Investigating the regulation of alternative splicing and its role in disease

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Project 2: Mapping gene regulatory networks that control alternative splicing Our understanding of transcriptomic complexity has advanced in parallel with the development of large-scale methodological approaches enabling the detection and mapping of RNA binding proteins (RBPs) across the transcriptome. Such studies have provided mechanistic insights into RNA processing regulation and its contribution to disease-states. Despite this remarkable progress, we are still lacking a complete understanding of the regulatory networks that control alternative splicing. These is particularly prevalent in the context of stress-related pathways that are exploited from cancer cells to generate transcript isoform with advantageous effects on tumor growth. It is thus crucial to elucidate the molecular pathways that enable stress signaling to converge on remodeling alternative splicing programs. Aim 1: Development and application of a massively parallel and quantitative CRISPR screening platform for mapping splicing regulatory networks. To enable large-scale combinatorial CRISPR perturbations coupled with alternative pre-mRNA splicing readouts, we have developed a novel methodology combining CHyMErA combinatorial genetic perturbations with deep-sequencing readouts of splicing reporters to quantitatively measure splicing phenotypes and associate them with each perturbation. To achieve this, we modified our lentiviral combinatorial CRISPR screening vector to enable the expression of splicing minigene reporters in an inducible manner while harboring gRNA expression cassette within the 3' end of the reporter. Lentiviral delivery of these constructs in cells expressing Cas nucleases results in programable genome editing as determined by the constitutively expressed gRNAs, which is followed by the doxycycline-dependent activation of reporter expression at the desired time. mRNA from these cells is purified and Illumina sequencing libraries are generated from splicing reporter cDNA. Paired-end sequencing is used to quantify splicing reporter phenotypes and link them with the corresponding genetic perturbations determined by the gRNA sequences, thus enabling genome-wide interrogation of splicing regulators. By applying different splicing reporters, we have identified several hundred genes that can impact alternative splicing, genes that are highly enriched for RNA splicing and spliceosome biogenesis functional terms. This demonstrates the feasibility and the power of our approach to identify bona-fide splicing regulatory genes. Aim 2: Identification and characterization of novel regulators of alternative splicing programs. Our genome-wide splicing reporter screens have identified several genes to impact the splicing of multiple reporters that have not been previously associated with splicing regulation. Some of these genes also display evidence of interacting with splicing related proteins based on BIOGRID and/or STRING databases. These include genes such as PAXBP1, ZNF41, ZNF207 and KBTBD2 all of which are nuclear and rather understudied genes that have been mostly implicated with transcription regulation. We hypothesize that these factors can directly impact alternative splicing of specific genes. To test this hypothesis, we will rapidly deplete these genes in human cells using degrons and assess their impact on newly transcribed RNA using Nascent-Seq. We will also apply affinity purification coupled to mass spectrometry as well as crosslinking and immunoprecipitation experiments to identify the direct protein and RNA interactions of these factors and gain mechanistic insights on how they can regulate alternative splicing decisions. These studies are expected to provide novel insights of alternative splicing regulation in human cells. Aim 3: Which are the molecular pathways that control alternative splicing responses to cellular stress? An important challenge in the splicing field is to understand how splicing regulatory pathways respond to environmental changes including stresses imposed by alterations in osmolarity or temperature. Indeed, hyperosmotic stress and heat shock both result in widespread splicing alterations. We have generated splicing reporters that are responsive to these stresses, and we have performed genome-wide screens as described above in cells subjected to the corresponding stress condition. These screens have identified several candidate genes critical for stress-dependent splicing changes.