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Next generation sequence analysis for transcriptome of Drosophila melanogaster third instar larval eye discs expressing activated Ras either alone or along with changes in hsromega lncRNA levels

ABSTRACT: Purpose: While studying possible interaction between ras and hsrω genes we found that down- or up- regulation hsrω RNA levels results in further enhancement in activity hyperactive Ras protein in eye discs of Drosophila melanogaster. In order to find out reason underlying this phenomena we checked difference in gene expression between eye discs expressing activated Ras along with alterations in hsrω RNA levels and those expressing activated Ras alone. Method: Eye disc RNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsrωRNAi, sev-GAL4>EP3037, sev-GAL4>UAS-RasV12, sev-GAL4>UAS-hsrωRNAi UAS-RasV12 and sev-GAL4>EP3037 UAS-RasV12 were generated by sequencing, in triplicate, using Illumina Hiseq2500 platform using 50bp pair-end reads, 12 samples per lane and each sample run across 2 lanes. This resulted in a sequencing depth of ~20 million reads. The resulting sequencing FastQ files were mapped to the Drosophila genome (dm6) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to Cuffmerge to get final transcriptome assembly across samples. In order to ascertain differential expression of gene transcripts between different samples, Cuffdiff 2.1.1 was used (Trapnell et al., 2012). Result: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the Drosophila genome (dm6) and identified 15,905 transcripts with TopHat workflow in each genotypes studied. The sev-GAL4 driven expression of activated Ras in eye discs resulted in differential expression of many genes, with 374 genes being down-regulated and 138 up-regulated, when compared with those in sev-GAL4>UAS-GFP eye discs. Compared to only Ras expressing eye discs in case of down regulation of hsrω RNA in activated Ras background 148 gene were up-regulated while 434 were down-regulated. In eye discs expressing activated Ras alongwith over expression of hsrω RNA, 192 genes were up-regulated and 470 down-regulated compared to only activated Ras expressing eye discs. Some of these (11 genes) were validated using qRT-PCR. Conclusion: In activated Ras expressing eye discs, besides the expected increase in levels of transcripts of genes involved in cell growth, proliferation and differentiation, transcripts of many genes involved in RNA biosynthesis, metabolism and processing were up-regulated when compared with sev-GAL4>UAS-GFP eye discs. Levels of transcripts of none of the known members of Ras/Raf/MAPK signaling pathway were found to be significantly affected by changes in hsrω transcript levels in activated Ras expression background. Interestingly, several sn/snoRNAs along with a scaRNAs and some members of the RNA processing machinery were differentially expressed in activated Ras background and were further affected when the hsrω RNA levels were down-or up-regulated. In addition, while down-regulation of hsrω activity resulted in up-regulation of a few positive modulators of Ras signaling pathway, up-regulation of these transcripts caused down-regulation of the negative regulators of Ras/Raf/MAPK pathway, and thus resulting in increase in Ras activity in either cases.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE107529 | GEO | 2018/11/30

REPOSITORIES: GEO

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Project description:Purpose: To compare transcriptomic changes in 3rd instar larval eye discs following sev-GAL4 driven down- or up- regulation of hsrω lncRNAs. Method: Eye disc total RNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsrωRNAi, sev-GAL4>EP3037 were generated by sequencing, in duplicate, using Illumina Hiseq2500 platform using 50bp pair-end reads, 6 samples per lane and each sample run across 2 lanes. This resulted in a sequencing depth of ~20 million reads. The resulting sequencing FastQ files were mapped to the Drosophila genome (dm6) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to Cuffmerge to get final transcriptome assembly across samples. In order to ascertain differential expression of gene transcripts between different samples, Cuffdiff 2.1.1 was used (Trapnell et al., 2012). Result: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the Drosophila genome (dm6) and identified 15,905 transcripts with TopHat workflow in each genotypes studied. The sev-GAL4 driven expression of RNAi for hsromega gene transcripts in eye discs resulted in differential expression of many genes, with 409 genes being down-regulated and 100 up-regulated, when compared with those in sev-GAL4>UAS-GFP eye discs. Compared to control eye discs in case of up regulation of hsrω RNA, 66 gene were up-regulated while 635 were down-regulated. Some of these (11 genes) were validated using qRT-PCR. Conclusion: Analysis of RNA-seq data revealed that a large proportion of transcripts were indeed similarly affected by down- or up-regulation of hsrω RNA in normal as well as activated Ras expression background. A comparison of transcriptomes of sev-GAL4>hsrωRNAi and sev-GAL4>EP3037 eye discs revealed that in each case the number of genes down regulated was more than those up-regulated. Interestingly, while 319 genes were commonly down regulated and 15 were commonly up-regulated in the two genotypes, only 2 genes showed opposing trends between sev-GAL4>UAS-hsrωRNAi and sev-GAL4>EP3037 eye discs.

Project description:Purpose: Feeding larvae of fly models of polyQ and other neurodegenerative disorders on AR or RS supplemented food substantially suppressed neurodegeneration. With a view to gain further insight into the suppression of polyQ pathology by AR or RS, we expressed the UAS-127Q transgene using the predominantly eye-specific GMR-GAL4 driver and examined the transcriptomic changes in wild type and 127Q-expressing eye discs following feeding on AR or RS. In order to find out reason underlying the suppression of neurodegeneration by feeding of AR and RS, we checked difference in gene expression between eye discs expressing GMR-GAL4>UAS-127Q and wild type. Method: Eye disc RNA profiles of wandering late third instar larvae of GMR-GAL4>UAS-127Q and wild type larvae, reared on normal food or food supplemented with AR or RS since after hatching, were generated by sequencing, in triplicate, using Illumina Hiseq2500 platform using 51bp pair-end reads, 12 samples per lane and each sample run across 2 lanes of flowcell (Flowcell ID: C7K52ACXX).This resulted in a sequencing depth of 19-25 million reads. The resulting sequencing fastq files were mapped to the Drosophila genome (dm6) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to merge with Cuffmerge to get final transcriptome assembly across samples. In order to ascertain differential expression of transcripts between different samples, Cuffdiff 2.1.1 was used (Trapnell et al., 2012). Result: Using an optimized data analysis workflow, we mapped about 19-25 million sequence reads per sample to the Drosophila melanogaster genome (dm6) and identified 15,904 transcripts with cufflink workflow in each genotypes studied. The GMR-GAL4 driven UAS-127Q expression in eye discs reared on AR supplemented food resulted in 259 differential expression, with 147 genes being down-regulated and 112 up-regulated, when compared with those reared on normal food, however, when larvae from same genotype reared on RS supplemented food resulted in 1641 DEGs, with 846 genes being down-regulated and 795 up-regulated compared with larvae reared on normal food. In eye disc of wild type larvae reared on AR supplemented food resulted in 680 DEGs, with 470 genes being down-regulated and 205 up-regulated compared with those reared on normal food. Larvae from wild type reared on RS supplemented food resulted in 427 DEGs, with 264 genes being down-regulated and 163 up-regulated compared with those reared on normal food. Compared to only GMR-GAL4>UAS-127Q and wild type larval eye discs reared on normal food, total of 1691, with 799 gene were up-regulated while 892 were down-regulated. Conclusion: The present results not only re-inforce earlier findings from our lab that the Ayurvedic Amalaki Rasayana and Rasa-Sindoor are potent suppressors of neurodegeneration but also provide direct evidence that these general health-promoting formulations significantly alter expression of many genes in protein-aggregation-clearance pathways and thus eliminate the cause of proteinopathy so that cell death is inhibited while other normal cellular activities continue. These formulations, which have been used in Ayurvedic practice for thousands of years as health-promoting as well as curative supplements, do not have any side-effects. Therefore, we believe that these Ayurvedic formulations have a good potential to ameliorate the sufferings of polyQ and other neurodegenerative disorders.

Dataset Information

Next generation sequence analysis for transcriptome of Drosophila melanogaster third instar larval eye discs expressing activated Ras either alone or along with changes in hsromega lncRNA levels

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