (E280A) causes early-onset Alzheimer's disease. Understanding the origin of this mutation will inform medical genetics. METHODS: We sequenced the genomes of 102 individuals from Antioquia, Colombia. We applied identity-by-descent analysis to identify regions of common ancestry. We estimated the age of the E280A mutation and the local ancestry of the haplotype harboring this mutation. RESULTS: All affected individuals share a minimal haplotype of 1.8 Mb containing E280A. We estimate a time to most recent common ancestor of E280A of 10 (95% credible interval, 7.2-12.6) generations. We date the de novo mutation event to 15 (95% credible interval, 11-25) generations ago. We infer a western European geographic origin of the shared haplotype. CONCLUSIONS: The age and geographic origin of E280A are consistent with a single founder dating from the time of the Spanish Conquistadors who began colonizing Colombia during the early 16th century.

Staged miRNA re-regulation patterns during reprogramming.

BACKGROUND: MiRNAs often operate in feedback loops with transcription factors and represent a key mechanism for fine-tuning gene expression. In transcription factor-induced reprogramming, miRNAs play a critical role; however, detailed analyses of miRNA expression changes during reprogramming at the level of deep sequencing have not been previously reported. RESULTS: We use four factor reprogramming to induce pluripotent stem cells from mouse fibroblasts and isolate FACS-sorted Thy1- and SSEA1+ intermediates and Oct4-GFP+ induced pluripotent stem cells (iPSCs). Small RNAs from these cells, and two partial-iPSC lines, another iPSC line, and mouse embryonic stem cells (mES cells) were deep sequenced. A comprehensive resetting of the miRNA profile occurs during reprogramming; however, analysis of miRNA co-expression patterns yields only a few patterns of change. Dlk1-Dio3 region miRNAs dominate the large pool of miRNAs experiencing small but significant fold changes early in reprogramming. Overexpression of Dlk1-Dio3 miRNAs early in reprogramming reduces reprogramming efficiency, suggesting the observed downregulation of these miRNAs may contribute to reprogramming. As reprogramming progresses, fewer miRNAs show changes in expression, but those changes are generally of greater magnitude. CONCLUSIONS: The broad resetting of the miRNA profile during reprogramming that we observe is due to small changes in gene expression in many miRNAs early in the process, and large changes in only a few miRNAs late in reprogramming. This corresponds with a previously observed transition from a stochastic to a more deterministic signal.

Exploratory data from complete genomes of familial alzheimer disease age-at-onset outliers.

Identifying genes that modify the age at onset (AAO) of Alzheimer disease and targeting them pharmacologically represent a potential treatment strategy. In this exploratory study, we sequenced the complete genomes of six individuals with familial Alzheimer disease due to the autosomal dominant mutation p.Glu280Ala in PSEN1 (MIM# 104311; NM_000021.3:c.839A>C). The disease and its AAO are highly heritable, motivating our search for genetic variants that modulate AAO. The median AAO of dementia in carriers of the mutant allele is 49 years. Extreme phenotypic outliers for AAO in this genetically isolated population with limited environmental variance are likely to harbor onset modifying genetic variants. A narrow distribution of AAO in this kindred suggests large effect sizes of genetic determinants of AAO in these outliers. Identity by descent (IBD) analysis and a combination of bioinformatics filters have suggested several candidate variants for AAO modifiers. Future work and replication studies on these variants may provide mechanistic insights into the etiopathology of Alzheimer disease.

Transcriptome profiling of the demosponge Amphimedon queenslandica reveals genome-wide events that accompany major life cycle transitions.

BACKGROUND: The biphasic life cycle with pelagic larva and benthic adult stages is widely observed in the animal kingdom, including the Porifera (sponges), which are the earliest branching metazoans. The demosponge, Amphimedon queenslandica, undergoes metamorphosis from a free-swimming larva into a sessile adult that bears no morphological resemblance to other animals. While the genome of A. queenslandica contains an extensive repertoire of genes very similar to that of complex bilaterians, it is as yet unclear how this is drawn upon to coordinate changing morphological features and ecological demands throughout the sponge life cycle. RESULTS: To identify genome-wide events that accompany the pelagobenthic transition in A. queenslandica, we compared global gene expression profiles at four key developmental stages by sequencing the poly(A) transcriptome using SOLiD technology. Large-scale changes in transcription were observed as sponge larvae settled on the benthos and began metamorphosis. Although previous systematics suggest that the only clear homology between Porifera and other animals is in the embryonic and larval stages, we observed extensive use of genes involved in metazoan-associated cellular processes throughout the sponge life cycle. Sponge-specific transcripts are not over-represented in the morphologically distinct adult; rather, many genes that encode typical metazoan features, such as cell adhesion and immunity, are upregulated. Our analysis further revealed gene families with candidate roles in competence, settlement, and metamorphosis in the sponge, including transcription factors, G-protein coupled receptors and other signaling molecules. CONCLUSIONS: This first genome-wide study of the developmental transcriptome in an early branching metazoan highlights major transcriptional events that accompany the pelagobenthic transition and point to a network of regulatory mechanisms that coordinate changes in morphology with shifting environmental demands. Metazoan developmental and structural gene orthologs are well-integrated into the expression profiles at every stage of sponge development, including the adult. The utilization of genes involved in metazoan-associated processes throughout sponge development emphasizes the potential of the genome of the last common ancestor of animals to generate phenotypic complexity.

Functionalization of a protosynaptic gene expression network.

Assembly of a functioning neuronal synapse requires the precisely coordinated synthesis of many proteins. To understand the evolution of this complex cellular machine, we tracked the developmental expression patterns of a core set of conserved synaptic genes across a representative sampling of the animal kingdom. Coregulation, as measured by correlation of gene expression over development, showed a marked increase as functional nervous systems emerged. In the earliest branching animal phyla (Porifera), in which a nearly complete set of synaptic genes exists in the absence of morphological synapses, these "protosynaptic" genes displayed a lack of global coregulation although small modules of coexpressed genes are readily detectable by using network analysis techniques. These findings suggest that functional synapses evolved by exapting preexisting cellular machines, likely through some modification of regulatory circuitry. Evolutionarily ancient modules continue to operate seamlessly within the synapses of modern animals. This work shows that the application of network techniques to emerging genomic and expression data can provide insights into the evolution of complex cellular machines such as the synapse.

Deep annotation of mouse iso-miR and iso-moR variation.

With a dataset of more than 600 million small RNAs deeply sequenced from mouse hippocampal and staged sets of mouse cells that underwent reprogramming to induced pluripotent stem cells, we annotated the stem-loop precursors of the known miRNAs to identify isomoRs (miRNA-offset RNAs), loops, non-preferred strands, and guide strands. Products from both strands were readily detectable for most miRNAs. Changes in the dominant isomiR occurred among the cell types, as did switches of the preferred strand. The terminal nucleotide of the dominant isomiR aligned well with the dominant off-set sequence suggesting that Drosha cleavage generates most miRNA reads without terminal modification. Among the terminal modifications detected, most were non-templated mono- or di-nucleotide additions to the 3'-end. Based on the relative enrichment or depletion of specific nucleotide additions in an Ago-IP fraction there may be differential effects of these modifications on RISC loading. Sequence variation of the two strands at their cleavage sites suggested higher fidelity of Drosha than Dicer. These studies demonstrated multiple patterns of miRNA processing and considerable versatility in miRNA target selection.

Identification of piRNAs in the central nervous system.

Piwi-interacting RNAs (piRNAs) are small noncoding RNAs generated by a conserved pathway. Their most widely studied function involves restricting transposable elements, particularly in the germline, where piRNAs are highly abundant. Increasingly, another set of piRNAs derived from intergenic regions appears to have a role in the regulation of mRNA from early embryos and gonads. We report a more widespread expression of a limited set of piRNAs and particularly focus on their expression in the hippocampus. Deep sequencing of extracted RNA from the mouse hippocampus revealed a set of small RNAs in the size range of piRNAs. These were confirmed by their presence in the piRNA database as well as coimmunoprecipitation with MIWI. Their expression was validated by Northern blot and in situ hybridization in cultured hippocampal neurons, where signal from one piRNA extended to the dendritic compartment. Antisense suppression of this piRNA suggested a role in spine morphogenesis. Possible targets include genes, which control spine shape by a distinctive mechanism in comparison to microRNAs.

Activation of apoptosis, but not necrosis, during Mycobacterium tuberculosis infection correlated with decreased bacterial growth: role of TNF-alpha, IL-10, caspases and phospholipase A2.

Monocyte/macrophage cell death is an important event during mycobacterial infection. To get insights about the influence of mononuclear phagocyte maturation in this event we compared the response to Mycobacterium tuberculosis (Mtb) infection of fresh isolated monocytes and monocyte-derived macrophages (MDM) from healthy tuberculin positive individuals. Both monocytes and MDM underwent apoptosis, however, there was a higher numbers of apoptotic macrophages with active Caspases 8 and 9. We also compared Mtb-induced cell death in U937 pro-monocytes and PMA-differentiated cells (U937D). In response to Mtb infection, U937D cells underwent apoptosis and promonocytes both apoptosis and necrosis. There were high number of U937D cells producing TNF-alpha and high number of IL-10+ promonocytes. These evidences suggest that U937 could be a valid model to study the mechanisms that rule Mtb-induced cell death. Experiments with the cell line and fresh isolated mononuclear cells with pharmacological inhibitors showed that induction of necrosis involved calcium and cAMP signals resulting in IL-10 production. Necrosis also correlated with Caspase 3, PLA2 activity and bacterial growth. In U937D cells and monocytes from healthy donors there was activation of calcium, TNF-alpha and Caspase 8 activation and decreased bacterial load. Understanding the mechanisms that control the dichotomy events between apoptosis and necrosis/oncosis associated with cell maturity might open new strategies to better control the course of mycobacterial infections.