In molecular biology, splicing is a modification of an RNA after transcription, in which introns are removed and exons are joined. Molecular biology is the study of Biology at a molecular level Ribonucleic acid ( RNA) is a Nucleic acid that consists of a long chain of Nucleotide units Transcription is the synthesis of RNA under the direction of DNA Introns, derived from the term "intragenic regions" and also called intervening sequence (IVS are DNA regions in a Gene that are not translated into An exon is a Nucleic acid sequence that is represented in the mature form of an RNA molecule after a portions of a precursor RNA Introns have been This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation. Animals Plants fungi, and Protists are eukaryotes (juːˈkærɪɒt or -oʊt Organisms whose cells are organized into complex Messenger ribonucleic acid ( mRNA) is a molecule of RNA encoding a chemical "blueprint" for a Protein product Translation is the first stage of Protein biosynthesis (part of the overall process of Gene expression) For many eukaryotic introns, splicing is done in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs), but there are also self-splicing introns. Catalysis is the process in which the rate of a Chemical reaction is increased by means of a Chemical substance known as a catalyst A spliceosome is a complex of specialized RNA and Protein subunits that removes Introns from a transcribed pre- mRNA ( HnRNA snRNP s (pronounced "snurps" or small nuclear ribonucleoproteins, are particles that combine with Pre-mRNA and various proteins to form Spliceosomes

Simple illustration of exons and introns in pre-mRNA and the formation of mature mRNA by splicing. The UTRs are non-coding parts of exons at the ends of the mRNA.

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. Catalysis is the process in which the rate of a Chemical reaction is increased by means of a Chemical substance known as a catalyst

Spliceosomal introns

Spliceosomal introns often reside in eukaryotic protein-coding genes. Animals Plants fungi, and Protists are eukaryotes (juːˈkærɪɒt or -oʊt Organisms whose cells are organized into complex 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' ). A spliceosome is a complex of specialized RNA and Protein subunits that removes Introns from a transcribed pre- mRNA ( HnRNA snRNP s (pronounced "snurps" or small nuclear ribonucleoproteins, are particles that combine with Pre-mRNA and various proteins to form Spliceosomes 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. snRNP s (pronounced "snurps" or small nuclear ribonucleoproteins, are particles that combine with Pre-mRNA and various proteins to form Spliceosomes

• 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 and is active in the nucleus. U1 spliceosomal RNA is a Small nuclear RNA (snRNA component of the Spliceosome (involved in pre- MRNA splicing U2 spliceosomal RNA is a Small nuclear RNA (snRNA component of the Spliceosome (involved in pre- MRNA splicing U4 small nuclear RNA (U4 snRNA is a Non-coding RNA component of the major U2-dependent Spliceosome. U5 RNA is a Non-coding RNA that is a component of both types of known Spliceosome. U6 snRNA is a Non-coding RNA that is a component of the Spliceosome which is involved in splicing pre-mRNA snRNP s (pronounced "snurps" or small nuclear ribonucleoproteins, are particles that combine with Pre-mRNA and various proteins to form Spliceosomes

• 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 out rare introns with different splice site sequences and is active in the cytosol ^[2]. The minor spliceosome is a Ribonucleoprotein complex that catalyses the removal ( splicing) of an atypical class of spliceosomal Introns (U12-type from 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. snRNP s (pronounced "snurps" or small nuclear ribonucleoproteins, are particles that combine with Pre-mRNA and various proteins to form Spliceosomes The U11 spliceosomal RNA is a Non-coding RNA that together with U4atac/U6atac U5 and U12 snRNAs and associated proteins forms a Spliceosome that cleaves a U12 minor spliceosomal RNA is formed from U12 small nuclear ( SnRNA) together with U4atac/U6atac U5, and U11 snRNAs and associated proteins forms ^[3]

• Trans-splicing

Trans-splicing is a form of splicing that joins two exons that are not within the same RNA transcript. Trans -splicing is a special form of RNA processing in Eukaryotes where Exons from two different primary RNA transcripts are joined end to end and

Self-splicing

Self-splicing occurs for rare introns that form a ribozyme, performing the functions of the spliceosome by RNA alone. A ribozyme (from ribo nucleic acid en' zyme', also called RNA Enzyme or catalytic RNA is an RNA Molecule that catalyzes There are three kinds of self-splicing introns, Group I, Group II and Group III. Group I catalytic introns are large self-splicing Ribozymes. They catalyze their own excision from MRNA, TRNA and RRNA precursors Group II intron is a class of Intron found in RRNA, TRNA, MRNA of organelles in Fungi, plants Protists and mRNA in Group III intron is a class of Introns found in mRNA genes of chloroplasts in euglenoid Protists They have a conventional group II-type dVI with a bulged 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. The RNA world hypothesis proposes that a world filled with life based on Ribonucleic acid (RNA predated current life based on Deoxyribonucleic acid (DNA 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 transesterifications 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'exon becomes a nucleophile and the second transesterification results in the joining of the two exons. The mechanism in which group II introns are spliced (two transesterification 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 transesterification 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. Transfer RNA (abbreviated tRNA) is a small RNA (usually about 74-95 nucleotides that transfers a specific Amino acid to a growing polypeptide chain at The splicing reaction involves a different biochemistry than the spliceomsomal and self-splicing pathways. Ribonucleases cleave the RNA and ligases join the exons together. Ribonuclease, abbreviated commonly as RNase, is a Nuclease that catalyzes the degradation of RNA into smaller components In Biochemistry, a ligase (from the Latin verb ligāre &mdash "to bind" or "to glue together" is an Enzyme that can catalyse

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. In biological Taxonomy, a kingdom or regnum is a Taxonomic rank in either (historically the highest rank or (in the new three-domain system In biological Taxonomy, a domain (also superregnum, superkingdom, or empire) is the highest Taxonomic rank of Organisms Eukaryotes splice many protein-coding messenger RNAs and some non-coding RNAs. Animals Plants fungi, and Protists are eukaryotes (juːˈkærɪɒt or -oʊt Organisms whose cells are organized into complex Messenger ribonucleic acid ( mRNA) is a molecule of RNA encoding a chemical "blueprint" for a Protein product A non-coding RNA ( ncRNA) is any RNA molecule that is not translated into a Protein. Prokaryotes, on the other hand, splice rarely, but mostly non-coding RNAs. The prokaryotes (proʊˈkærioʊts singular prokaryote /proʊˈkæriət/ are a group of Organisms that lack a Cell nucleus (= karyon or any other 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). Introns, derived from the term "intragenic regions" and also called intervening sequence (IVS are DNA regions in a Gene that are not translated into

Biochemical mechanism

Diagram illustrating the two-step biochemistry of splicing

Spliceosomal splicing and self-splicing involves a two-step biochemical process. Both steps involve transesterification reactions that occur between RNA nucleotides. In Organic chemistry, transesterification is the process of exchanging the Alcohol group of an Ester compound with another Alcohol. 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. In Chemistry, a nucleophile (literally nucleus lover as in nucleus and phile) is a Reagent that forms a Chemical bond to 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. Alternative splicing is the RNA splicing variation mechanism in which the Exons of the primary gene transcript the Pre-mRNA, are separated and reconnected This phenomenon is then called alternative splicing. Alternative splicing is the RNA splicing variation mechanism in which the Exons of the primary gene transcript the Pre-mRNA, are separated and reconnected

Experimental manipulation of splicing

Splicing events can be experimentally altered^[4] 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,^[5] or to splice-regulatory element binding sites. In Molecular biology, a Morpholino is a Molecule used to modify Gene expression. Peptide nucleic acid (PNA is an artificially synthesized Polymer similar to DNA or RNA and is used in biological research and medical treatments ^[6]

Splicing errors

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, leading to inclusion or exclusion of more RNA than expected, resulting in longer or shorter exons.

Protein splicing

Main article: Protein splicing

Not only pre-mRNA but also proteins can undergo splicing. Protein splicing is an intramolecular reaction of a particular Protein in which an internal protein segment (called an Intein) is removed from a precursor protein Although the biomolecular mechanisms are different, the principle is the same, that parts of the protein, called inteins instead of introns, are removed. An intein is a segment of a Protein that is able to excise itself and rejoin the remaining portions (the exteins with a Peptide bond. The remaining parts, called exteins instead of exons, are fused together. Protein splicing has been observed in lower organisms, yeast, plants and animals, including in humans. ^[7]

References

• 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.  

See also

• cDNA
• Exon
• Intron
• Primary transcript
• Spliceosome
• Minor spliceosome