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Next-generation sequencing facilitates quantitative analysis of transcriptomes of pro-inflammatory cytokines stimulated mouse dorsal root ganglionic neurons infected with lentivirus expressing GFP or a SNAP-25 cleaving protease

ABSTRACT: Purpose: The goals of this study are to investigate the influence of viral mediated cleavage of SNAP-25 in mouse dorsal root ganglionic neurons (mDRGs) on transcriptome profiling (RNA-seq). Methods: A total of 6 samples were tested using the DNBSEQ platform, 3 samples with viral mediated expression of GFP in mDRGs, the other 3 samples with viral mediated expression of the light chain protease of botulinum toxin A (LCA) in mDRGs, with an average yield of 1.12 G data per sample. The average alignment ratio of the sample comparison genome was 96.00%, which was comparative. The average alignment of the gene set was 83.78%; a total of 19963 genes were detected. Results: Using an optimized data analysis workflow, RNA-seq data confirmed cleavage of SNAP-25 affects the mRNA levels of certain neuropeptide genes, receptors and transducers. Altered expression of 5 genes were validated using RT–qPCR. RNA-seq data were consistent with RT-qPCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study show that viral mediated cleavage of SNAP-25 robustly downregulated mRNA levels of certain neuropeptide genes, receptors and transducers.

ORGANISM(S): Mus musculus

PROVIDER: GSE176059 | GEO | 2021/08/31

REPOSITORIES: GEO

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Project description:One of the major players controlling RNA decay is the cytoplasmic 5’ to 3’ exoribonuclease, which is conserved among eukaryotic organisms. In Arabidopsis, the 5’ to 3’ exoribonuclease XRN4 is involved in disease resistance, the response to ethylene, RNAi and miRNA-mediated RNA decay. Curiously, XRN4 appears to display selectivity among its substrates because certain 3’ cleavage products formed by miRNA-mediated decay, such as from ARF10 mRNA, accumulate in the xrn4 mutant, whereas others, such as from AGO1, do not. To examine the nature of this selectivity, transcripts that differentially accumulate in xrn4 were identified by combining PARE and Affymetrix arrays. Certain functional categories, such as stamen-associated proteins and hydrolases, were over-represented among transcripts decreased in xrn4, whereas transcripts encoding nuclear-encoded chloroplast-targeted proteins and nucleic acid-binding proteins were over-represented in transcripts increased in xrn4. To ascertain if RNA sequence influences the apparent XRN4 selectivity, a series of chimeric constructs were generated where the miRNA-complementary sites and different portions of the surrounding sequences from AGO1 and ARF10 were interchanged. Analysis of the resulting transgenic plants revealed that the presence of a 150 nucleotide sequence downstream of the ARF10 miRNA-complementary site conferred strong accumulation of the 3’ cleavage products in xrn4. In addition, sequence analysis of differentially accumulating transcripts led to the identification of 27 hexamer motifs that were over-represented in transcripts or miRNA-cleavage products accumulating in xrn4. Taken together, the data indicate that specific mRNA sequences, like those in ARF10, and mRNAs from select functional categories are attractive targets for XRN4-mediated decay. Mixed-stage inflorescences from Col-0 and xrn4-5 plants were harvested. Total RNA was extracted and hybridized to the ATH1 array to determine genes differentially expressed in xrn4-5 compared to Col-0. 2 replicates each.

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Next-generation sequencing facilitates quantitative analysis of transcriptomes of pro-inflammatory cytokines stimulated mouse dorsal root ganglionic neurons infected with lentivirus expressing GFP or a SNAP-25 cleaving protease

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