Unit 4: Post Transcriptional Modifications, Processing of Eukaryotic RNA and Gene Regulation
Gene Splicing
Splicing is a key process in gene expression that involves removing introns and joining exons to generate the mature mRNA. This is a highly regulated process that takes place within the nucleus of eukaryotic cells and is mediated by large and complex macromolecular machinery called spliceosomes.
Gene Splicing Introduction
Gene splicing is a post-transcriptional modification in which a single gene can code for multiple proteins. Gene Splicing is done in eukaryotes, prior to mRNA translation, by the differential inclusion or exclusion of regions of pre-mRNA. Gene splicing is an important source of protein diversity. During a typical gene splicing event, the pre-mRNA transcribed from one gene can lead to different mature mRNA molecules that generate multiple functional proteins. Thus, gene splicing enables a single gene to increase its coding capacity, allowing the synthesis of protein isoforms that are structurally and functionally distinct. Gene splicing is observed in high proportion of genes. In human cells, about 40-60% of the genes are known to exhibit alternative splicing.
Gene Splicing: Various forms of gene splicing
There are several types of common gene splicing events. These are the events that can simultaneously occur in the genes after the mRNA is formed from the transcription step of the central dogma of molecular biology.
Exon Skipping: This is the most common known gene splicing mechanism in which exon(s) are included or excluded from the final gene transcript leading to extended or shortened mRNA variants. The exons are the coding regions of a gene and are responsible for producing proteins that are utilized in various cell types for a number of functions.
Intron Retention: An event in which an intron is retained in the final transcript. In humans 2-5 % of the genes have been reported to retain introns. The gene splicing mechanism retains the non-coding (junk) portions of the gene and leads to a demornity in the protein structure and functionality.
Alternative 3' Splice Site and 5' Splice Site: Alternative gene splicing includes joining of different 5' and 3' splice site. In this kind of gene splicing, two or more alternative 5' splice site compete for joining to two or more alternate 3' splice site.
Splice Variant Detection Methods
Gene splicing leads to the synthesis of alternate proteins that play an important role in the human physiology and disease. Currently, the most efficient methods for large scale detection of splice variants include computational prediction methods and microarray analysis. Microarray based splice variant detection is the most popular method currently in use. The highly parallel and sensitive nature of microarrays make them ideal for monitoring gene expression on a tissue-specific, genome-wide level. Microarray based methods for detecting splice variants provide a robust, scalable platform for high-throughput discovery of alternative gene splicing. A number of novel gene transcripts were detected using microarray based methods that were not detected by ESTs using computational methods. Another commonly used method for discovering of novel gene isoforms is RT-PCR followed by sequencing. This is a powerful approach and can be effectively used for analyzing a small number of genes. However, it only provides only a limited view of the gene structure, is labor-intensive, and does not easily scale to thousands of genes or hundreds of tissues.
Challenges in Microarray Design for Splice Variant Detection
Microarray based gene splicing detection poses some unique challenges in designing probes for isoforms that show a high degree of homology. In order to differentiate between these isoforms, a microarray that uses a combination of probes for exons and exon-exon junctions is used. Exon skipping events or other deletions can be monitored by using junction probes. For example, a probe spanning the exon 1 and exon 3 of the gene will detect the skipping of exon 2 from the gene that is translated into a protein.
Splicing takes place in two stages
The first step involves recognition and cleavage of the 5' and 3' splice sites of introns, and the second step joins exons together to form the mature mRNA. A detailed overview of the splicing mechanism follows.
Step 1: Detect and Cut Splices
The first step in splicing involves recognition and cleavage of splice junctions flanking introns. The spliceosome complex recognizes these sites by binding to specific sequences in the pre-mRNA located at the 5' and 3' ends of introns. These sequences, called 5' and 3' splice sites, respectively, usually consist of short consensus sequences that are highly conserved between species.
Splice site recognition involves a series of interactions between the spliceosome complex and the pre-mRNA to form the spliceosome complex. Spliceosomes are large macromolecular machines composed of snRNAs (small nuclear RNAs) and proteins. The snRNA functions as the catalytic subunit of the spliceosome, and the protein provides structural support and helps stabilize the complex.
Once assembled, the spliceosome complex cleaves the mRNA at the 5' and 3' splice sites. This cleavage creates short RNA sequences called introns that are removed from the pre-mRNA and degraded.
Step 2: Exon Ligation
The second step of splicing joins the exons to form the mature mRNA. This process is carried out by a spliceosome complex that catalyzes the joining of two exons.
After the intron is removed, the spliceosome complex undergoes a series of conformational changes that join the two exons. A spliceosome bridge is formed that spans the two exons, bringing the two exons into close proximity. When the exons join, the spliceosome catalyzes the formation of phosphodiester bonds between the two exons, joining them together to form the mature mRNA. After the exons are ligated, the spliceosome is degraded and the mature mRNA is transported from the nucleus to the cytoplasm and translated into protein.
alternate splicing
An important aspect of splicing is alternative splicing, which can generate multiple protein variants from a single gene. Alternative splicing occurs when different combinations of exons are included or excluded in the mature mRNA. This is achieved by using alternative splice sites within the pre-mRNA that can be recognized by the spliceosome complex to generate different mRNA isoforms.
Alternative splicing is a highly regulated process that is controlled by a variety of factors, including the sequence of the pre-mRNA, the availability of spliceosome components, and the presence of regulatory proteins that can bind to the pre-mRNA and modulate splicing.
Conclusion
Splicing is a critical process in gene expression that is responsible for the removal of introns and the joining together of exons to produce mature mRNA. This process is highly regulated and occurs in the nucleus of eukaryotic cells, where it is mediated by a large and complex macromolecular machine called the spliceosome.
Alternative splicing
Alternative splicing is the process by which different combinations of exons within the pre-mRNA molecule are spliced together to generate multiple mRNA transcripts, each of which can be translated into a different protein. In other words, a single gene can generate multiple protein isoforms with different structures and functions by altering the way pre-mRNA is spliced.
In alternative splicing, the pre-mRNA molecule is processed by a spliceosome, a complex of RNA and protein that removes introns and joins exons. Depending on the specific combination of splice sites recognized by the spliceosome, different exons are included or excluded from the final mRNA transcript, giving rise to different protein isoforms.
Alternative splicing is a common mechanism of gene regulation in eukaryotes, allowing increased proteomic diversity and specialization of cellular functions. It is estimated that up to 95% of human genes are alternatively spliced.
