GSE115442
Coordinate regulation of alternative pre-mRNA splicing events by the human RNA chaperone proteins hnRNPA1 and DDX5
Alternative pre-messenger RNA (pre-mRNA) splicing is a post-transcriptional mechanism for controlling gene expression. Splicing patterns are determined by both RNA binding proteins and nuclear pre-mRNA structure. Here, we analyze pre-mRNA splicing patterns, RNA binding sites and RNA structures near these binding sites coordinately controlled by two splicing factors, the heterogeneous nuclear ribonucleoprotein hnRNPA1 and the RNA helicase DDX5. We identified thousands of alternative pre-mRNA splicing events controlled by these factors by RNA-seq following RNA interference. Enhanced CLIP (eCLIP) on nuclear extracts was used to identify protein-RNA binding sites for both proteins in the nuclear transcriptome. We found a significant overlap between hnRNPA1 and DDX5 splicing targets and that they share many closely linked binding sites as determined by eCLIP analysis. In vivo SHAPE chemical RNA structure probing data was used to model RNA structures near several exons controlled and bound by both proteins. Both sequence motifs and in vivo UV crosslinking sites for hnRNPA1 and DDX5 were used to map binding sites in their RNA targets and often these sites flanked regions of higher chemical reactivity suggesting an organized nature to nuclear pre-mRNPs. This work provides a first glimpse into the possible RNA structures surrounding pre-mRNA splicing factor binding sites. Overall design: A human myeloid leukemia cell line, K562 cells, were treated with hnRNPA1 and DDX5 siRNA. Two replicate samples of hnRNPA1 knockdown and DDX5 knockdown were prepared for RNA-seq for differential pre-mRNA splicing analysis, along with control scramble siRNA samples. Triplicate eCLIP samples were prepared for hnRNPA1 and DDX5 using nuclear extracts in K562 cells, with input control and no UV treatment control samples.