Genes affected upon dsRNA knockdown treatment for nbr/CG9247 in Drosophila DL1 cells and small RNA profiling in Drosophila wild-type and nbr[f02257] mutants
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE32564 | GEO | 2011/11/01
SECONDARY ACCESSION(S): PRJNA147121
REPOSITORIES: GEO
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Project description:nbr/CG9247 gene function regulates the length of the 3'end of miRNAs. Such 3'end heterogeneity of miRNAs may impact miRNA target gene silencing. To address this, we performed microarray analysis for genes altered upon nbr knockdown in Drosophila DL1 cells. This approach revealed a subset of genes upregulated upon nbr knockdown.
2011-11-01 | GSE32683 | GEO
Project description:This SuperSeries is composed of the following subset Series: GSE32683: Genes affected upon dsRNA knockdown treatment for nbr/CG9247 in Drosophila DL1 cells GSE32684: Small RNA profiling in Drosophila wild-type and nbr[f02257] mutants Refer to individual Series
2011-11-01 | E-GEOD-32564 | biostudies-arrayexpress
Project description:nbr/CG9247 gene function regulates the length of the 3'end of miRNAs. Such 3'end heterogeneity of miRNAs may impact miRNA target gene silencing. To address this, we performed microarray analysis for genes altered upon nbr knockdown in Drosophila DL1 cells. This approach revealed a subset of genes upregulated upon nbr knockdown. Five independent replicates of DL1 cells treated with control (dsRNA to renilla) or nbr (dsRNA RNA to nbr/CG9247) were collected 5 days after dsRNA bathing. Total RNA from from each replicate was extracted using TRIzol reagent (Invitrogen). A portion of total RNA for each replicate was used to confirm the effect of nbr knockdown on one of the nbr-dependent miRNAs, miR-34, by small RNA northern.
2011-11-01 | E-GEOD-32683 | biostudies-arrayexpress
Project description:nbr/CG9247 gene regulates 3'end heterogeneity of a subset of miRNAs. It is not clear how broad this effect is on small RNA population. To address this, we compared small RNA population in wild-type and tmr[f02257] mutants. This approach identified more miRNAs whose 3'end heterogeneity was affected in nbr[f02257] mutants.
2011-11-01 | GSE32684 | GEO
Project description:nbr/CG9247 gene regulates 3'end heterogeneity of a subset of miRNAs. It is not clear how broad this effect is on small RNA population. To address this, we compared small RNA population in wild-type and tmr[f02257] mutants. This approach identified more miRNAs whose 3'end heterogeneity was affected in nbr[f02257] mutants. 2-3 day old control (w homogeneous strain Bloomington stock center 5905) and nbr[f02257] null mutant flies were collected. nbr[f02257] line was in the homogenous (Bloomington stock center stock 5905) background through a minimum of 5 backcrosses. Total RNA from whole flies was extracted using TRIzol reagent (Invitrogen). 40ug of total RNA from each genotype was used for small RNA library preparation with Small RNA Sample Prep kit (v1.5) (Illumina).
2011-11-01 | E-GEOD-32684 | biostudies-arrayexpress
Project description:Light is the source of energy for photosynthetic organisms; when in excess, however, it also drives the formation of reactive oxygen species and, consequently, photoinhibition. Plants and algae have evolved mechanisms to regulate light harvesting efficiency in response to variable light intensity so as to avoid oxidative damage. Nonphotochemical quenching (NPQ) consists of the rapid dissipation of excess excitation energy as heat. Although widespread among oxygenic photosynthetic organisms, NPQ shows important differences in its machinery. In land plants, such as Arabidopsis thaliana, NPQ depends on the presence of PSBS, whereas in the green alga Chlamydomonas reinhardtii it requires a different protein called LHCSR. In this work, we show that both proteins are present in the moss Physcomitrella patens. By generating KO mutants lacking PSBS and/or LHCSR, we also demonstrate that both gene products are active in NPQ. Plants lacking both proteins are more susceptible to high light stress than WT, implying that they are active in photoprotection. These results suggest that NPQ is a fundamental mechanism for survival in excess light and that upon land colonization, photosynthetic organisms evolved a unique mechanism for excess energy dissipation before losing the ancestral one found in algae.
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Project description:House flies (Musca domestica) are worldwide agricultural pests with estimated control costs at $375 million annually in the U.S. Non-target effects and widespread resistance challenge the efficacy of traditional chemical control. Double stranded RNA (dsRNA) has been suggested as a biopesticide for M. domestica but a phenotypic response due to the induction of the RNAi pathway has not been demonstrated in adults. In this study female house flies were injected with dsRNA targeting actin-5C or ribosomal protein (RP) transcripts RPL26 and RPS6. Ovaries showed highly reduced provisioning and clutch reductions of 94-99% in RP dsRNA treated flies but not in actin-5C or GFP treated flies. Gene expression levels were significantly and specifically reduced in dsRNA injected groups but remained unchanged in the control dsGFP treated group. Furthermore, injections with an Aedes aegypti conspecific dsRNA designed against RPS6 did not impact fecundity, demonstrating species specificity of the RNAi response. Analysis of M. domestica tissues following RPS6 dsRNA injection showed significant reduction of transcript levels in the head, thorax, and abdomen but increased expression in ovarian tissues. This study demonstrates that exogenous dsRNA is specifically effective and has potential efficacy as a highly specific biocontrol intervention in adult house flies. Further work is required to develop effective methods for delivery of dsRNA to adult flies.
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Project description:RNA interference (RNAi) is a powerful tool for studying functions of candidate genes in both model and nonmodel organisms and a promising technique for therapeutic applications. Successful application of this technique relies on the accuracy and reliability of methods used to quantify gene knockdown. With the limitation in the availability of antibodies for detecting proteins, quantitative PCR (qPCR) remains the preferred method for quantifying target gene knockdown after dsRNA treatment. We evaluated how qPCR primer binding site and target gene expression levels affect quantification of intact mRNA transcripts following dsRNA-mediated RNAi. The use of primer pairs targeting the mRNA sequence within the dsRNA target region failed to reveal a significant decrease in target mRNA transcripts for genes with low expression levels, but not for a highly expressed gene. By contrast, significant knockdown was detected in all cases with primer pairs targeting the mRNA sequence extending beyond the dsRNA target region, regardless of the expression levels of the target gene. Our results suggest that at least for genes with low expression levels, quantifying the efficiency of dsRNA-mediated RNAi with primers amplifying sequences completely contained in the dsRNA target region should be avoided due to the risk of false-negative results. Instead, primer pairs extending beyond the dsRNA target region of the mRNA transcript sequences should be used for accurate and reliable quantification of silencing efficiency.
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Project description:PELDOR (pulsed electron-electron double resonance) is an established method to study intramolecular distances and can give evidence for conformational changes and flexibilities. However, it can also be used to study intermolecular interactions as for example oligerimization. Here, we used PELDOR to study the "end-to-end" stacking of small double-stranded (ds) RNAs. For this study, the dsRNA molecules were only singly labeled with the spin label TPA to avoid multispin effects and to measure only the intermolecular stacking interactions. It can be shown that small dsRNAs tend to assemble to rod-like structures due to π-π interactions between the base pairs at the end of the strands. On the one hand, these interactions can influence or complicate measurements aimed at the determining of the structure and dynamics of the dsRNA molecule itself. On the other hand, it can be interesting to study such intermolecular stacking interactions in more detail, as for example their dependence on ion concentration. We quantitatively determined the stacking probability as a function of the monovalent NaCl salt and the dsRNA concentration. From these data, the dissociation constant K d was deduced and found to depend on the ratio between the NaCl salt and dsRNA concentrations. Additionally, the distances and distance distributions obtained predict a model for the stacking geometry of dsRNAs. Introducing a nucleotide overhangs at one end of the dsRNA molecule restricts the stacking to the other end, leading only to dimer formations. Introducing such an overhang at both ends of the dsRNA molecule fully suppresses stacking, as we demonstrate by PELDOR experiments quantitatively.
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Project description:The dsRNA binding protein (dsRBP) PACT was first described as an activator of the dsRNA dependent protein kinase PKR in response to stress signals. Additionally, it has been identified as a component of the small RNA processing pathway. A role for PACT in this pathway represents an important interplay between two modes of post-transcriptional gene regulation. The function of PACT in this context is poorly understood. Thus, additional approaches are required to clarify the mechanism by which PACT functions. In this study, the genetic utility of Drosophila melanogaster was employed to identify dsRNA-binding proteins that are functionally orthologous to PACT. Transgenic Drosophila expressing human PACT were generated to determine whether PACT is capable of functionally substituting for the Drosophila dsRBP R2D2, which has a well-defined role in small RNA biogenesis. Results presented here indicate that PACT is unable to substitute for R2D2 at the whole organism level.
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