Project description:UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), relies on SILAC labelling to quantify RNA-associated protein recovery in the presence and absence of irradiation. We utilised TRAPP to study alterations in RNA-protein interactions upon exposure to weak acid stress in yeast. In addition, as UV irradiation induces short distance crosslinks between proteins and nucleic acids, the identity of crosslinked amino acids reveals the exact protein-RNA interacting interface. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified following phospho-peptide enrichment combined with a bioinformatic pipeline (Xi).
Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was performed to generate genome-wide binding maps of two U1-snRNP proteins: U1C and U1-70K in Trypanosoma brucei. 3 (2) biological replicates of U1C (U1-70K) -specific co-immunoprecipitated RNA after UV-crosslinking
Project description:The RNA helicase BRR2 (SNRNP200) is one of the key remodeling factors of the spliceosome. Here we show its direct interaction with C9ORF78, a poorly characterized protein predicted to be largely intrinsically disordered. We present cryo-EM structures showing how C9ORF78 and the spliceosomal B-complex protein FBP21 wrap around the C-terminal helicase cassette of BRR2 and that binding of the two proteins is mutually exclusive. C9ORF78 associates with the spliceosome, as we confirm via proteomics and RNA UV-crosslinking. An siRNA mediated C9ORF78 knockdown reveals changes in alternative splicing of specific target pre-mRNAs, which in part depend on its interaction with BRR2. In particular, C9ORF78 regulates a substantial number of alternative 3’ splice sites, which might be facilitated through an additional interaction with human PRP22 (DHX8).
Project description:The RNA helicase BRR2 (SNRNP200) is one of the key remodeling factors of the spliceosome. Here we show its direct interaction with C9ORF78, a poorly characterized protein predicted to be largely intrinsically disordered. We present cryo-EM structures showing how C9ORF78 and the spliceosomal B-complex protein FBP21 wrap around the C-terminal helicase cassette of BRR2 and that binding of the two proteins is mutually exclusive. C9ORF78 associates with the spliceosome, as we confirm via proteomics and RNA UV-crosslinking. An siRNA mediated C9ORF78 knockdown reveals changes in alternative splicing of specific target pre-mRNAs, which in part depend on its interaction with BRR2. In particular, C9ORF78 regulates a substantial number of alternative 3’ splice sites, which might be facilitated through an additional interaction with human PRP22 (DHX8).
Project description:The RNA helicase BRR2 (SNRNP200) is one of the key remodeling factors of the spliceosome. Here we show its direct interaction with C9ORF78, a poorly characterized protein predicted to be largely intrinsically disordered. We present cryo-EM structures showing how C9ORF78 and the spliceosomal B-complex protein FBP21 wrap around the C-terminal helicase cassette of BRR2 and that binding of the two proteins is mutually exclusive. C9ORF78 associates with the spliceosome, as we confirm via proteomics and RNA UV-crosslinking. An siRNA mediated C9ORF78 knockdown reveals changes in alternative splicing of specific target pre-mRNAs, which in part depend on its interaction with BRR2. In particular, C9ORF78 regulates a substantial number of alternative 3’ splice sites, which might be facilitated through an additional interaction with human PRP22 (DHX8).
Project description:Individual-nucleotide resolution UV-crosslinking and immunoprecipitation (iCLIP) combined with high-throughput sequencing was used to generate a transcriptome-wide binding map of hnRNP L. Supplementary file GSE37560_hnRNPL_crosslink_site.bed includes filtered crosslink sites of hnRNPL: combining data from all 3 experiments. 3 biological replicates of hnRNP L-specific and control (Flag) co-immunoprecipitated RNA after UV-crosslinking in HeLa cells
Project description:We report the identification of potential novel RNA targets for box C/D snoRNAs in human HEK293 cells, using the approaches of UV crosslinking and sequencing of hybrids (CLASH), and formaldehyde crosslinking and sequencing of hybrids (FLASH).
Project description:Mass spectrometry based screening for interaction of lncRNA mitolnc with mitochondrial proteins using UV crosslinking and hydrofluoric acid mediated (HF) degradation of RNA