Global analysis of physical and functional RNA targets of hnRNP L reveals distinct sequence and epigenetic features of repressed and enhanced exons

Complementary high-throughput sequencing approaches identify hnRNP L-dependent alternative splicing events in Jurkat T cells. (A) Experimental design in which Jurkat T cells were depleted of hnRNP L by transfection of antisense oligonucleotides (AMO) or transduction with a lentiviral-based shRNA, then grown under unstimulated conditions or stimulated with PMA for 48 h prior to RNA and protein extraction. (B) Western blot of hnRNP L depletion by AMO or lentiviral shRNA. HnRNP A1 is used as a loading control. (C) Regression analysis comparing the ΔPSI (hnRNP L KD-WT) determined by RASL-seq versus rMATS for cassette exons queried by both methods. Only ΔPSI with P < 0.05 are shown. In C–E, the gray lines in graph indicate an absolute value of ΔPSI = 10. (D) Scatterplot of significant (P < 0.05) inclusion level changes in unstimulated and stimulated conditions, as calculated by either sequencing methods. (E) Regression analysis comparing RASL/rMATS inferred values of ΔPSI versus RT-PCR-determined values for 48 cassette exons exhibiting either stringent non-responsiveness (ΔPSI < 3, P > 0.05) or significant responsiveness (ΔPSI > 10, P > 0.05) upon hnRNP L depletion (see also Supplemental Table S4). In cases in which both rMATS and RASL-seq provide evidence of a significant change, the ΔPSI value from the RASL-seq analysis is used. (F) GO categories for genes housing cassette exons that are significantly enhanced or repressed by hnRNP L (as defined in E). Top five categories that meet the threshold of P < 0.05, fold enrichment >1.5 are shown (see also Supplemental Table S5). (G) Regression analysis comparing RPKM for introns in wild-type versus hnRNP L-depleted cells. Slope of best-fit line is 0.98 (R² = 0.92) for unstimulated conditions and 1.08 (R² = 0.86) for stimulated conditions. Shown are only values for ∼800 introns identified in Braunschweig et al. (2014) to be retained in 20%–80% of messages. See Supplemental Figure S1B for regression analysis of all ∼200,000 introns (slope is 1.1 [R² = 0.99] for both cell conditions).