ABSTRACT: Purpose: This study was performed to determine the RNA binding targets of the RNA binding protein CELF6 in the mouse brain. Method: 4 pools of CELF6-HA/YFP brains (cortices & cerebella removed) were snap frozen in liquid nitrogen, powdered, crosslinked (254 nm, 400 J/cm^2, 3 pulses), and homogenized and immunoprecipitated with anti-EGFP antibodies. Control samples consistent of input RNA from immunoprecipitates as well as anti-EGFP immunoprecipitates of wild-type (WT, HA/YFP negative) tissue. Sequencing libraries were prepared from harvested RNA with 9-mer unique molecular identifiers (UMIs). Procedure uses Trimmomatic to catalog UMIs as well as trim low quality bases, STAR to align to mm10 reference genome, Fulcrum Genomics FGBio to annotate BAM files with UMI information, Picard Tools to remove PCR duplicates, and Subread featureCounts to count reads mapping to features in a GTF in a strand specific manner. GTFs were prepared to contain annotated: 3'UTRs, 5'UTRs, CDS, as introns+/-10 bp overlap into surrounding exons. Additionally, BAMs were counted against features in a GTF corresponding to peaks identified by Piranha. To do this, an example BAM file (merge of all HA/YFP+ samples) was windowed using Bedtools, counted in a strand-specific manner using Subread featureCounts, and converted to BED format for peak calling with Piranha. The final called peaks were edited to a width of of 100 bp around significant maxima, and compiled into a GTF. This GTF was used for recounting individual sample files for immunoprecipitates and controls. Final counts were tested for significant enrichment in the HA/YFP-immunoprecipitated sample compared to (a) input samples, and (b) WT controls using edgeR. Samples were sequenced on 4 lanes of an 2xruns on an Illumina NextSeq, and BAMs were prepared on data for each lane separately and then merged across lanes by sample before counting. Results: Features showing nominal edgeR p values <0.05 in both YFP vs. Input and YFP vs. WT comparisons, as well as positive log2 fold change in YFP vs. Input and YFP vs. WT input comparison showed a predominance of significant comparisons falling in counts from 3'UTR regions. This was also true when restricting to only high confidence targets which showed FDR <0.05 by Benjamini-Hochberg. This was the case whether testing across sums under Piranha peaks in YFP vs. controls, as well as summing all counts across annotated 3'UTR, 5'UTR, introns, and CDS regardless of the presence of a called peak. Conclusions: CELF6 shows enriched binding in 3'UTR regions in YFP+ samples vs. controls. Nominally bound regions were used downstream to validate findings and explore the consequence of CELF6 associate in a massively parallel reporter assay. This is the first exploration of the binding targets of this RNA binding protein, linked to behavioral changes in knock-out mice (Dougherty et al. J. Neurosci 2013)
