Massively Parallel Splicing Assay to Examine Splicing Errors Caused by Disease-Related Intronic Variants

Summary

Here, we present a detailed protocol for performing massively parallel splicing assays (MaPSy), which employ minigene constructs to systematically evaluate intronic variants in bulk. This approach enables high-throughput analysis of variant-induced splicing changes in cells through amplicon sequencing, providing functional assessments of their impact on pre-mRNA splicing.

Abstract

Splicing errors represent 10-30% of the pathogenic mutations responsible for rare genetic disorders. RNA splicing ensures proper gene expression by selectively joining exons and removing introns, with key regulatory sequences being located within the introns. The 5' splice site and branch site interact with small nuclear RNAs to form the spliceosome's recognition complex, while elements such as the polypyrimidine tract and splicing enhancers/silencers recruit proteins to regulate spliceosome assembly. Predicting splicing disruptions from intronic variants is challenging due to the complexity of these interactions.

Intronic variants, comprising 90% of natural human gene variations, may disrupt canonical splicing and give rise to disease. To investigate this possibility, we developed a massively parallel splicing assay (MaPSy) to assess patient-identified intronic variants. Synthesized oligonucleotides with reference or variant sequences were ligated into splicing minigenes containing promoter and polyadenylation signals. Each construct included two constant exons flanking a middle exon that harbored the variable intron-exon junction sequence of interest. The cellular splicing efficiency of the variant sequences was compared to reference counterparts, allowing us to identify significant disruptions as splicing variants.

The results of the MaPSy can be validated through additional approaches, such as minigene assays or CRISPR-mediated genome editing in vivo. Furthermore, aggregate analysis of the disrupted junctions can provide deeper insights into splicing mechanisms and the molecular basis of diseases associated with splicing errors.

Introduction

RNA splicing is a crucial process that joins exons for translation and removes introns, facilitating RNA export and maintaining nucleic acid homeostasis. This tightly regulated mechanism operates in a temporal and spatial manner, contributing to transcriptome diversity and complexity¹. Splicing is guided by key signals, including the 5' splice site (5'ss), branch site, and 3' splice site (3'ss), along with additional regulatory elements such as the polypyrimidine tract downstream of the branch site and the AG dinucleotide exclusion zone, which aid in 3'ss recognition². Mechanistically, U1 small nuclear RNA (snRNA) pairs ....

Protocol

1. Synthesis of the MaPSy oligonucleotide (oligo) library

1. Basic oligo structure: Design each oligo in the 155-nuncleotide (nt) pool to include a 35-nt exonic sequence and an 80-nt intronic sequence, forming a 115-nt gene-specific region flanked by 20-nt common priming sequences at each end (Figure 2A).
2. Variant collection: To retain motif integrity and focus on regulatory regions affecting splicing, collect the intronic variants that are:
   -78 to +10 nt from the 3'ss
   -3 to +30 nt from the 5'ss
   NOTE: Clinical variants can be sourced from ClinVar¹⁹, low-frequency en....

Results

Following cellular splicing of MaPSy constructs, both spliced and unspliced products are present as a mixture. Due to the library's size diversity and potential for noncanonical splicing, both types of products may appear somewhat diffuse on a gel. In constructs targeting the 3' end, the second intron, containing partial adenoviral sequences, tends to splice very robustly (Figure 4A).

In MaPSy experiments, approximately 10-30% of disease-relevant variants exhibit .......

Discussion

The intrinsic EGFP signal in the MaPSy construct enables fluorescence-based detection of exon skipping. If the sequence in the middle exon or introns promotes exon skipping, ligation of the first and third exons produces an EGFP signal detectable by FACS, making this a valuable method for identifying variants that influence exon skipping and facilitating microscopy-based visualization of splicing variants¹³. However, the amplicon-sequencing approach described herein does not capture exon-skipping .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Direct-zol RNA MiniPrep Plus kit	Zymo Research	R2072
Dulbecco’s Modified Eagle’s Medium (DMEM)	Thermo Fisher Scientific	11965084
Fetal Bovine Serum (FBS)	Thermo Fisher Scientific	26140079
L-Glutamine	Thermo Fisher Scientific	A2916801
Lipofectamine 3000	Thermo Fisher Scientific	L3000015
Penicillin-Streptomycin	Thermo Fisher Scientific	15140122
pGint plasmid	Addgene	24217
Phusion High-Fidelity DNA Polymerase	Thermo Fisher Scientific	F530L
QIAquick Gel Extraction Kit	Qiagen	28706
QIAquick PCR Purification Kit	Qiagen	28106
QIAxcel DNA Screening Kit (2400)	Qiagen	929004
SuperScript IV reverse transcriptase	Thermo Fisher Scientific	18090010