No description available.

Mechanisms of post-transcriptional control are critical to deploy RNAs and proteins asymmetrically to a discrete region of cytoplasm at the posterior of the Drosophila oocyte and embryo, called the pole plasm and thus allow differentiation of the germline. Research presented in this thesis investigates the post-transcriptional control of Drosophila pole plasm component germ cell-less (gcl ). Maternal gcl activity is required for germ cell specification and gcl RNA and protein accumulate asymmetrically in the pole plasm. gcl RNA, but not Gcl protein, is also detected in somatic regions of the embryo, and ectopic expression of Gcl in the soma causes repression of somatic patterning genes suggesting that gcl RNA is subject to translational control. I find that Gcl is expressed during oogenesis, where its expression is regulated by translational repressor Bruno (Bru). Increased levels of Gcl are observed in the oocyte when Bru is reduced (i.e., in an arrest heterozygote) and Bru overexpression reduces the amount of Gcl. Consistent with this, reduction of the maternal dosage of Bru leads to ectopic Gcl expression in the embryo, which, in turn, causes repression of anterior huckebein RNA expression. Bruno binds directly to the gcl3'UTR in vitro, but surprisingly, this binding is largely independent of a Bruno Response Element (BRE) in the gcl 3'UTR and depends upon a novel site. Furthermore, the gcl BRE-like region is not required to repress Gcl expression during oogenesis or embryogenesis. I concluded that Bru regulates gcl translation in a BRE-independent manner. In addition, I established the role of the gcl 3'UTR in gcl RNA localization and translation using transgenes that replace the endogenous 3'UTR with the alpha-tubulin 3'UTR or place it in tandem to the bicoid 3'UTR. I find that accumulation of gcl RNA in the embryonic pole plasm requires the gcl 3'UTR. Moreover, Gel is restricted to the pole plasm by translational repression mediated by the gcl 3'UTR and a limiting pool of trans-acting translational repressors. The phenotypic consequences of loss of this translational control are relatively mild, suggesting that gcl translation does not require stringent repression in the soma.

Diss. (sammanfattning) Umeå : Umeå universitet, 2008. / Härtill 5 uppsatser.

In 1990, Richard Jorgensen’s lab initiated a study to test if they could create a more vivid color petunia (Napoli et al. 1990). Their plan was to transform plants with the chalcone synthase transgene––the predicted rate limiting factor in the production of purple pigmentation. Much to their surprise, the transgenic plants, as well as their progeny, displayed a great reduction in pigmentation. This loss of endogenous function was termed “cosuppression” and it was thought that sequence-specific repression resulted from over-expression of the homologous transgene sequence. In 1998, Andrew Fire and Craig Mello described a phenomenon in which double stranded RNA (dsRNA) can trigger silencing of cognate sequences when injected into the nematode, Caenorhabditis elegans (Fire et al. 1998). This data explained observations seen years earlier by other worm researchers, and suggested that repression of pigmentation in plants was caused by a dsRNA-intermediate (Guo and Kemphues 1995; Napoli et al. 1990). The phenomenon––which soon after was coined RNA interference (RNAi)––was soon discovered to be a post-transcriptional surveillance system in plants and animals to remove foreign nucleic acids.

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.

Signal transduction by transforming growth factor β (TGF-β) cytokines is mediated by an evolutionarily conserved mechanism that depends on the Smad proteins to transduce an extracellular stimulus into the nucleus. In the unstimulated state, Smads spontaneously shuttle across the nuclear envelope and distribute throughout the cell. Upon TGF-β or bone morphogenetic protein (BMP) stimulation, the receptor-activated Smads are phosphorylated, assemble into complexes with Smad4, and become mostly localized in the nucleus. Such signal-induced nuclear translocation of activated Smads is essential for TGF-β–dependent gene regulation that is critical for embryonic development and homeostasis. The molecular machinery responsible for this process, especially how the activated Smads are imported as complexes, is not entirely clear. Thus, I became interested in investigating the molecular requirements for nuclear targeting of Smads upon stimulation. Recently, whole-genome RNAi screening offers a complementary cell-based approach to functionally identify molecules that mediate nuclear accumulation of Smads in response to TGF-β. In the first part of this dissertation, I performed a genome-wide RNAi screen that uncovered the importin moleskin (Msk) required in nuclear import of Dpp-activated MAD. Both genetic and biochemical studies further confirmed this finding. I also investigated Smad interactions with the Msk mammalian orthologues, Importin7 and 8 and validated that Smads are bona fide cargos of Imp7/8. Besides the importin Msk, the screen also uncovered a subset of nucleoporins as required factors in signal-induced nuclear accumulation of MAD. Thus in the second part of this thesis, I focused on how the NPC mediates this Msk-dependent nuclear import of activated MAD. Most of these nucleoporins, including Sec13, Nup75, Nup93 and Nup205, were thought to be structural nucleoporins without known cargo-specific functions. We, however, demonstrated that this subset of nucleoporins was specifically used in the Msk-dependent nuclear import of activated MAD but not the constitutive import of cargos containing a classic nuclear localization signal (cNLS). I also uncovered novel pathway-specific functions of Sec13 and Nup93. Regulation of TGF-β signaling can be achieved not only by modulating Smad nuclear translocation but also by modifying Smad phosphorylation status. Previously we identified a kinase, Misshapen (Msn), that caused the linker phosphorylation of MAD, resulting in negative regulation of Dpp signaling (Drosophila BMP). In the third part of this thesis, I investigated the biological relevance of Msn kinase to Dpp signaling in Drosophila wings. Both over-expression and RNAi studies suggest that Msn is a negative regulator of the Dpp/MAD pathway in vivo. As a whole, my findings delineated two critical requirements for MAD nuclear import: the importin Msk and a unique subset of nucleoporins. For the first time, structural Nups are implicated in the direct involvement of cargo import, providing a unique trans-NPC mechanism.

Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs from premicroRNA. My thesis focuses on the functional characteristics of two Drosophila Dicers that makes them specific for their biological substrates. We found that RNA binding protein partners of Dicers and two small molecules, ATP and phosphate are key in regulating Drosophila Dicers’ specificity. Without any additional factor, recombinant Dicer-2 cleaves pre-miRNA, but its product is shorter than the authentic miRNA. However, the protein R2D2 and inorganic phosphate block pre-miRNA processing by Dicer-2. In contrast, Dicer-1 is inherently capable of processing the substrates of Dicer, long dsRNAs. Yet, partner protein of Dicer-1, Loqs-PB and ATP increase the efficiency of miRNA production from pre-miRNAs by Dicer-1, therefore enhance substrate specificity of Dicer-1. Our data highlight the role of ATP and regulatory dsRNA-binding partner proteins to achieve substrate specificity in Drosophila RNA silencing. Our study also sheds light onto the function of the helicase domain in Drosophila Dicers. Although Dicer-1 doesn’t hydrolyze ATP, ATP enhances miRNA production by increasing Dicer-1’s substrate specificity through lowering its KM. On the other hand, Dicer-2 is a dsRNA-stimulated ATPase that hydrolyzes ATP to ADP, and ATP hydrolysis is required for Dicer-2 to process long dsRNA. Wild-type Dicer-2, but not a mutant defective in ATP hydrolysis, is processive; generating siRNAs faster than it can dissociate from a long dsRNA substrate. We propose that the Dicer-2 helicase domain uses ATP to generate many siRNAs from a single molecule of dsRNA before dissociating from its substrate. Piwi-dependent small RNAs, namely piRNAs, are a third class of small RNAs that are distinct from miRNAs and siRNAs. Their primary function is to repress transposons in the animal germline. piRNAs are Dicer-independent, and require Piwi family proteins for their biogenesis and function. Recently in addition to their presence in animal germlines, the presence and function of piRNA-like RNAs in the somatic tissues have been suggested (Yan et al. 2011; Morazzani et al. 2012; Rajasethupathy et al. 2012). We have investigated whether the piRNA-like reads in our many Drosophila head libraries come from the germline as a contaminant or are soma-specific. Most of the piRNA reads in our published head libraries show high similarity to germline piRNAs. However, piRNA-like reads from manually dissected heads are distinct from germline piRNAs, proving the presence of somatic piRNA-like small RNAs. We are currently asking the question whether these distinct piRNA-like reads in the heads are dependent on the Piwi family proteins, like the germline piRNAs.

The innate immune system is the first line of defense against infectious agents. It is essential for protection against pathogens and stimulation of long-term adaptive immune responses. Therefore, deciphering the mechanisms of the innate immune system is crucial for understanding the integrated systems of host defense against microbial infections, which is conserved from insects to humans. Despite lacking a conventional adaptive immune system, insects can mount a robust immune response against a wide array of microbial pathogens. These innate immune mechanisms have been widely studied in Drosophila melanogaster, because of the model system’s powerful genetic, genomic and molecular tools. The Drosophila immunity relies on cellular and humoral innate immune responses to fight pathogens. The hallmark of the Drosophilahumoral immune response is the rapid induction of antimicrobial peptide genes in the fat body, the homolog of the mammalian liver. Expression of these antimicrobial peptide genes is controlled by two distinct immune signaling pathways, the Toll pathway and the IMD (immune deficiency) pathway. The Toll pathway is activated by fungal and Gram-positive bacterial infections, whereas the IMD pathway responds to Gram-negative bacteria. Both pathways culminate in activation of the Rel/NF-кB transcription factors DIF (Dorsal-related immunity factor), Dorsal and Relish, which in turn translocate to the nucleus to induce the antimicrobial peptide genes. DIF and Dorsal are activated by the Toll pathway and control induction of antimicrobial peptide genes such as Drosomycin. The NF-кB precursor Relish, which is composed of an N-terminal Rel homology domain and a C-terminal IкB-like domain, is activated by the IMD pathway and initiates transcription of antimicrobial peptide genes such as Diptericin. Although many components of the Drosophila immune signaling pathways have been identified, the detailed mechanisms of signal trans

Neurons exhibit highly polarized structures, including two morphologically and functionally distinct domains, axons and dendrites. Dendrites and axons receive versus send information, and proper execution of each requires different sets of molecules. Differential distribution of membrane proteins in distinct neuronal compartments plays essential roles in neuronal functions. The major goal of my doctoral thesis was to study the molecular mechanisms that govern the differential distribution of membrane proteins in neurons, using the Drosophilalarval mushroom body (MB) as a model system. My work was initiated by an observation of differential distribution of distinct Dscam isoforms in neurons. Dscam stands for Down Syndrome Cell Adhesion Molecule, which is a Drosophila homolog of human DSCAM. According to genomic analysis, DrosophilaDscam gene can generate more than 38,000 isoforms through alternative splicing in its exons 4, 6, 9 and 17. All Dscam isoforms share similar domain structures, with 10 immunoglobulin domains and 6 fibronectin type III repeats in the ectodomain, a single transmembrane domain and a cytoplasmic endodomain. There are two alternative exons in exon 17 (17.1 and 17.2), which encodes Dscam’s transmembrane domain. Interestingly, in ectopic expression, Dscam isoforms carrying exon 17.1 (Dscam[TM1]) can be preferentially localized to dendrites and cell bodies, while Dscam isoforms carrying exon 17.2 (Dscam[TM2]) are distributed throughout the entire neuron including axons and dendrites. To unravel the mechanisms involved in the differential distribution of Dscam[TM1] versus Dscam[TM2], I conducted a mosaic genetic screening to identify the possible factors affecting dendritic distribution of Dscam[TM1], established an in vivoTARGET system to better distinguish the differential distribution of Dscam, identified the axonal and dendritic targeting motifs of Dscam molecules and further showed that Dscam’s differential roles in dendrites versus axons are correlated with its localization. Several mutants affecting dendritic distribution of Dscam[TM1] have been identified using a MARCM genetic screen. Three of these mutants (Dlis1, Dmn and p24) are components of the dynein/dynactin complex. Silencing of other dynein/dynactin subunits and blocking dynein function with a dominant-negative Glued mutant also resulted in mislocalization of Dscam[TM1] from dendrites to axons. However, microtubule polarity in the mutant axons was maintained. Taken together, this was the first demonstration that the dynein/dynactin complex is involved in the polarized distribution of membrane proteins in neurons. To further examine how dynein/dynactin is involved in the dendritic distribution of Dscam[TM1], I compromised dynenin/dynactin function with dominant-negative Glued and transiently induced Dscam[TM1] expression. The results suggested that dynein/dynactin may not be directly involved in the targeting of newly synthesized Dscam[TM1] to dendrites. Instead, it plays a role in maintaining dendritic restriction of Dscam[TM1]. Notably, dynein/dynactin dysfunction did not alter distribution of another dendritic transmembrane protein Rdl (Resistant to Dieldrin), supporting involvement of diverse mechanisms in distributing distinct molecules to the dendritic membrane. To identify the targeting motifs of Dscam, I incorporated the TARGET (Temporal and regional gene expression targeting) system into mushroom body (MB) neurons, and this allowed the demonstration of the differential distribution of Dscam[TM1] and Dscam[TM2] with more clarity than conventional overexpression techniques. Using the TARGET system, I identified an axonal targeting motif located in the cytoplasmic juxtamemebrane domain of Dscam[TM2]. This axonal targeting motif is dominant over the dendritic targeting motif located in Dscam’s ectodomain. Scanning alanine mutagenesis demonstrated that two amino acids in the axonal targeting motif were essential for Dscam’s axonal distribution. Interestingly, swapping the cytoplasmic juxtamembrane portions between TM1 and TM2 not only reversed TM1’s and TM2’s differential distribution patterns but also their functional properties in dendrites versus axons. My thesis research also involved studying endodomain diversity of Dscam isoforms. Besides the diversity originally found in the ectodomain and transmembrane domain of Dscam, my colleagues and I further demonstrated the existence of four additional endodomain variants. These four variants are generated by skipping or retaining exon 19 or exon 23 through independent alternative splicing. Interestingly, different Dscam endodomain isoforms are expressed at different developmental stages and in different areas of the nervous system. Through isoform-specific RNA interference, we showed the differential involvement of distinct Dscam endodomains in specific neuronal morphogenetic processes. Analysis of the primary sequence of the Dscam endodomain indicated that endodomain variants may confer activation of different signaling pathways and functional roles in neuronal morphogenesis. In Summary, my thesis work identified and characterized several previously unknown mechanisms related to the differential distribution of membrane proteins in neurons. I showed that there may be a dynein/dynactin-independent mechanism for selective transport of dendritic membrane proteins to dendrites. Second, dynein/dynactin plays a maintenance role in dendritic restriction of Dscam[TM1]. Third, different membrane proteins may require distinct combinations of mechanisms to be properly targeted and maintained in certain neuronal compartments. Further analysis of the mutants indentified from my genetic screen will definitely help to resolve the missing pieces of the puzzle. These findings provide novel mechanistic insight into the differential distribution of membrane proteins in polarized neurons.

This thesis focuses on several aspects of olfactory processing in Drosophila. In chapter I and II, I will discuss how odorants are encoded in the brain. In both insects and mammals, olfactory receptor neurons (ORNs) expressing the same odorant receptor gene converge onto the same glomerulus. This topographical organization segregates incoming odor information into combinatorial maps. One prominent theory suggests that insects and mammals discriminate odors based on these distinct combinatorial spatial codes. I tested the combinatorial coding hypothesis by engineering flies that have only one class of functional ORNs and therefore cannot support combinatorial maps. These files can be taught to discriminate between two odorants that activate the single functional class of ORN and identify an odorant across a range of concentrations, demonstrating that a combinatorial code is not required to support learned odor discrimination. In addition, these data suggest that odorant identity can be encoded as temporal patterns of ORN activity. Behaviors are influenced by motivational states of the animal. Chapter III of this thesis focuses on understanding how motivational states control behavior. Appetitive memory in Drosophilaprovides an excellent system for such studies because the motivational state of hunger promotes reliance on learned appetitive cues whereas satiety suppresses it. We found that activation of neuropeptide F (dNPF) neurons in fed flies releases appetitive memory performance from satiety-mediated suppression. Through a GAL4 screen, we identified six dopaminergic neurons that are a substrate for dNPF regulation. In satiated flies, these neurons inhibit mushroom body output, thereby suppressing appetitive memory performance. Hunger promotes dNPF release, which blocks the inhibitory dopaminergic neurons. The motivational drive of hunger thus affects behavior through a hierarchical inhibitory control mechanism: satiety inhibits memory performance through a subset of dopaminergic neurons, and hunger promotes appetitive memory retrieval via dNPF-mediated disinhibition of these neurons. The aforementioned studies utilize sophisticated genetic tools for Drosophila. In chapter IV, I will talk about two new genetic tools. We developed a new technique to restrict gene expression to different subsets of mushroom body neurons with unprecedented precision. We also adapted the light-activated adenylyl cyclase (PAC) from Euglena gracilis as a light-inducable cAMP system for Drosophila. This system can be used to induce cAMP synthesis in targeted neurons in live, behaving preparations.