E-GEOD-40468 - Cold induced RNA-binding proteins influence alternative polyadenylation
Released on 1 May 2013, last updated on 9 May 2013
We employed PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify Cirbp and Rbm3 binding sites at transcriptome-level. Genome-wide RNA-seq analysis indicated that cold temperature leads to extensive 3’UTR lengthening whereas the loss of Cirbp or Rbm3 resulted in 3’UTR shortening. Combining with PAR-CLIP results, we found that these two RBPs repress the polyadenylation adjacent to their binding sites. Examination of the binding sites of Cirbp and Rbm3 by PAR-CLIP and their influence on the transcriptome by RNA-seq. PARCLIP was performed as in Hafner et al. 2010 Cell 141, 129–141, with MEFs cell lines stably expressing FLAG-tagged Cirbp and Rbm3. We used 4-thiouridine (4SU) to enhance the crosslink. For RNA-seq, polyadenylated RNA from the mock-transfected cells at 37°C or 32°C with two replicates, siCirbp-1, siCirbp-2, siRbm3-1 and siRbm3-2 MEFs at 37°C were sequenced on Solexa GAII using 76bp single-end kits according to the manufacturer’s instructions.
other, RNA-seq of coding RNA
Jun Yan, Yuting Liu