Dissecting splicing decisions and cell-to-cell variability with designed sequence libraries

Abstract

Most human genes are alternatively spliced, allowing for a large expansion of the proteome. The multitude of regulatory inputs to splicing limits the potential to infer general principles from investigating native sequences. Here, we created a rationally designed library of >32,000 splicing events to dissect the complexity of splicing regulation through systematic sequence alterations. Measuring RNA and protein splice isoforms allowed us to investigate both cause and effect of splicing decisions, quantify diverse regulatory inputs and accurately predict (R2=0.75-0.85) isoform ratios from sequence and secondary structure. By profiling individual cells, we measure the cell-to-cell variability of splicing decisions and show that it can be encoded in the DNA and influenced by regulatory inputs, opening the door for a novel, single-cell perspective on splicing regulation.

Gene Architecture and Sequence Composition Underpin Selective Dependency of Nuclear Export of Long RNAs on NXF1 and the TREX Complex

“…To examine the relationship between NXF1 sensitivity and splicing in a better controlled setting, we used a library of thousands of reporters carrying splicing events based on a set of 38 native introns with various systematic splicing-related sequence changes and hence variable splicing efficiencies stably integrated into K562 cells (Mikl et al, 2019). We perturbed NXF1 with RNAi in these cells and measured subcellular localization and splicing efficiency of the reporter mRNAs (Figures 6C, S6C, and S6D; Table S3).…”

“…Fractionation and library construction for massively parallel splicing reporter assay We employed the existing collection of designed and genomically integrated intron retention library, composed of $8500 variants of a retained intron flanked by exonic sequences as described in Mikl et al(2019). K562 cells with integrated library were grown in IMDM (GIBCO) containing 10% fetal bovine serum and Penicillin-Streptomycin mixture (1%) at 37 C in a humidified incubator with 5% CO 2 .…”

High-throughput mutagenesis identifies mutations and RNA-binding proteins controlling CD19 splicing and CART-19 therapy resistance

“…Massively parallel reporter assays such as our high-throughput mutagenesis screen provide comprehensive insights into the regulatory code of splicing, as they characterise the complete set of cis-acting sequence mutations and reveal the binding sites of trans-acting RNA-binding proteins (e.g., [18][19][20][32][33][34]). The interpretation of these datasets is challenging due to nonlinear interactions of individual mutation effects.…”

MTSplice predicts effects of genetic variants on tissue-specific splicing

“…One approach to overcome the issue of observational data is to perform massively parallel reporter assays (MPRA) for different cell types. MPRA for human splicing have been performed in HEK293 cells [25,27,[56][57][58], K562 cells [59,60], HepG2 cells [59], and HELA and MCF7 cells [61]. These data provide powerful resources to train complex models on splicing, but tissue and cell-type diversity is still lacking.…”