Project description:We report that AUF1 modulates global mRNA stability and translation, in turn promoting the maintenance of DNA integrity. Please see individual series. In short, for AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.
Project description:We report that AUF1 modulates global mRNA stability and translation, in turn promoting the maintenance of DNA integrity. Please see individual series. In short, For AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.
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:We report that AUF1 modulates global mRNA stability and translation, in turn promoting the maintenance of DNA integrity. Please see individual series. In short, for AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.
Project description:We report that AUF1 modulates global mRNA stability and translation, in turn promoting the maintenance of DNA integrity. Please see individual series. For AUF1 PAR-CLIP, the four isoforms of AUF1 (p37, p40, p42, and p45) tagged with a Flag epitope were expressed in HEK293 cells. For total RNA-Seq HEK293 cells were transfected with Control siRNA, AUF1 siRNA, Empty Vector, Flag-AUF1 p37, p40, p42, or p45 as well as WI-38 cells were collected at PDL 15 and 55 and also transfected with Control siRNA, AUF1 siRNA, HuR siRNA. For Ribo-Seq HeLa cells were transfected with Control siRNA, AUF1 siRNA, or HuR siRNA.
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: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.