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Journal of Statistical Software, 40, 10.18637/jss.v040.i01. To discriminate between individual Y RNA copies we used CLIP reads, aligned with novoalign. This molecule then undergoes a process called splicing which removes certain segments, and the resulting ‘messenger RNA’ molecule is then translated into protein. Filelinked codes latest 2020-2021 Journal of Statistical Software, 21, 10.18637/jss.v021.i12. Gene products in clusters with at least 10 nodes were examined for an enrichment of functional annotations with Enrichr (In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. Prior to UV treatment, cells were transferred to poly-D-lysine-coated 10-cm culture dishes (BD Biosciences, San Jose, CA) and allowed to adhere for at least 24 hr. CLIP allows the identification of functional RNA-protein interactions in vivo by using UV-irradiation of intact tissues to covalently crosslink and then purify RNA-protein complexes present in vivo (To gain insight into nELAVL-mediated RNA regulation in human brain we performed CLIP on postmortem brain samples of eight human subjects (nELAVL-crosslinked RNA tags were sequenced and mapped to the hg18 build of the human genome. They show that nELAVL knockdown in human neuroblastoma cells causes reduced expression of mRNAs that exhibit 3' UTR binding. Using loss-of-function and gain-of-function genetic screening in human cancer cells followed by biochemical reconstitution, we show that HQ461 acts by promoting an interaction between CDK12 and DDB1-CUL4-RBX1 E3 ubiquitin ligase, leading to polyubiquitination and degradation of CDK12-interacting protein Cyclin K (CCNK). nELAVL/Y RNA complexes were specifically remodeled in AD and after acute UV stress in neuroblastoma cells. Nonetheless, we were able to estimate that up to 6% of nELAVL was bound by Y RNAs, which is shown in We used a stringent definition of the nELAVL binding motif when searching Y RNA sequences (a stretch of 6 U’s, allowing a G at any position). The nELAV proteins have been widely studied. We performed crosslinking-immunoprecipitation and RNAseq on human brain, and identified nELAVL binding sites on 8681 transcripts. If we have no further questions, you will not hear from us. Scheckel, Drapeau et al. 31 Aug 2017 Notepad++ v7.5.1. This database has both common and human-specific features that confirm and enhance previous work in mice and underscore its value as a resource for scientific inquiry. We have linked the data to genome-wide measurements of both mRNA levels and splicing suggesting specific functions for these important interactions. The reviewers all felt that the authors should work to make some of these mechanistic connections between the Y RNAs and AD and thus strengthen this most interesting finding.1) The peak finding in the CLIP analysis needs to be better described. Structure-activity relationship analysis of HQ461 revealed the importance of a 5-methylthiazol-2-amine pharmacophore and resulted in an HQ461 derivate with improved potency. ShanaEncoder v4.5 29 Mar 2017. We therefore re-mapped CLIP tags, allowing multiple alignments, but reporting only the best match, permitting a more accurate estimate of overall Y RNA binding. We examined the 118 Y RNAs that did not fit this consensus in more detail. These copies are relatively short and very similar, yet too different to be detected by one Taqman probe or one qPCR primer set. Yet the RNA targets of nELAVL in human brain were completely unknown. To address this point, we have added cell fractionation experiments. 0.25% trypsin and 1% EDTA (Invitrogen) were used to passage cells every three days. nELAVL binding at these sites correlated between individuals (To relate nELAVL binding to mRNA abundance, we performed RNAseq on the same brain samples used for CLIP analysis (To identify genes most likely to be impacted by nELAVL, we defined the top 1000 nELAVL targets (colored in green in To investigate if nELAVL-mediated RNA regulation is conserved, we compared our dataset with previously published nELAVL targets in mice (We additionally investigated the overlap of nELAVL binding at individual binding sites. Most deleterious variants to date have been identified by exome sequencing while as many as 50% of disease-causing mutations are thought to affect splicing (To analyze the functional consequences of nELAVL binding, we used two different loss-of-function models: nELAVL 3’UTR binding has been implicated in increasing mRNA abundance in vivo (nELAVL has also been shown to regulate splicing in mouse brain by binding to intronic sequences (Splicing defects have been associated with many neurological diseases (While Y RNAs have not been linked to AD before, they have been implicated in various types of stress responses. ShanaEncoder v2.8 20 … Prime members enjoy fast & free shipping, unlimited streaming of movies and TV shows with Prime Video and many more exclusive benefits. ShanaEncoder v4.1.2 20 Jan 2017.