Project description:RNA interference (RNAi) is an evolutionarily conserved mechanism in plant and animal cells that directs the degradation of messenger RNAs homologous to short double-stranded RNAs termed small interfering RNA (siRNA). The ability of siRNA to direct gene silencing in mammalian cells has raised the possibility that siRNA might be used to investigate gene function in a high throughput fashion or to modulate gene expression in human diseases. The specificity of siRNA-mediated silencing, a critical consideration in these applications, has not been addressed on a genomewide scale. Here we show that siRNA-induced gene silencing of transient or stably expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genomewide expression profiling. A test for transitive RNAi, extension of the RNAi effect to sequences 5' of the target region that has been observed in Caenorhabditis elegans, was unable to detect this phenomenon in human cells. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
Project description:RNA interference (RNAi) is an evolutionarily conserved mechanism in plant and animal cells that directs the degradation of messenger RNAs homologous to short double-stranded RNAs termed small interfering RNA (siRNA). The ability of siRNA to direct gene silencing in mammalian cells has raised the possibility that siRNA might be used to investigate gene function in a high throughput fashion or to modulate gene expression in human diseases. The specificity of siRNA-mediated silencing, a critical consideration in these applications, has not been addressed on a genomewide scale. Here we show that siRNA-induced gene silencing of transient or stably expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genomewide expression profiling. A test for transitive RNAi, extension of the RNAi effect to sequences 5' of the target region that has been observed in Caenorhabditis elegans, was unable to detect this phenomenon in human cells. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
Project description:In flies, repeat-associated small interfering RNAs (rasiRNAs) ensure genomic stability by silencing endogenous selfish genetic elements such as retrotransposons and repetitive sequences. Here, we show that while small interfering RNAs (siRNAs) derive from both the sense and antisense strands of their double-stranded RNA precursors, rasiRNAs arise mainly from the antisense strand. rasiRNA production appear not to require Dicer-1, which makes microRNAs, or Dicer-2, which makes siRNAs, and rasiRNAs lack the 2´,3´ hydroxy termini characteristic of animal siRNA and miRNA. Unlike siRNAs and miRNAs, rasiRNAs function through the Piwi, rather than the Ago, Argonaute protein subfamily. Thus, rasiRNAs define a third RNA silencing pathway distinct from both the miRNA and RNAi pathways. Keywords: gene silencing; post-transcriptional gene regulation; short RNAs; RNAi; rasiRNAs; rasiRNA; microRNAs; microRNA; siRNAs; siRNA
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:The Argonautes (AGOs) are widely expressed, evolutionarily conserved RNA binding proteins that play an important role in gene expression regulation. The AGOs bind to small regulatory noncoding RNAs such as micro RNAs (miRNAs), short interfering RNAs (siRNAs), Piwi-interacting RNAs (piRNAs) etc. The small regulatory noncoding RNAs serve the function of guiding the AGOs to the right target RNAs by complementary base pairing. Additionally, the AGOs interact with GW182 (TNRC6A/-B/-C) proteins and together with small RNAs, they form an effector ribonucleo protein complex named, RNA Induced Silencing Complex (RISC) that regulates several aspects of transcriptional and post-transcriptional gene expression. ALG-1 (Argonaute Like Gene) and ALG-2 are the AGO proteins in C. elegans that are required for miRNA mediated gene expression regulation. Our efforts towards the characterization of the protein complexes comprised of ALG-1 led to the identification of DPF-3, a conserved protease belonging to clinically relevant Di Peptidyl Peptidase IV family, as the novel interacting partner of ALG-1. We have further explored the role of DPF-3 in AGO regulation.
Project description:Secondary metabolites are involved in the plant stress response. Among these are scopolin and its active form scopoletin, which are coumarin derivatives associated with reactive oxygen species scavenging and pathogen defence. Here we show that in Arabidopsis thaliana, scopolin accumulation can be induced in the root by osmotic stress and in the leaf by low temperature stress. A genetic screen for altered scopolin levels in Arabidopsis thaliana identified a mutant compromised for scopolin accumulation in response to stress; the lesion was present in a homologue of THO1, the product of which contributes to the THO/TREX complex. The THO/TREX complex contributes to RNA silencing, supposedly by trafficking precursors of small RNAs. Mutants carrying defective THO and RDR6 genes were impaired with respect to scopolin accumulation in response to stress, suggesting a mechanism based on RNA silencing like the transacting small interfering RNA pathway which requires THO/TREX and RDR6 function.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.