Project description:Goal: Identification of RNAs that are associated with the novel Ferry complex Methods: We performed a GST pulldown experiment, where GST-Ferry and GST were incubated with a HEK 293 cell lysate and extensively washed prior to elution. Subsequently, RNA was extracted from the elution and sequenced. Results: We identifed around 17 588 different transcripts and found a set of 252 mRNAs that are specifically enriched in the GST-Ferry sample. Conclusions: The Ferry complex seems to interact with a specific subset of mRNAs
Project description:As important roles of small RNA pathways, AGO proteins mediate interaction of incorporated small RNAs with their targets. The resolution of AGO associated small RNAs showed a significant landscape of AGO proteins and their binding small RNAs. To characterize small RNAs that associated with BmAGO2 protein in Bombyx mori, the small RNA population associated with BmAGO2 in BmN cells was extracted from the AGO immunoprecipitated complex and the small RNAs between 17nt to 50nt separated by a polyacrylamide gel electrophoresis were subjected to library construction and deep sequencing.The high throughput sequencing yielded a total of 11691441 reads, representing 813,702 unique reads with a abundance from 5731905 to 1.
Project description:Introduction: Glioblastoma (GBM) invasion studies have focused on coding genes, while few studies evaluate long non-coding RNAs (lncRNAs), transcripts without protein-coding potential, for role in GBM invasion. We leveraged CRISPR-interference (CRISPRi) to evaluate invasive function of GBM-associated lncRNAs in an unbiased functional screen, characterizing and exploring the mechanism of identified candidates. Methods: We implemented a CRISPRi lncRNA loss-of-function screen evaluating association of lncRNA knockdown (KD) with invasion capacity in Matrigel. Top screen candidates were validated using CRISPRi and oligonucleotide(ASO)-mediated knockdown in three tumor lines. Clinical relevance of candidates was assessed via The Cancer Genome Atlas(TCGA) and Genotype-Tissue Expression(GTEx) survival analysis. Mediators of lncRNA effect were identified via differential expression analysis following lncRNA KD and assessed for tumor invasion using knockdown and rescue experiments. Results: Forty-eight lncRNAs were significantly associated with 33-83% decrease in invasion (p<0.01) upon knockdown. The top candidate, LINC03045, identified from effect size and p-value, demonstrated 82.7% decrease in tumor cell invasion upon knockdown, while LINC03045 expression was significantly associated with patient survival and tumor grade(p<0.0001). RNAseq analysis of LINC03045 knockdown revealed that WASF3, previously implicated in tumor invasion studies, was highly correlated with lncRNA expression, while WASF3 KD was associated with significant decrease in invasion. Finally, WASF3 overexpression demonstrated rescue of invasive function lost with LINC03045 KD. Conclusion: CRISPRi screening identified LINC03045, a previously unannotated lncRNA, as critical to GBM invasion. Gene expression is significantly associated with tumor grade and survival. RNA-seq and mechanistic studies suggest that this novel lncRNA may regulate invasion via WASF3.
