Introduction

Posttranscriptional modifications on newly synthesized mRNA are imperative for the translation into a functional protein. mRNA splicing is an integral part of pre-mRNA processing that involves removing non-coding stretches and merging the coding sequences before transport to the cytoplasm.

Why Does pre-mRNA Need Splicing: Introns and Exons

The segmentation of the genome into coding and non-coding regions was discovered in 1977, earning the Nobel Prize in Medicine in 1993¹. According to this split gene structure, DNA and its newly transcribed premature mRNA contain introns and exons. The word exon refers to a coding DNA sequence and its mRNA transcript that carries the information to encode amino acids. Exons are interrupted by non-coding sequences called introns. Interestingly, exons only make up 1% of the entire genome, while the vast majority consists of introns².

Although introns may seem redundant, they play a significant role in regulating gene expression and translational variation. For example, some introns can be processed further into non-coding RNA, such as miRNA and siRNA, which are responsible for gene regulation and silencing³. Furthermore, as we cover later in the article, the presence of introns between coding sequences allows for the synthesis of multiple proteins from a single gene. Research also suggests that some introns may be involved in mRNA export⁴, nonsense-mediated decay⁵, crossover between sister chromosomes, and recombination.

Spliceosomes

In many eukaryotes, splicing is mediated by a ribonucleoprotein (RNP) complex in the nucleus called the spliceosome. It is an assembly of small nuclear RNP complexes (snRNPs) that are, in turn, made up of small nuclear RNA (snRNAs) and protein factors.

Introns carry conserved sequences, such as a 5' splice site, a 3' splice site, and a branch site in between, which guide the splicing process. The snRNPs in the spliceosome recognize and bind these sequences to begin mRNA splicing.

The Spliceosome Working Mechanism

The Spliceosome complex is formed during and immediately after pre-mRNA synthesis.

The first step involves the sequential attachment of uridine-rich snRNPs to the conserved intron sequences. U1 binds to the 5' splice site, while U2 attaches to the branch site, followed by the binding of other snRNPs, such as U4, U5, and U6. Splicing factors or enhancers facilitate this assembly⁶.

Other Types of Splicing

Self-splicing

In rare cases, an intron can initiate the reaction mechanism described above on its own; hence, this is called self-splicing. This type of splicing occurs in specific types of introns called Groups I and II. These introns are present in organelle genomes of bacteria, fungi, lichens, and plants, constituting mRNA, ribosomal RNA (rRNA), and transfer RNA (tRNA)⁷'⁸.

t-RNA splicing

A rare form of splicing occurs in the tRNA of Saccharomyces cerevisiae with the catalysis of tRNA splicing endonucleases (TSEN)⁹.

TSENs cleave the pre-tRNA at two splice sites to generate two half-tRNAs, which are further processed by tRNA kinase and cyclic phosphodiesterase, followed by tRNA ligase that joins them together¹⁰.

Alternative Splicing

Alternative splicing is the process of varying the combination of exons from an mRNA fragment to form multiple proteins with different functions. From this perspective, alternative splicing is one of the key factors leading to structural and functional diversity, especially in eukaryotic organisms. High-throughput screening of the human transcriptome revealed that alternative splicing occurred in 95% of the genes containing multiple exons¹¹.

There are several mechanisms of alternative splicing. Exon skipping is the most common mechanism in mammals, determining which exons are excluded or retained in the final combination. Furthermore, an exon may contain multiple 5' or 3' splice sites, generating several ways of joining two exons. Another possible mechanism is intron retention, where an intron is included in the coding region with its surrounding exons.

Significance of Splicing in Evolution and Disease

Alternative splicing is often associated with evolutionary flexibility, where the same gene can give rise to protein isoforms with a variety of functions¹². Research suggests that this mechanism of functional diversity predates multicellularity and might have assisted the development of multicellular organisms¹³. Comparative genomic studies show that the genome size of vertebrates is not significantly larger than that of invertebrates, implicating alternative splicing as a mechanism for the increased complexity of vertebrates¹⁴.

On the other hand, dysregulation of RNA splicing mechanisms is strongly correlated with disease onset. Research suggests that more than 60% of all disease-causing mutations in humans involve erroneous splicing¹⁵ and that one-third of all hereditary diseases can be attributed to mutations in the splicing mechanism¹⁶. Splice site mutations can alter the exon combination or cause unwanted incorporation of an intron, leading to loss of function in the protein.

Splice site mutations are one of the most common causes of cancer, influencing the composition of the splice isoforms of many proteins partaking in cellular pathways and causing transcriptome instability¹⁷'¹⁸. A well-studied effect of splicing mutations occurs in the DNA methyltransferase (DNMT) genes that regulate gene expression through DNA methylation. Abnormal splicing of the DNMT mRNAs caused significant disruption in DNA methylation pathways, leading to more rapid growth and proliferation in cancer cells than in control cells¹⁹. Another example is the proto-oncogene Ron for the macrophage-stimulating protein receptor (MST1R). Overexpression of the splicing factor SF2/ASF resulted in an increase in the amounts of a Ron isoform that enhanced cancer cell motility, increasing the invasive capabilities of the cells²⁰.

mRNA Splicing and Applications in Gene Therapy

The potential of mRNA splicing in correcting disease-causing mutations has come to attention with the discovery of trans-splicing mechanisms in mammals in 1995. Here, the researchers observed that the spliceosome complex could join exons from two pre-mRNA complexes to form a hybrid mRNA in murine models²¹.

This phenomenon was leveraged to give rise to a gene therapy method called RNA exon editing or Spliceosomal-Mediated RNA trans-splicing (SMaRT), where faulty exons in the pre-mRNA are replaced with the correct ones through the introduction of synthetic RNA²²'²³. This is achieved by a pre-trans-splicing molecule (PTM) plasmid. PTM contains an antisense oligonucleotide that can target the mutated intron splice site, a synthetic splice site, and the synthetic RNA sequence²⁴. This combination ensures that the mutated splice site is ignored during splicing and that the synthesized protein isoform is functional without altering the expression levels in the genome. Furthermore, it is possible to design the synthetic RNA to correct multiple exons, enabling the centralized correction of genes with mutational variances²⁵.

Over the last two decades, several cell and animal studies have explored the effectiveness of trans-splicing in treating rare genetic disorders²⁶, hemophilia²⁷, sickle cell disease²⁸, HIV²⁹, and cancer³⁰. One example of a genetic disorder is Leber congenital amaurosis, which causes severely impaired vision in infants due to an abnormal intron inclusion during the splicing of the pre-mRNA of the gene CEP290. Dooley et al. used a synthetic RNA that targeted the intron binding site and introduced wild-type exon sequences upstream to abrogate intron retention and restore its function³¹.

In contrast to restoring functional proteins in genetic disorders, cancer research with RNA exon editing followed a different approach called suicide gene therapy. This method involves the introduction of synthetic RNA encoding cytotoxic genes and inducing cell death. The RNA exon editing can specifically target cancer cells and spare healthy cells the unwanted effects. The cancer-type organic anion transporting polypeptide 1B3 (Ct-OOATP1B3) gene is an ideal target for suicide gene therapy. Sun et al. synthesized a pre-mRNA construct containing exons for a herpes simplex virus 1 thymidine kinase (HSV-tk), which gets fused with Ct-OOATP1B3 pre-mRNA, resulting in a fusion protein that can be targeted by the antiviral agent ganciclovir. The system exhibited cytotoxic effects on colorectal cancer cells in mouse models³².

Despite the promising results, the challenge remains to improve the delivery and efficacy of the SMaRT construct so that trans-splicing is favored over mutagenic cis-splicing. The correct design should account for the secondary structures of the endogenous pre-mRNA and the target intron sequence, which can impact the specificity of trans-splicing. Bioinformatic and computational approaches must be explored to analyze the 3D structures of the target pre-mRNA to improve specificity and prevent off-target binding²².

References

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