Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Area of Science:

• Molecular Biology
• RNA Biology
• Gene Regulation

Background:

• RNA Binding Proteins (RBPs) regulate eukaryotic RNA processing through modular domains.
• The Pumilio (PUF) domain offers a programmable RNA binding scaffold for engineering artificial RBPs.
• Previous work established PUF-based scaffolds for manipulating RNA metabolism.

Purpose of the Study:

• To describe a detailed protocol for constructing Engineered Splicing Factors (ESFs).
• To enable specific modulation of alternative splicing (AS) in target genes.
• To provide a new strategy for studying splicing regulation and isoform function.

Main Methods:

• Designing and constructing customized PUF scaffolds for specific RNA targets.
• Creating ESF expression plasmids by fusing designer PUF domains with effector domains.
• Utilizing splicing reporters and cultured human cells to assay ESF activity and splicing changes.

Main Results:

• Demonstrated the successful design and construction of ESFs.
• Showcased the application of ESFs in manipulating alternative splicing in human cells.
• Validated ESF-induced splicing changes and their downstream effects.

Conclusions:

• The developed protocol enables the design and generation of ESFs for targeted alternative splicing regulation.
• ESFs offer a versatile platform for studying splicing mechanisms and RNA processing.
• This approach facilitates the engineering of artificial factors for diverse RNA manipulation applications.