Targeting RNA Splicing: Mechanisms and Strategies

What Is RNA Splicing?

RNA splicing is a post-transcriptional modification process that occurs in eukaryotic cells. When a gene is transcribed, the initial RNA product is called pre-mRNA, which contains both coding regions (exons) and non-coding regions (introns). RNA splicing removes the introns from the pre-mRNA molecule and joins together the exons to form a mature mRNA molecule that can be translated into protein.

What Is the Process of RNA Splicing?

The process of splicing is carried out by a complex called the spliceosome, which is composed of small nuclear ribonucleoproteins (snRNPs) and other protein factors. The spliceosome recognizes specific sequences at the boundaries between exons and introns, known as splice sites.

During splicing, the spliceosome assembles on the pre-mRNA molecule and brings together the donor site at the 5' end of the intron and the acceptor site at the 3' end of the intron. The intron is then removed in a two-step process. First, the donor site is cleaved, and the 5' end of the intron is joined to a branch site within the intron, forming a looped structure called a lariat. Then, the free 3' end of the upstream exon is joined to the 5' end of the downstream exon, resulting in the removal of the intron and the splicing together of the exons.

The splicing process can be complex, and alternative splicing allows for the production of multiple mRNA isoforms from a single gene. Different combinations of exons can be included or excluded during splicing, resulting in the generation of different protein variants with potentially distinct functions.

What Is Alternative Splicing?

Alternative splicing refers to the process by which different combinations of exons within a pre-mRNA molecule can be included or excluded during RNA splicing, leading to the generation of multiple mRNA isoforms from a single gene. This phenomenon allows a single gene to produce different types of mRNA molecules and, consequently, different protein isoforms.

What Is the Process of Alternative Splicing?

The process of alternative splicing provides a way to increase the protein diversity encoded by the genome without the need for a corresponding increase in the number of genes. By selectively including or excluding specific exons during splicing, cells can generate mRNA molecules with different exon compositions. Each mRNA variant can then be translated into a distinct protein isoform, thereby expanding the functional repertoire of the genome.

This mechanism is often referred to as a "space-saving" or "economical" process because it allows cells to achieve greater protein diversity using a relatively limited number of genes. Instead of having separate genes for each protein variant, alternative splicing enables the creation of multiple protein isoforms from a single gene.

The specific combination of exons included in the mature mRNA molecule can vary depending on factors such as cell type, developmental stage, or environmental conditions. Alternative splicing plays a significant role in various biological processes, including tissue-specific functions, cell signaling, and the regulation of gene expression.

There are different modes of alternative splicing, including:

• Exon skipping: In this mode, an exon is skipped during splicing, resulting in the exclusion of that exon from the mature mRNA. This leads to the production of a shorter protein isoform.
• Intron retention: Instead of being completely removed, an intron is retained in the mature mRNA. The retained intron can contain important regulatory elements, such as protein-binding sites or microRNA target sites, which can influence gene expression or protein function.
• Alternative 5' or 3' splice sites: Different splice sites at the 5' or 3' ends of an exon can be used, resulting in the inclusion or exclusion of specific portions of the exon in the mature mRNA.
• Mutually exclusive exons: In this mode, two or more exons are positioned in such a way that only one of them can be included in the mature mRNA, while the others are excluded.

The specific combination of exons included in the mature mRNA depends on various factors, including the tissue or cell type, developmental stage, and environmental conditions.

How To Target RNA Splicing?

RNA-Seq plays a crucial role in characterizing the splicing alterations, identifying specific targets, and assessing the impact of the interventions on splicing patterns. It provides a comprehensive and high-resolution view of the transcriptome, allowing researchers to investigate the effects of targeted approaches on RNA splicing.

Antisense Oligonucleotides (ASOs) and RNA-Seq

RNA-Seq can be used to identify aberrant splicing events or alternative splicing isoforms in a particular cellular context or disease. By analyzing RNA-Seq data, one can identify specific target exons or splice sites that need to be modified. ASOs can then be designed to target these regions and redirect splicing to desired outcomes. Following ASO treatment, RNA-Seq can be used again to assess the impact of ASOs on splicing patterns and validate the desired changes.

Small Molecules and RNA-Seq

Similarly, RNA-Seq can be used to identify splicing alterations and differentially expressed splicing factors. Small molecules can be screened using high-throughput methods to identify compounds that specifically modulate splicing factors or the spliceosome. After treatment with small molecules, RNA Sequencing can be used to analyze changes in splicing patterns and identify specific splice events affected by the compound.

RNA Interference (RNAi)

RNA-Seq can be used to identify splicing factors or regulatory proteins whose knockdown or inhibition can alter splicing patterns. By silencing the expression of specific splicing factors using siRNAs or shRNAs, Full-length RNA-Seq can then be used to analyze the changes in splicing patterns resulting from the downregulation of these factors. This approach can provide insights into the specific splicing events regulated by the targeted splicing factors.

CRISPR-Cas9 and RNA-Seq

RNA-Seq can be used to identify key splice sites or regulatory elements that govern splicing outcomes. By designing gRNAs to target these specific sites, CRISPR-Cas9 can be utilized to introduce precise modifications and manipulate splicing. Following CRISPR-Cas9 treatment, RNA-Seq can be performed to assess the impact of the modifications on splicing patterns and validate the desired changes.

Modulating Splicing Factors and RNA-Seq

RNA-Seq can be used to identify differentially expressed splicing factors or regulatory proteins in specific cellular contexts or diseases. By modulating the activity of these splicing factors, either through small molecules or gene therapies, RNA-Seq can be employed to analyze the resulting changes in splicing patterns and identify specific splice events affected by the modulation.

Reference:

1. Cerasuolo, Andrea, et al. "The role of RNA splicing factors in cancer: regulation of viral and human gene expression in human papillomavirus-related cervical cancer." Frontiers in cell and developmental biology 8 (2020): 474.