Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Summary

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

Abstract

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

Introduction

Most human genes undergo Alternative Splicing (AS) to produce multiple isoforms with distinct activities, which has greatly increased the coding complexity of the genome^1,2. AS provides a major mechanism to regulate gene function, and it is tightly regulated through diverse pathways in different cellular and developmental stages^3,4. Because splicing misregulation is a common cause of human disease^5,6,7,8, targeting splicing r....

Protocol

1. Construction of a PUF Scaffold with Customized RNA-binding Specificity by Overlapping PCR

1. Design a series of PCR primers containing the PUF sequences that specifically recognize different RNA nucleotides in each position¹² (see Table 1 for the primer sequences and see Figure 1A for the RNA:PUF recognition code). For each PUF repeat, design four different primers to recognize a different base on each position.
   NOTE: These primers will be used in a series of four rounds of PCR reactions to generate PUF fragments that are joined together (Fig....

Results

This report describes the complete protocol for the design and construction of ESFs and splicing reporters. It also outlines the further application of ESFs in manipulating the AS of endogenous genes¹⁶. To illustrate typical results of ESF-mediated splicing changes, we use the data from our previous work as an example. The ESFs with different functional domains can be used to promote or inhibit the inclusion of the target cassette exon (Figure .......

Discussion

This report provides a detailed description for the design and construction of artificial splicing factors that can specifically manipulate the alternative splicing of a target gene. This method takes advantage of the unique RNA binding mode of PUF repeats to produce an RNA-binding scaffold with customized specificity. It can be used to either activate or repress splicing.

The critical step in this protocol is the generation of the reprogramed PUF domain that defines the specificity of ESFs. A.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
High-fidelity DNA polymerase (Phusion High-Fidelity) with PCR buffer	New England Biolabs	M0530L
DNA ligase (T4 DNA ligase)	New England Biolabs	M0202L
Liposomal transfection reagent (Lipofectamine 2000)	Invitrogen	11668-019
Reduced serum medium (Opti-MEM)	Gibco	31985-062
RNA extraction buffer (TRIzol Reagent)	ambion	15596018	TRIzol reagent includes phenol, which can cause burns. Wear gloves when handling
PBS (1x)	Life Technologies	10010-031
SuperScript III Reverse Transcriptase (RT)	Invitrogen	18080044
Caspase-3 antibody	Cell Signaling Technology	9668
PARP antibody	Cell Signaling Technology	9542
Bcl-x antibody	BD Bioscience	610211
beta-actin antibody	Sigma-Aldrich	A5441
alpha-tubulin antibody	Sigma-Aldrich	T5168
FLAG antibody	Sigma-Aldrich	F4042
Nitrocellulose membrane	Amersham-Pharmacia	RPN203D
ECL Western Blotting detection reagents	Invitrogen	WP20005
Cy5-dCTP	GE Healthcare	PA55021
Fluorescence-activated Cell Sorter (FACS)	BD Bioscience	FACSCalibur
Dulbecco’s Modified Eagle’s Medium (DMEM)	GE Healthcare	SH30243.01
Fetal Bovine Serum (FBS)	Invitrogen	26140079
Propidium iodide (PI)	Sigma	P4170
Bovine Serum Albumin (BSA)	Sigma	A7638-5G
Triton-X100	Promega	H5142
Poly-L-Lysine	Sigma	P-4832	Filter-sterilize and store at 4 °C
Vector pWPXLd	Addgene	12258
Vector pMD2.G	Addgene	12259
Vector psPAX2	Addgene	12260
DNase I (RNase-free)	New England Biolabs	M0303S
Oligo(dT)18 Primer	Thermo Scientific	SO131
Anti-mouse secondary antibody (Anti-mouse IgG, HRP-linked Antibody)	Cell Signaling Technology	7076S