Project description:We constructed a DBR1 knockout cell line (C22) using CRISPR in HEK293T cells. Through mapping of lariat reads, lariat levels in the DBR1 - samples are shown to increase dramatically (~20x) relative to wild type cells. Over 60% of this increase in lariat levels is abrogated upon rescue of DBR1 - cells with a DBR1 expression vector
Project description:We used CRISPR in HEK293T cells to create two DBR1 knockout cell lines (C19 and C22). After high-throughput sequencing of total RNA extracted from these cells, we performed lariat read mapping using the method described in "Large-scale mapping of branchpoints in human pre-mRNA transcripts in vivo" (Taggart et al, 2012). We found lariats in the two DBR1- cell lines to be ~20-fold enriched relative to the levels observed in the HEK293T control samples.
Project description:AGO-PAR-CLIP was employed to identify microRNA binding sites in BCBL-1, a Kaposi's sarcoma-associated herpesvirus (KSHV) infected B-cell line and DG75, a KSHV negative B-cell line as a control. By using our novel computational method (PARma) and differential analysis of PAR-CLIP data, highly accurate target sites of KSHV microRNAs can be defined. Examination of microRNA target sites in two different cell lines using replicate PAR-CLIP experiments
Project description:The identification of RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as Crosslinking and Immunoprecipitation (CLIP) has revolutionized the genome-wide discovery of RNA-BP RNA targets. Among the different versions of CLIP that have been developed, the use of photoactivable nucleoside analogs has resulted in high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP) in vivo. Nonetheless, PAR-CLIP has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we determined suitable conditions for the incorporation of 4-thiouridine (4SU), a photoactivable nucleoside, into E. coli RNA and for the isolation of RNA crosslinked to RNA-BPs of interest. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA)-messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq, as well as functionally relevant sites of Hfq-mRNA interactions at nucleotide resolution. Based on our findings, PAR-CLIP represents an improved method to identify both the RNAs and the specific regulatory sites that are recognized by RNA-BPs in prokaryotes.
Project description:Probes flanking all predicted Candida albicans ORFs are used to detect intron sequences that accumulate in a debranchase (DBR1) mutant. We synthesized cDNA from RNA from both wild-type and dbr1-delta mutant strains grown under standard (YEPD) or various mixed conditions, labeled the two populations differentially, and hybridized them together on our microarray.