Project description:We present results of RNA-Seq, Ribo-Seq, and RIP-Seq (YB-1, YB-3) experiments performed in HEK293T cells, as well as in HEK293T cells with YB-1 knockout and overexpression. The data shows YB-1 function as a global translation inhibitor and YB-3 ability to substitute YB-1 in its function in YB-1 knockout mutant.
Project description:piRNAs direct Piwi to repress transposons to maintain genome integrity in Drosophila ovarian somatic cells. piRNA maturation and association with Piwi occur at perinuclear Yb bodies, the centers of piRNA biogenesis. Here, we show that piRNA intermediates arising from the piRNA cluster flamenco (flam) concentrate into perinuclear foci adjacent to Yb bodies, termed Flam bodies. Although flam expression is not required for Yb body formation, Yb, the core component of Yb bodies, is required for Flam body formation. Abolishment of the RNA-binding activity of Yb disrupts both Yb bodies and Flam bodies. Loss of Zucchini, an endoribonuclease necessary for piRNA maturation, enlarges Flam bodies, which now superimpose with Yb bodies. Yb directly binds flam, but not neighboring protein-coding gene, transcripts. Thus, Yb integrates piRNA processing factors and piRNA intermediates into Yb bodies and Flam bodies, respectively, through direct binding to enhance piRNA biogenesis and formation of piRNA-inducing silencing complexes. HITS-CLIP was performed using OSC (Ovarian Somatic Cells). The antibody for Drosophila Yb, which was generated in this study, was used. Obtained CLIP tags were analyzed using illumina HiSeq200.
Project description:This SuperSeries is composed of the following subset Series: GSE21574: Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: QKI data GSE21575: Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: IGF2BP data GSE21577: Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: miRNA inhibition data GSE21918: Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP: sequencing data Refer to individual Series
Project description:In this study, we generated transcriptome profiles from HEK293T, HEK293T ΔYB-1ΔYB-3, and HEK293T ΔYB-1ΔYB-3 with the restored expression of YB-1 and/or YB-3. The data are used to explore YB-1 and YB-3 influence on transcriptome and translatome and their functional interchangeability in transcription and translation.
Project description:We developed a method for measuring non-specific background in PAR-CLIP data demonstrating that covalently crosslinked background binding is common, reproducible and apparently universal. Furthermore, we show that quantitative determination of background is essential for identifying targets of weakly binding RNA-binding proteins and can substantially improve motif analysis. To define background binding events in PAR-CLIP data we performed the standard PAR-CLIP protocol (Hafner et al., Cell 2010.) on lysates expressing a commonly used non-RBP control, FLAG-GFP. After FLAG-tag immunopurification of UV 365nm irradiated lysates prepared from cells supplemented with 4-thiouridine (4SU), RNA was partially digested with RNase T1, radiolabeled and separated by SDS-PAGE. Reads were sequenced by Illumina HiSeq. PAR-CLIP was also performed for HuR. Included as well is a total from lysates treated like PAR-CLIP, but without immunoprecipitation (see sample description for more detail).
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: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.