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Splicing (genetics)

In genetics, splicing is a modification of genetic information after transcription, in which introns of precursor messenger RNA (pre-mRNA) are removed and exons of it are joined. Since in prokaryotic genomes introns do not exist, splicing naturally only occurs in eukaryotes. The splicing prepares the pre-mRNA to produce the mature messenger RNA (mRNA), which then undergoes translation as part of the protein synthesis to produce proteins. Splicing includes a series of biochemical reactions, which are catalyzed by the spliceosome, a complex of small nuclear ribonucleo-proteins (snRNPs).

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Splicing pathways

Several methods of RNA splicing occur in nature.
The type of splicing depends on the structure of the spliced intron and the catalysts required for splicing to occur.
Regardless of which pathway is used, the excised introns are discarded.

Spliceosomal introns

Spliceosomal introns often reside in eukaryotic protein-coding genes. Within the intron, a 3' splice site, 5' splice site, and branch site are required for splicing. Splicing is catalyzed by the spliceosome which is a large RNA-protein complex composed of five small nuclear ribonucleoproteins (snRNPs, pronounced 'snurps' ). The RNA components of snRNPs interact with the intron and may be involved in catalysis. Two types of spliceosomes have been identified (the major and minor) which contain different snRNPs.

Major

The major spliceosome splices introns containing GU at the 5' splice site and AG at the 3' splice site. It is composed of the U1, U2, U4, U5, and U6 snRNPs.

E Complex-U1 binds to the GU sequence at the 5' splice site, along with accessory proteins/enzymes ASF/SF2, U2AF (binds at the Py-AG site), SF1/BBP (BBP=Branch Binding Protein);
A Complex-U2 binds to the branch site, and ATP is hydrolyzed;
B1 Complex-U5/U4/U6 trimer binds, and the U5 binds exons at the 5' site, with U6 binding to U2;
B2 Complex-U1 is released, U5 shifts from exon to intron and the U6 binds at the 5' splice site;
C1 Complex-U4 is released, U6/U2 catalyzes transesterification, U5 binds exon at 3' splice site, and the 5' site is cleaved, resulting in the formation of the lariat;
C2 Complex-U2/U5/U6 remain bound to the lariat, and the 3' site is cleaved and exons are ligated using ATP hydrolysis. The spliced RNA is released and the lariat debranches.

This type of splicing is termed canonical splicing or termed the lariat pathway, which accounts for more than 99% of splicing. By contrast, when the intronic flanking sequences do not follow the GU-AG rule, noncanonical splicing is said to occur (see "minor spliceosome" below). ^[1]

Minor

The minor spliceosome is very similar to the major spliceosome, however it splices rare introns with different splice site sequences. While the minor and major spliceosomes contain the same U5 snRNP, the minor spliceosome has different, but functionally analogous snRNPs for U1, U2, U4, and U6, which are respectively called U11, U12, U4atac, and U6atac. ^[2]

Trans-splicing

Trans-splicing is a form of splicing that joins two exons that are not within the same RNA transcript.

Self-splicing

Self-splicing occurs for rare introns that form a ribozyme, performing the functions of the spliceosome by RNA alone. There are two kinds of self-splicing introns, Group I and Group II. Group I and II introns perform splicing similar to the spliceosome without requiring any protein. This similarity suggests that Group I and II introns may be evolutionarily related to the spliceosome. Self-splicing may also be very ancient, and may have existed in an RNA world that was present before protein. Although the two splicing mechanisms described below do not require any proteins to occur, 5 additional RNA molecules and over 50 proteins are used and hydrolyzes many ATP molecules. The splicing mechanisms use ATP in order to accurately splice mRNA's. If the cell were to not use any ATP's, the process would be highly inaccurate and many mistakes would occur. Two transesterfications characterize the mechanism in which group I introns are sliced: 1) 3'OH of a free guanine nucleoside (or one located in the intron) or a nucleotide cofactor (GMP, GDP, GTP) attacks phosphate at the 5' splice site. 2) 3'OH of the 5'OH becomes a nucleophile and the second transesterfication results in the joining of the two exons. The mechanism in which group II introns are spliced (two transesterfication reaction like group I introns) is as follows: 1)The 2'OH of a specific adenosine in the intron attacks the 5' splice site, thereby forming the lariat 2) The 3'OH of the 5' exon triggers the second transesterfication at the 3' splice site thereby joining the exons together.

tRNA splicing

tRNA (also tRNA-like) splicing is another rare form of splicing that usually occurs in tRNA. The splicing reaction involves a different biochemistry than the spliceomsomal and self-splicing pathways. Ribonucleases cleave the RNA and ligases join the exons together. This form of splicing does also not require any RNA components for catalysis.

Evolution

Splicing occurs in all the kingdoms or domains of life, however, the extent and types of splicing can be very different between the major divisions. Eukaryotes splice many protein-coding messenger RNAs and some non-coding RNAs. Prokaryotes, on the other hand, splice rarely, but mostly non-coding RNAs. Another important difference between these two groups of organisms is that prokaryotes completely lack the spliceosomal pathway.

Because spliceosomal introns are not conserved in all species, there is debate concerning when spliceosomal splicing evolved. Two models have been proposed: the intron late and intron early models (see intron evolution).

**Splicing Diversity**
	Eukaryotes	Prokaryotes
Spliceosomal	+	-
Self-splicing	+	+
tRNA	+	+

Biochemical mechanism

Spliceosomal splicing and self-splicing involves a two-step biochemical process. Both steps involve transesterification reactions that occur between RNA nucleotides. tRNA splicing, however, is an exception and does not occur by transesterification.

Spliceosomal and self-splicing transesterification reactions occur via two sequential transesterification reactions. First, the 2'OH of a specific branch-point nucleotide within the intron that is defined during spliceosome assembly performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site forming the lariat intermediate. Second, the 3'OH of the released 5' exon then performs a nucleophilic attack at the last nucleotide of the intron at the 3' splice site thus joining the exons and releasing the intron lariat.

Alternative splicing

Main article: Alternative splicing

In many cases, the splicing process can create a range of unique proteins by varying the exon composition of the same messenger RNA. This phenomenon is then called alternative splicing.

Experimental manipulation of splicing

Splicing events can be experimentally altered^[3] by binding steric-blocking antisense oligos such as Morpholinos or Peptide nucleic acids to snRNP binding sites, to the branchpoint nucleotide that closes the lariat,^[4] or to splice-regulatory element binding sites.^[5]

Splicing errors

Mutations in the introns or exons can prevent splicing and thus may prevent protein biosynthesis.

Common errors:

Mutation of a splice site resulting in loss of function of that site. Results in exposure of a premature stop codon, loss of an exon, or inclusion of an intron.
Mutation of a splice site reducing specificity. May result in variation in the splice location, causing insertion or deletion of amino acids, or most likely, a loss of the reading frame.
Transposition of a splice site, resulting in inclusion or exclusion of more DNA than expected. Results in longer or shorter exons.

Protein splicing

Main article: Protein splicing

Not only pre-mRNA but also proteins can undergo splicing. Although the biomolecular mechanisms are different, the principle is the same, that parts of the protein, called inteins instead of introns, are removed. The remaining parts, called exteins instead of exons, are fused together. However, protein splicing has so far not been observed in humans, but in yeast.

References

^ Ng B, Yang F, Huston DP, et al (Dec 2004). "Increased noncanonical splicing of autoantigen transcripts provides the structural basis for expression of untolerized epitopes". J. Allergy Clin. Immunol. 114 (6): 1463-70. doi:10.1016/j.jaci.2004.09.006. PMID 15577853.
^ Patel AA, Steitz JA (2003). "Splicing double: insights from the second spliceosome". Nat. Rev. Mol. Cell Biol. 4 (12): 960-70. doi:10.1038/nrm1259. PMID 14685174.
^ Draper BW, Morcos PA, Kimmel CB (2001). "Inhibition of zebrafish fgf8 pre-mRNA splicing with morpholino oligos: a quantifiable method for gene knockdown". Genesis 30 (3): 154-6. doi:10.1002/gene.1053. PMID 11477696.
Sazani P, Kang SH, Maier MA, et al (Oct 2001). "Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs". Nucleic Acids Res. 29 (19): 3965-74. PMID 11574678.
^ Morcos, PA (2007). "Achieving targeted and quantifiable alteration of mRNA splicing with Morpholino oligos.". Biochem Biophys Res Commun 358 (2): 521-7. doi:10.1016/j.bbrc.2007.04.172. PMID 17493584.
^ Bruno IG, Jin W, Cote GJ (2004 Oct 15). "Correction of aberrant FGFR1 alternative RNA splicing through targeting of intronic regulatory elements". Hum. Mol. Genet. 13 (20): 2409-20. doi:10.1093/hmg/ddh272. PMID 15333583.(Epub August 27, 2004)

Lodish, Harvey; Berk, Arnold; Zipursky, S. Lawrence; Matsudaira, Paul; Baltimore, David; Darnell, James E. (1999). Molecular Cell Biology. New York: W. H. Freeman & Co.. ISBN 0-7167-3706-X.
Hartl, Daniel L.; Elizabeth W. Jones (2005). Genetics: Analysis of Genes and Genomes. Jones & Bartlett Publishers. ISBN 0763715115.