Small RNA populations revealed by blocking rRNA fragments in Drosophila melanogaster reproductive tissues

RNA interference (RNAi) is a complex and highly conserved regulatory mechanism mediated via small RNAs (sRNAs). Recent technical advances in high throughput sequencing have enabled an increasingly detailed analysis of sRNA abundances and profiles in specific body parts and tissues. This enables investigations of the localized roles of microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, variation in the proportions of non-coding RNAs in the samples being compared can hinder these analyses. Specific tissues may vary significantly in the proportions of fragments of longer non-coding RNAs (such as ribosomal RNA or transfer RNA) present, potentially reflecting tissue-specific differences in biological functions. For example, in Drosophila, some tissues contain a highly abundant 30nt rRNA fragment (the 2S rRNA) as well as abundant 5’ and 3’ terminal rRNA fragments. These can pose difficulties for the construction of sRNA libraries as they can swamp the sequencing space and obscure sRNA abundances. Here we addressed this problem and present a modified “rRNA blocking” protocol for the construction of high-definition (HD) adapter sRNA libraries, in D. melanogaster reproductive tissues. The results showed that 2S rRNAs targeted by blocking oligos were reduced from >80% to < 0.01% total reads. In addition, the use of multiple rRNA blocking oligos to bind the most abundant rRNA fragments allowed us to reveal the underlying sRNA populations at increased resolution. Side-by-side comparisons of sequencing libraries of blocked and non-blocked samples revealed that rRNA blocking did not change the miRNA populations present, but instead enhanced their abundances. We suggest that this rRNA blocking procedure offers the potential to improve the in-depth analysis of differentially expressed sRNAs within and across different tissues

Mutations in Wnt2 Alter Presynaptic Motor Neuron Morphology and Presynaptic Protein Localization at the Drosophila Neuromuscular Junction

Wnt proteins are secreted proteins involved in a number of developmental processes including neural development and synaptogenesis. We sought to determine the role of the Drosophila Wnt7b ortholog, Wnt2, using the neuromuscular junction (NMJ). Mutations in wnt2 produce an increase in the number of presynaptic branches and a reduction in immunolabeling of the active zone proteins, Bruchpilot and synaptobrevin, at the NMJ. There was no change, however, in immunolabeling for the presynaptic proteins cysteine-string protein (CSP) and synaptotagmin, nor the postsynaptic proteins GluRIIA and DLG at the NMJ. Consistent with the presynaptic defects, wnt2 mutants exhibit approximately a 50% reduction in evoked excitatory junctional currents. Rescue, RNAi, and tissue-specific qRT-PCR experiments indicate that Wnt2 is expressed by the postsynaptic cell where it may serve as a retrograde signal that regulates presynaptic morphology and the localization of presynaptic proteins

Epsin 1 Promotes Synaptic Growth by Enhancing BMP Signal Levels in Motoneuron Nuclei

We thank Carl-Henrik Heldin (Uppsala University, Sweden) for his generous gift of the PS1 pMad antibody, Hugo Bellen, Corey Goodman, Janis Fischer, Graeme Davis, Guillermo Marques, Michael O'Connor, Kate O'Connor-Giles, and the Bloomington Drosophila Stock Center for flies strains, the Developmental Studies Hybridoma Bank at the University of Iowa for antibodies to Wit and CSP; Marie Phillips for advice on membrane fractionation; Avital Rodal, Kate O'Connor-Giles, Ela Serpe, Kristi Wharton, Mojgan Padash-Barmchi for discussions or comments on the manuscript. We also thank Jody Summers at OUHSC for her generosity in letting us to use her confocal microscope.Conceived and designed the experiments: PAV TRF LRC BZ. Performed the experiments: PAV TRF LRC SMR HB NER BZ. Analyzed the data: PAV TRF LRC SMR HB NER BZ. Wrote the paper: PAV TRF BZ.Bone morphogenetic protein (BMP) retrograde signaling is crucial for neuronal development and synaptic plasticity. However, how the BMP effector phospho-Mother against decapentaplegic (pMad) is processed following receptor activation remains poorly understood. Here we show that Drosophila Epsin1/Liquid facets (Lqf) positively regulates synaptic growth through post-endocytotic processing of pMad signaling complex. Lqf and the BMP receptor Wishful thinking (Wit) interact genetically and biochemically. lqf loss of function (LOF) reduces bouton number whereas overexpression of lqf stimulates bouton growth. Lqf-stimulated synaptic overgrowth is suppressed by genetic reduction of wit. Further, synaptic pMad fails to accumulate inside the motoneuron nuclei in lqf mutants and lqf suppresses synaptic overgrowth in spinster (spin) mutants with enhanced BMP signaling by reducing accumulation of nuclear pMad. Interestingly, lqf mutations reduce nuclear pMad levels without causing an apparent blockage of axonal transport itself. Finally, overexpression of Lqf significantly increases the number of multivesicular bodies (MVBs) in the synapse whereas lqf LOF reduces MVB formation, indicating that Lqf may function in signaling endosome recycling or maturation. Based on these observations, we propose that Lqf plays a novel endosomal role to ensure efficient retrograde transport of BMP signaling endosomes into motoneuron nuclei.Yeshttp://www.plosone.org/static/editorial#pee

A unique cluster of roo insertions in the promoter region of a stress response gene in Drosophila melanogaster

Transposable elements (TEs) are not randomly distributed in the genome. A genome-wide analysis of the D. melanogaster genome found that differences in TE density across 50 kb genomic regions was due both to transposition and duplication. At smaller genomic scales, promoter regions of hsp genes and the promoter region of CG18446 have been shown to accumulate TE insertions. In this work, we have further analyzed the promoter region of CG18446. We screened 218 strains collected in 15 natural populations, and we found that the CG18446 promoter region contains 20 independent roo insertions. Based on phylogenetic analysis, we suggest that the presence of multiple roo insertions in this region is likely to be the result of several bursts of transposition. Moreover, we found that the roo insertional cluster in the CG18446 promoter region is unique: no other promoter region in the genome contains a similar number of roo insertions. We found that, similar to hsp gene promoters, chromatin accessibility could be one of the factors explaining the recurrent insertions of roo elements in CG18446 promoter region.This work was funded by the European Commission (H2020-ERC-2014-CoG-647900). C.I. was funded by an ERASMUS+ fellowship. We acknowledge the support of the Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de laGeneralitat de Catalunya (GRC 2017 SGR 880). We also acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)