piRNA Function and Biogenesis in the \u3cem\u3eDrosophila\u3c/em\u3e Female Germline: A Dissertation

The studies presented in this thesis addressed mainly two aspects of Piwi-interacting RNA (piRNA) biology in the Drosophilagermline. We investigated the role of the piRNA pathway in embryonic axis specification. piRNAs mediate silencing of retrotransposons and the Stellate locus. Mutations in the Drosophila piRNA pathway genes armitage and aubergine disrupt embryonic axis specification, triggering defects in microtubule polarization and asymmetric localization of mRNA and protein determinants in the developing oocyte. Mutations in the ATR/Chk2 DNA damage signal transduction pathway dramatically suppress these axis specification defects, but do not restore retrotransposon or Stellatesilencing. Furthermore, piRNA pathway mutations lead to germline-specific accumulation of γ-H2Av foci characteristic of DNA damage. We conclude that piRNA based gene silencing is not required for axis specification, and that the critical developmental function for this pathway is to suppress DNA damage signaling in the germline. We have also identified a new member of the piRNA pathway. We show that mutations in rhino, which encodes a rapidly evolving Heterochromatin Protein 1 (HP1) chromo box protein, lead to germline specific DNA break accumulation, trigger Chk2 kinase dependent defects in axis specification, and disrupt germline localization of Piwi proteins. Mutations in rhino and the piRNA pathway gene armitage disrupt silencing of all major transposon families, but do not alter expression of euchromatic or heterochromatic protein coding genes. Deep sequencing studies show that rhino mutations significantly reduce or eliminate anti-sense piRNAs derived from the majority of transposable elements in the Drosophila genome, and lead to a dramatic reduction in piRNAs derived from major piRNA production clusters on chromosomes 2R and 4. Rhino protein localizes to distinct nuclear foci, and associates with the chromosome 2R and 4 clusters by chromatin immunoprecipitation. The Rhino HP1 homologue is therefore required for piRNA biogenesis, transposon silencing, and maintenance of germline genome integrity

Parameter Identification of Pressure Sensors by Static and Dynamic Measurements

Fast identification methods of pressure sensors are investigated. With regard to a complete accurate sensor parameter identification two different measurement methods are combined. The approach consists on one hand in performing static measurements - an applied pressure results in a membrane deformation measured interferometrically and the corresponding output voltage. On the other hand optical measurements of the modal responses of the sensor membranes are performed. This information is used in an inverse identification algorithm to identify geometrical and material parameters based on a FE model. The number of parameters to be identified is thereby generally limited only by the number of measurable modal frequencies. A quantitative evaluation of the identification results permits furthermore the classification of processing errors like etching errors. Algorithms and identification results for membrane thickness, intrinsic stress and output voltage will be discussed in this contribution on the basis of the parameter identification of relative pressure sensors.Comment: Submitted on behalf of EDA Publishing Association (http://irevues.inist.fr/EDA-Publishing

Biogenesis and germline functions of piRNAs

Small interfering RNAs and microRNAs are generated from double-stranded RNA precursors by the Dicer endonucleases, and function with Argonaute-family proteins to target transcript destruction or to silence translation. A distinct class of 24- to 30-nucleotide-long RNAs, produced by a Dicer-independent mechanism, associates with Piwi-class Argonaute proteins. Studies in flies, fish and mice implicate these Piwi-associated RNAs (piRNAs) in germline development, silencing of selfish DNA elements, and in maintaining germline DNA integrity. However, whether piRNAs primarily control chromatin organization, gene transcription, RNA stability or RNA translation is not well understood, neither is piRNA biogenesis. Here, we review recent studies of piRNA production and function, and discuss unanswered questions about this intriguing new class of small RNAs

Novel trends in optical non-destructive testing methods

Non-destructive testing (NDT) describes a wide range of methods for measuring and comparing physical quantities against a nominal condition. In this paper we describe and compare different optical NdT (ONDT)-methods with respect to their characteristics and capability for different measurement tasks. ONDT may be specified in two categories, passive and active. The NDT principles of the first category just use a measurement method like view inspection, elipsometry or reflectometry to detect defects which are easily accessible. The principles of the second category use an excitation force, such as heat or mechanical vibration introduced by transducers to detect hidden defects. This category can be specified into two subcategories. The first subcategory "time-/depth-resolved" includes measurement methods delivering detailed information of the geometric features of a hidden defect. Therefore the excitation of the material and the detection of the reaction have to provide a time step which enables depth-solved measurements. Phase-resolved thermography and laser ultrasound are examples for this category. The second subcategory "Integrating" includes measurement technique coupled with an excitation that enables detection of defects but not evaluation of their geometric features. Examples for these measurement techniques are shearography, reflectometry, vibrometry and thermography coupled with excitation method like simple heating or loading with a constant force. We demonstrate experimental results obtained using methods developed in our institute and highlight directions of further development

Normal microRNA Maturation and Germ-Line Stem Cell Maintenance Requires Loquacious, a Double-Stranded RNA-Binding Domain Protein

microRNAs (miRNAs) are single-stranded, 21- to 23-nucleotide cellular RNAs that control the expression of cognate target genes. Primary miRNA (pri-miRNA) transcripts are transformed to mature miRNA by the successive actions of two RNase III endonucleases. Drosha converts pri-miRNA transcripts to precursor miRNA (pre-miRNA); Dicer, in turn, converts pre-miRNA to mature miRNA. Here, we show that normal processing of Drosophila pre-miRNAs by Dicer-1 requires the double-stranded RNA-binding domain (dsRBD) protein Loquacious (Loqs), a homolog of human TRBP, a protein first identified as binding the HIV trans-activator RNA (TAR). Efficient miRNA-directed silencing of a reporter transgene, complete repression of white by a dsRNA trigger, and silencing of the endogenous Stellate locus by Suppressor of Stellate, all require Loqs. In loqs (f00791) mutant ovaries, germ-line stem cells are not appropriately maintained. Loqs associates with Dcr-1, the Drosophila RNase III enzyme that processes pre-miRNA into mature miRNA. Thus, every known Drosophila RNase-III endonuclease is paired with a dsRBD protein that facilitates its function in small RNA biogenesis

Braveheart, a Long Noncoding RNA Required for Cardiovascular Lineage Commitment

Long noncoding RNAs (lncRNAs) are often expressed in a development-specific manner, yet little is known about their roles in lineage commitment. Here, we identified Braveheart (Bvht), a heart-associated lncRNA in mouse. Using multiple embryonic stem cell (ESC) differentiation strategies, we show that Bvht is required for progression of nascent mesoderm toward a cardiac fate. We find that Bvht is necessary for activation of a core cardiovascular gene network and functions upstream of mesoderm posterior 1 (MesP1), a master regulator of a common multipotent cardiovascular progenitor. We also show that Bvht interacts with SUZ12, a component of polycomb-repressive complex 2 (PRC2), during cardiomyocyte differentiation, suggesting that Bvht mediates epigenetic regulation of cardiac commitment. Finally, we demonstrate a role for Bvht in maintaining cardiac fate in neonatal cardiomyocytes. Together, our work provides evidence for a long noncoding RNA with critical roles in the establishment of the cardiovascular lineage during mammalian development.Damon Runyon Cancer Research Foundation (DRG 2032-09)Damon Runyon Cancer Research Foundation (DFS 04-12)European Molecular Biology Organization (Long-term Fellowship)National Heart, Lung, and Blood Institute. Bench to Bassinet Program (U01HL098179)National Heart, Lung, and Blood Institute. Bench to Bassinet Program (U01HL098188)Smith Family FoundationPew Charitable Trusts. Program in the Biomedical Science

piRNAs, transposon silencing, and Drosophila germline development

Transposons are prominent features of most eukaryotic genomes and mobilization of these elements triggers genetic instability. Transposon silencing is particularly critical in the germline, which maintains the heritable genetic complement. Piwi-interacting RNAs (piRNAs) have emerged as central players in transposon silencing and genome maintenance during germline development. In particular, research on Drosophila oogenesis has provided critical insights into piRNA biogenesis and transposon silencing. In this system, the ability to place piRNA mutant phenotypes within a well-defined developmental framework has been instrumental in elucidating the molecular mechanisms underlying the connection between piRNAs and transposon control

COOLAIR Antisense RNAs Form Evolutionarily Conserved Elaborate Secondary Structures

SummaryThere is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. We investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription

Integrative Deep Sequencing of the Mouse Lung Transcriptome Reveals Differential Expression of Diverse Classes of Small RNAs in Response to Respiratory Virus Infection

We previously reported widespread differential expression of long non-protein-coding RNAs (ncRNAs) in response to virus infection. Here, we expanded the study through small RNA transcriptome sequencing analysis of the host response to both severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza virus infections across four founder mouse strains of the Collaborative Cross, a recombinant inbred mouse resource for mapping complex traits. We observed differential expression of over 200 small RNAs of diverse classes during infection. A majority of identified microRNAs (miRNAs) showed divergent changes in expression across mouse strains with respect to SARS-CoV and influenza virus infections and responded differently to a highly pathogenic reconstructed 1918 virus compared to a minimally pathogenic seasonal influenza virus isolate. Novel insights into miRNA expression changes, including the association with pathogenic outcomes and large differences between in vivo and in vitro experimental systems, were further elucidated by a survey of selected miRNAs across diverse virus infections. The small RNAs identified also included many non-miRNA small RNAs, such as small nucleolar RNAs (snoRNAs), in addition to nonannotated small RNAs. An integrative sequencing analysis of both small RNAs and long transcripts from the same samples showed that the results revealing differential expression of miRNAs during infection were largely due to transcriptional regulation and that the predicted miRNA-mRNA network could modulate global host responses to virus infection in a combinatorial fashion. These findings represent the first integrated sequencing analysis of the response of host small RNAs to virus infection and show that small RNAs are an integrated component of complex networks involved in regulating the host response to infection

MicroRNAs in pulmonary arterial remodeling

Pulmonary arterial remodeling is a presently irreversible pathologic hallmark of pulmonary arterial hypertension (PAH). This complex disease involves pathogenic dysregulation of all cell types within the small pulmonary arteries contributing to vascular remodeling leading to intimal lesions, resulting in elevated pulmonary vascular resistance and right heart dysfunction. Mutations within the bone morphogenetic protein receptor 2 gene, leading to dysregulated proliferation of pulmonary artery smooth muscle cells, have been identified as being responsible for heritable PAH. Indeed, the disease is characterized by excessive cellular proliferation and resistance to apoptosis of smooth muscle and endothelial cells. Significant gene dysregulation at the transcriptional and signaling level has been identified. MicroRNAs are small non-coding RNA molecules that negatively regulate gene expression and have the ability to target numerous genes, therefore potentially controlling a host of gene regulatory and signaling pathways. The major role of miRNAs in pulmonary arterial remodeling is still relatively unknown although research data is emerging apace. Modulation of miRNAs represents a possible therapeutic target for altering the remodeling phenotype in the pulmonary vasculature. This review will focus on the role of miRNAs in regulating smooth muscle and endothelial cell phenotypes and their influence on pulmonary remodeling in the setting of PAH