Project description:iCLIP experiments tomap the RNA binding sites of the RNA-binding protein Unkempt across the transcriptome in SH-SY5Y cells, HeLa cells with ectopic Unk expression and mouse E15 embryonic brain samples. Expression of Unk is normally largely restricted to the nervous system. We therefore mapped the binding sites in human SH-SY5Y and mouse E15 brain to detect its physiological binding sites (in SH-SY5Y, we also performed the RNAseq experiment upon Unk knockdown). HeLa cells on the other hand normally don't express Unk, but convert to neuron-like shape when the protein is ectopically expressed. So, here we hoped to identify those binding events (and hence target transcripts) that are critical for this morphological transformation.
Project description:We identified the precise genome-wide binding sites for all SR proteins, using iCLIP-seq SR proteins were encoded on stable transgenes, transfected in S2 cells, FLAG-tag immunopurified, and the bound RNA purified and subjected to RNA-seq. The resulting reads (CLIP tags) were aligned to the Drosophila genome and generated 38,695-5,900,000 unique CLIP tags for each SR-protein replicate.
Project description:About half of all human and mouse miRNA genes are located within introns of protein-coding genes. Despite this, little is known about functional interactions between miRNAs and their host genes. The intronic miRNA miR-128 regulates neuronal excitability and controls dendrite outgrowth of projection neurons during development of the mouse cerebral cortex. Its host genes R3hdm1 and Arpp21 encode highly conserved, putative RNA-binding proteins. Here we use iCLIP to describe the RNA-binding activity of ARPP21, which recognizes uridine-rich sequences with exquisite sensitivity for 3UTRs. Surprisingly, ARPP21 antagonizes miR-128 activity by co-regulating a subset of miR-128 target mRNAs enriched for neurodevelopmental functions. In contrast to miR-128, we show that ARPP21 acts as a positive post-transcriptional regulator, at least in part through interaction with the eukaryotic translation initiation complex eIF4F. This molecular antagonism is also reflected in inverse activities during dendritogenesis: miR-128 overexpression or knockdown of ARPP21 reduces dendritic complexity; ectopic ARPP21 leads to an increase. The regulatory interaction between ARPP21 and miR-128 is a unique example of convergent function by two products of a single gene.
Project description:Stau2 iCLIP of mouse brain was performed to identify RNA binding sites of Stau2 protein in mouse brain cells. The experiment was performed in triplicate, and each of the replicates was split into two separate halves at the cDNA stage, which together led to 6 separate datasets. The iCLIP protocol includes the following steps; Embryonic day 18 whole mouse brain was dissociated and irradiated with UV-C light, and the cells were then lysed using a buffer containing detergents. The RNAs were partially digested, and Stau2 and the cross-linked RNA fragments were immunoprecipitated using anti-Stau2 antibody. A DNA adaptor was then ligated to the RNA fragments and the cross-linked RNAs were further purified by SDS-PAGE and nitrocellulose membrane transfer. The RNAs were extracted from the membrane by proteinase K treatment, and converted into a high-throughput DNA sequencing compatible library by reverse transcription and PCR. For further details, see the methods of the associated manuscript. Note that the sequence reads start with (3 nucleotides of unique molecular identifiers) + (4 nucleotides of experimental barcode) + (2 nucleotides of unique molecular identifiers) followed by the sequence of the cross-linked RNA fragments.
Project description:Dynamic RNA-protein interactions govern the co-transcriptional packaging of RNA polymerase II (RNAPII)-derived transcripts. Yet, our current understanding of this process in vivo primarily stems from steady state analysis. To remedy this, we here conduct temporal-iCLIP (tiCLIP), combining RNAPII transcriptional synchronisation with UV cross-linking of RNA-protein complexes at serial timepoints. We apply tiCLIP to the RNA export adaptor, ALYREF; a component of the Nuclear Exosome Targeting (NEXT) complex, RBM7; and the nuclear cap binding complex (CBC). Regardless of function, all tested factors interact with nascent RNA as it exits RNAPII. Moreover, we demonstrate that the two transesterification steps of pre-mRNA splicing temporally separate ALYREF and RBM7 binding to splicing intermediates, and that exon-exon junction density drives RNA 5’end binding of ALYREF. Finally, we identify underappreciated steps in snoRNA 3’end processing performed by RBM7. Altogether, our data provide an unprecedented temporal view of RNA-protein interactions during the early phases of transcription.