Advancements in Genetic Engineering
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Overview of Alternative Pre-mRNA Splicing Mechanisms and Regulation

Keith T Gagnon^{* *}Correspondence: Keith T Gagnon, Department of Biochemistry and Molecular Biology, Southern Illinois University, School of Medicine, Carbondale, Illinois, USA, Tel: +44 (0)300 019 6175, Email:

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Description

The first RNA transcribed from a gene of DNA template has to be processed before it becomes a mature messenger RNA (mRNA) that can control protein synthesis in most eukaryotic genes (and some prokaryotic genes). The removal or "splicing out" of particular regions known as intervening sequences, or introns, is one of the phases in this activity termed RNA splicing. The remaining sequences, known as exons, are then combined to one another during the splicing process to form the final mRNA. In the 1970s, RNA splicing was discovered, causing a paradigm shift in the study of gene expression.

Gene regulation was initially addressed in bacterial systems that were quite basic. Most bacterial RNA transcripts are collinear, meaning they are encoded directly by DNA rather than undergoing splicing. In other words, the gene and the mRNA generated from the gene (with exception of the 5′ and 3′ noncoding regions) have a one-to-one correlation of bases. In 1977, however, multiple groups of researchers working with adenoviruses that infect and reproduce in mammalian cells discovered some unexpected findings. These researchers discovered a series of RNA molecules known as "mosaics," each of which comprised sequences from noncontiguous viral genome regions. These mosaics were discovered late in the viral infection process. Early infection studies revealed lengthy primary RNA transcripts that included all of the late RNA sequences as well as what became known as the intermediate sequences (introns). Introns were discovered in many additional viral and eukaryotic genes after the adenoviral discovery, including those for hemoglobin and immunoglobulin. In vitro systems generated from eukaryotic cells were then used to investigate RNA transcript splicing, including the removal of introns from transfer RNA in yeast cell-free extracts. These findings supported the concept that big initial transcripts were spliced to produce mature mRNA. Other theories suggested that the DNA template looped or developed a secondary structure that facilitated transcription from non-contiguous areas.

RNA splicing biochemical mechanism has been researched in a variety of situations and is now pretty well described. Splice sites, which are conserved sequences situated at the 5′ and 3′ ends of major transcripts, cleave introns out of them. The most commonly deleted RNA sequence begins with the dinucleotide GU at the 5′ end and ends with AG at the 3′ end. These consensus sequences are recognized to be crucial because changing one of the conserved nucleotides hinders splicing. Splice sites, which have conserved sequences, are used to remove introns from main transcripts. The 5′ and 3′ ends of introns include these sites. The most common RNA deletion sequence starts at the 5′ end with the dinucleotide GU and ends at the 3′ end with AG. These consensus sequences are known to be crucial because changing one of the conserved nucleotides hinders splicing. The branch point, which can be located anywhere between 18 and 40 nucleotides upstream from the 3′ end of an intron, is another important area. The adenine at the branch point is always present, while the remainder of the sequence is just a little bit conserved. YNYYRAY is a common sequence, with Y denoting pyrimidine, N denoting any nucleotide, R denoting any purine, and A denoting adenine. Alternate splice site sequences that begin with the dinucleotide AU and conclude with AC are encountered seldom and are spliced in a similar manner.

Splicing is performed out during numerous phases, with tiny nuclear ribonucleoproteins acting as catalysts (snRNPs, commonly pronounced "snurps"). Following the attachment of a snRNP termed U1 to its corresponding sequence inside the intron, the pre-mRNA is cleaved at the 5′ end of the intron. The cut end then joins the conserved branch point area downstream by guanine and adenine nucleotide pairing from the 5′ end and branch point, respectively, to produce a looping structure known as a lariat. The guanine and adenine bases are joined by a chemical mechanism called trans esterification, in which a hydroxyl (OH) group on an adenine carbon atom "attacks" the guanine nucleotide's link at the splice site. The guanine residue on the RNA strand is therefore cleaved and helps to establish connection with the adenine. Then after snRNPs U2 and U4/U6 then appear to play a role in bringing the 5′ end and branch point closer together. With the help of U5, the intron's 3′ end is pushed closer to the 5′ end, chopped, and connected. An OH group at the 3′ end of the exon assaults the phosphodiester link at the 3′ splice site in this phase, which is known as trans esterification. The neighboring exons are covalently linked, resulting in a lariat that is released with U2, U5, and U6 connected to it. Exon splicing enhancers are found in eukaryotic genes with lengthy introns in addition to consensus sequences at their splice sites (ESEs). These sequences, which aid in the positioning of the splicing apparatus, are present in the exons of genes and bind proteins that aid in the recruitment of the splicing machinery to the proper location. The majority of splicing happens between exons on a single RNA transcript, however trans-splicing, in which exons from separate pre-mRNAs are ligated together, does happen rarely.

Splicing takes place in cellular machinery called spliceosomes, which include snRNPs as well as other proteins. The primary form of spliceosome is one of the most abundant structures in the cell, and a secondary kind of spliceosome that processes a small category of introns was recently discovered. Because they depend on the activity of a snRNP termed U12, these introns are referred to as U12-type introns (the common introns described above are called U2-type introns). Although the function of U12-type introns is unknown, their existence throughout evolution and conservation across homologous genes from widely separated species implies that they serve a significant functional purpose.