mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA-binding protein

mutants deleted for TDP-1, an ortholog of the neurodegeneration-associated RNA-binding protein TDP-43, display only mild phenotypes. nuclear dsRNA accumulation in multiple tissues. Immunoprecipitation using a dsRNA-specific antibody revealed that mutant animals accumulate a variety of double-stranded transcripts indicating a global effect of TDP-1 on RNA structure or stability. Analysis of TDP-1 binding buy Prasugrel (Effient) by deep sequencing of anti-TDP-1 chromatin immunoprecipitation (ChIP) indicated that TDP-1 associates with highly structured regions co-transcriptionally. The reduction Mouse monoclonal to CD21.transduction complex containing CD19, CD81and other molecules as regulator of complement activation of dsRNA accumulation is likely to be a conserved function of TDP-43 proteins because knockdown of mammalian TDP-43 in HeLa cells and M17 neuronal cells also causes dsRNA accumulation. Results RNA transcripts aberrantly represented in mutant animals buy Prasugrel (Effient) to identify changes in RNA metabolism or large quantity. While two deletion alleles exist for and allele appeared to be a clear null (Supplementary Fig S1B and C); therefore, our study is focused on this allele. We produced and sequenced poly(A)-selected cDNA libraries from wild-type and buy Prasugrel (Effient) animals. Mapping of sequenced reads showed over 50% of all amplifiable annotated genes were well represented (roughly 14,000 transcripts). A differential gene expression comparison (RPKMs) between mutants and wild-type revealed over 1,700 transcripts aberrantly represented with close to an equal quantity of transcripts underrepresented as overrepresented (Fig?(Fig1A;1A; Supplementary Table S1). qRTCPCR verification of a select set of these large quantity changes is usually shown in Supplementary Fig S2. Gene ontology analysis of aberrantly represented transcripts in mutants indicated very buy Prasugrel (Effient) few pathways were enriched among over- and underexpressed genes (Supplementary Table S2). Most enriched pathways were associated with developmental processes and stage-specific molting, which may be an artifact due to the moderate growth delay reported in mutant animals (Zhang < 0.001) changes in splice site representation (Supplementary Table S3, independent verification in Supplementary Fig S3); however, the majority of transcripts altered in splicing were not altered in abundance compared to wild-type. While splicing abnormalities may contribute to loss of function defects, splicing differences do not readily explain changes in transcript large quantity in deletion using the IGV genome browser and online databases (wormbase, aceview) for common characteristics within the RNA molecule. Interestingly, we noticed that a large proportion of altered transcripts experienced potential double-stranded structure. Specifically, many over/underexpressed transcripts contained either antisense overlap with another gene or multiple inverted repeats within intronic regions. To quantify these effects, we first analyzed the percentage of altered genes with antisense overlap to another coding gene. While about 8% of the worm genome is usually arranged antisense to another gene (Thierry-Mieg & Thierry-Mieg, 2006), approximately 35% of increased and decreased transcripts were arranged in this manner, representing a highly significant enrichment (= 5.3 10?181, hypergeometric distribution (hgd)) (Fig?(Fig1B).1B). We also decided the percentage of genes made up of intronic inverted repeats that were aberrantly represented in deletion. We limited our analysis to inverted repeats contained within introns greater than 1 kb. While about 25% of all expressed genes contained an intron inverted repeat (2,641), 40% of underrepresented genes in RNA-seq contained inverted repeats, representing a significant enrichment (= 4 10?5, hgd, = 3.2 10?3, chi-square test) (Fig?(Fig1B).1B). Among overexpressed transcripts, inverted repeat made up of introns were not significantly enriched, suggesting that intronic RNA structure in mutants results in reduction of mRNA large quantity. Transcripts with antisense overlap to another RNA and transcripts made up of inverted repeats are capable of forming either inter- or intra-molecular dsRNA. Because deletion of perturbed the large quantity of these transcripts, we hypothesized that TDP-1 has a fundamental function in the formation, or metabolism, of dsRNA. TDP-1 limits the accumulation of double-stranded RNA To address the hypothesis that TDP-1 affects dsRNA metabolism, we looked directly at the amount and localization of dsRNA in mutant worms by immunostaining with a dsRNA-specific antibody, J2. The.