Editing Alternative splicing (section)

==Mechanisms==

===General splicing mechanism===
{{Main|RNA splicing}}
[[File:A complex.jpg|thumb|Spliceosome A complex defines the 5' and 3' ends of the intron before removal<ref name=Matlin/>]]
When the pre-mRNA has been transcribed from the [[DNA]], it includes several [[intron]]s and [[exon]]s. (In [[nematode]]s, the mean is 4–5 exons and introns; in the fruit fly ''[[Drosophila melanogaster|Drosophila]]'' there can be more than 100 introns and exons in one transcribed pre-mRNA.) The exons to be retained in the [[mRNA]] are determined during the splicing process. The regulation and selection of splice sites are done by trans-acting splicing activator and splicing repressor proteins as well as cis-acting elements within the pre-mRNA itself such as exonic splicing enhancers and exonic splicing silencers.

The typical eukaryotic nuclear intron has consensus sequences defining important regions. Each intron has the sequence GU at its 5' end. Near the 3' end there is a branch site. The nucleotide at the branchpoint is always an A; the consensus around this sequence varies somewhat. In humans the branch site consensus sequence is yUnAy.<ref name=Gao>{{cite journal | vauthors = Gao K, Masuda A, Matsuura T, Ohno K | title = Human branch point consensus sequence is yUnAy | journal = Nucleic Acids Research | volume = 36 | issue = 7 | pages = 2257–67 | date = April 2008 | pmid = 18285363 | pmc = 2367711 | doi = 10.1093/nar/gkn073 }}</ref> The branch site is followed by a series of [[pyrimidine]]s – the [[polypyrimidine tract]] – then by AG at the 3' end.<ref name=Matlin/>

Splicing of mRNA is performed by an RNA and protein complex known as the [[spliceosome]], containing [[snRNP]]s designated U1, [[U2]], U4, U5, and U6 (U3 is not involved in mRNA splicing).<ref name=Clark>{{cite book | vauthors = Clark D |title=Molecular biology |publisher=Elsevier Academic Press |location=Amsterdam |year=2005 |isbn=978-0-12-175551-5}}</ref> U1 binds to the 5' GU and U2, with the assistance of the [[U2AF2|U2AF]] protein factors, binds to the branchpoint A within the branch site. The complex at this stage is known as the spliceosome A complex. Formation of the A complex is usually the key step in determining the ends of the intron to be spliced out, and defining the ends of the exon to be retained.<ref name=Matlin/> (The U nomenclature derives from their high uridine content).

The U4,U5,U6 complex binds, and U6 replaces the U1 position. U1 and U4 leave. The remaining complex then performs two [[transesterification]] reactions. In the first transesterification, 5' end of the intron is cleaved from the upstream exon and joined to the branch site A by a 2',5'-[[phosphodiester]] linkage. In the second transesterification, the 3' end of the intron is cleaved from the downstream exon, and the two exons are joined by a phosphodiester bond. The intron is then released in lariat form and degraded.<ref name=Black>{{cite journal | vauthors = Black DL | title = Mechanisms of alternative pre-messenger RNA splicing | journal = Annual Review of Biochemistry | volume = 72 | issue = 1 | pages = 291–336 | year = 2003 | pmid = 12626338 | doi = 10.1146/annurev.biochem.72.121801.161720 | s2cid = 23576288 | url = https://cloudfront.escholarship.org/dist/prd/content/qt2hg605wm/qt2hg605wm.pdf }}</ref>

===Regulatory elements and proteins===

[[File:Splicing repression.jpg|thumb|left|Splicing repression]]
Splicing is regulated by [[trans-acting]] proteins (repressors and activators) and corresponding [[cis-acting]] regulatory sites (silencers and enhancers) on the pre-mRNA. However, as part of the complexity of alternative splicing, it is noted that the effects of a splicing factor are frequently position-dependent. That is, a splicing factor that serves as a splicing activator when bound to an intronic enhancer element may serve as a repressor when bound to its splicing element in the context of an exon, and vice versa.<ref name="Lim">{{cite journal | vauthors = Lim KH, Ferraris L, Filloux ME, Raphael BJ, Fairbrother WG | title = Using positional distribution to identify splicing elements and predict pre-mRNA processing defects in human genes | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 27 | pages = 11093–8 | date = July 2011 | pmid = 21685335 | pmc = 3131313 | doi = 10.1073/pnas.1101135108 | bibcode = 2011PNAS..10811093H | doi-access = free }}</ref> The secondary structure of the pre-mRNA transcript also plays a role in regulating splicing, such as by bringing together splicing elements or by masking a sequence that would otherwise serve as a binding element for a splicing factor.<ref>{{cite journal | vauthors = Warf MB, Berglund JA | title = Role of RNA structure in regulating pre-mRNA splicing | journal = Trends in Biochemical Sciences | volume = 35 | issue = 3 | pages = 169–78 | date = March 2010 | pmid = 19959365 | pmc = 2834840 | doi = 10.1016/j.tibs.2009.10.004 }}</ref><ref name="ncbi.nlm.nih.gov">{{cite journal | vauthors = Reid DC, Chang BL, Gunderson SI, Alpert L, Thompson WA, Fairbrother WG | title = Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA sequence | journal = RNA | volume = 15 | issue = 12 | pages = 2385–97 | date = December 2009 | pmid = 19861426 | pmc = 2779669 | doi = 10.1261/rna.1821809 }}</ref> Together, these elements form a "splicing code" that governs how splicing will occur under different cellular conditions.<ref name=Wang>{{cite journal | vauthors = Wang Z, Burge CB | title = Splicing regulation: from a parts list of regulatory elements to an integrated splicing code | journal = RNA | volume = 14 | issue = 5 | pages = 802–13 | date = May 2008 | pmid = 18369186 | pmc = 2327353 | doi = 10.1261/rna.876308 | format = Free full text }}</ref><ref name=Barash>{{cite journal | vauthors = Barash Y, Calarco JA, Gao W, Pan Q, Wang X, Shai O, Blencowe BJ, Frey BJ | display-authors = 6 | title = Deciphering the splicing code | journal = Nature | volume = 465 | issue = 7294 | pages = 53–9 | date = May 2010 | pmid = 20445623 | doi = 10.1038/nature09000 | s2cid = 2398858 | bibcode = 2010Natur.465...53B }}</ref>

There are two major types of cis-acting RNA sequence elements present in pre-mRNAs and they have corresponding trans-acting [[RNA-binding protein]]s. Splicing ''silencers'' are sites to which splicing repressor proteins bind, reducing the probability that a nearby site will be used as a splice junction. These can be located in the intron itself (intronic splicing silencers, ISS) or in a neighboring exon ([[exonic splicing silencer]]s, ESS). They vary in sequence, as well as in the types of proteins that bind to them. The majority of splicing repressors are [[heterogeneous nuclear ribonucleoprotein]]s (hnRNPs) such as hnRNPA1 and polypyrimidine tract binding protein (PTB).<ref name=Matlin/><ref name=Wang/> Splicing ''enhancers'' are sites to which splicing activator proteins bind, increasing the probability that a nearby site will be used as a splice junction. These also may occur in the intron (intronic splicing enhancers, ISE) or exon ([[exonic splicing enhancer]]s, ESE). Most of the activator proteins that bind to ISEs and ESEs are members of the [[SR protein]] family. Such proteins contain RNA recognition motifs and arginine and serine-rich (RS) domains.<ref name=Matlin/><ref name=Wang/>

[[File:Splicing activation.jpg|thumb|left|Splicing activation]]

In general, the determinants of splicing work in an inter-dependent manner that depends on context, so that the rules governing how splicing is regulated form a splicing code.<ref name=Barash/> The presence of a particular cis-acting RNA sequence element may increase the probability that a nearby site will be spliced in some cases, but decrease the probability in other cases, depending on context. The context within which regulatory elements act includes cis-acting context that is established by the presence of other RNA sequence features, and trans-acting context that is established by cellular conditions. For example, some cis-acting RNA sequence elements influence splicing only if multiple elements are present in the same region so as to establish context. As another example, a cis-acting element can have opposite effects on splicing, depending on which proteins are expressed in the cell (e.g., neuronal versus non-neuronal PTB). The adaptive significance of splicing silencers and enhancers is attested by studies showing that there is strong selection in human genes against mutations that produce new silencers or disrupt existing enhancers.<ref name="Ke">{{cite journal | vauthors = Ke S, Zhang XH, Chasin LA | title = Positive selection acting on splicing motifs reflects compensatory evolution | journal = Genome Research | volume = 18 | issue = 4 | pages = 533–43 | date = April 2008 | pmid = 18204002 | pmc = 2279241 | doi = 10.1101/gr.070268.107 }}</ref><ref>{{cite journal | vauthors = Fairbrother WG, Holste D, Burge CB, Sharp PA | title = Single nucleotide polymorphism-based validation of exonic splicing enhancers | journal = PLOS Biology | volume = 2 | issue = 9 | pages = E268 | date = September 2004 | pmid = 15340491 | pmc = 514884 | doi = 10.1371/journal.pbio.0020268 | doi-access = free }}</ref>

===Examples===

====Exon skipping: ''Drosophila'' ''dsx''====
[[File:Dsx splicing.jpg|thumb|Alternative splicing of ''dsx'' pre-mRNA]]
Pre-mRNAs from the ''D. melanogaster'' gene ''[[doublesex|dsx]]'' contain 6 exons. In males, exons 1,2,3,5,and 6 are joined to form the mRNA, which encodes a transcriptional regulatory protein required for male development. In females, exons 1,2,3, and 4 are joined, and a [[polyadenylation]] signal in exon 4 causes cleavage of the mRNA at that point. The resulting mRNA is a transcriptional regulatory protein required for female development.<ref name=Lynch>{{cite journal | vauthors = Lynch KW, Maniatis T | title = Assembly of specific SR protein complexes on distinct regulatory elements of the Drosophila doublesex splicing enhancer | journal = Genes & Development | volume = 10 | issue = 16 | pages = 2089–101 | date = August 1996 | pmid = 8769651 | doi = 10.1101/gad.10.16.2089 | doi-access = free }}</ref>

This is an example of exon skipping. The intron upstream from exon 4 has a [[polypyrimidine tract]] that doesn't match the [[consensus sequence]] well, so that U2AF proteins bind poorly to it without assistance from splicing activators. This 3' splice acceptor site is therefore not used in males. Females, however, produce the splicing activator Transformer (Tra) (see below). The SR protein Tra2 is produced in both sexes and binds to an ESE in exon 4; if Tra is present, it binds to Tra2 and, along with another SR protein, forms a complex that assists U2AF proteins in binding to the weak polypyrimidine tract. U2 is recruited to the associated branchpoint, and this leads to inclusion of exon 4 in the mRNA.<ref name=Lynch/><ref name=Graveley>{{cite journal | vauthors = Graveley BR, Hertel KJ, Maniatis T | title = The role of U2AF35 and U2AF65 in enhancer-dependent splicing | journal = RNA | volume = 7 | issue = 6 | pages = 806–18 | date = June 2001 | pmid = 11421359 | pmc = 1370132 | doi = 10.1017/S1355838201010317 }}</ref>

====Alternative acceptor sites: ''Drosophila'' ''{{vanchor|Transformer}}''====
[[File:transformer splicing.gif|left|thumb|Alternative splicing of the ''Drosophila'' ''Transformer'' gene product.]]
Pre-mRNAs of the ''[[Transformer (gene)|Transformer]]'' (Tra) gene of ''[[Drosophila melanogaster]]'' undergo alternative splicing via the alternative acceptor site mode. The gene Tra encodes a protein that is expressed only in females. The primary transcript of this gene contains an intron with two possible acceptor sites. In males, the upstream acceptor site is used. This causes a longer version of exon 2 to be included in the processed transcript, including an early [[stop codon]]. The resulting mRNA encodes a truncated protein product that is inactive. Females produce the master sex determination protein [[Sex lethal]] (Sxl). The Sxl protein is a splicing repressor that binds to an ISS in the RNA of the Tra transcript near the upstream acceptor site, preventing [[U2AF2|U2AF]] protein from binding to the polypyrimidine tract. This prevents the use of this junction, shifting the spliceosome binding to the downstream acceptor site. Splicing at this point bypasses the stop codon, which is excised as part of the intron. The resulting mRNA encodes an active Tra protein, which itself is a regulator of alternative splicing of other sex-related genes (see ''dsx'' above).<ref name=Black/>

====Exon definition: Fas receptor====
[[File:Fas alternative splicing.jpg|thumb|right|Alternative splicing of the Fas receptor pre-mRNA]]
Multiple isoforms of the [[Fas receptor]] protein are produced by alternative splicing. Two normally occurring isoforms in humans are produced by an exon-skipping mechanism. An mRNA including exon 6 encodes the membrane-bound form of the Fas receptor, which promotes [[apoptosis]], or programmed cell death. Increased expression of Fas receptor in skin cells chronically exposed to the sun, and absence of expression in skin cancer cells, suggests that this mechanism may be important in elimination of pre-cancerous cells in humans.<ref name=Filipowicz>{{cite journal | vauthors = Filipowicz E, Adegboyega P, Sanchez RL, Gatalica Z | title = Expression of CD95 (Fas) in sun-exposed human skin and cutaneous carcinomas | journal = Cancer | volume = 94 | issue = 3 | pages = 814–9 | date = February 2002 | pmid = 11857317 | doi = 10.1002/cncr.10277 | s2cid = 23772719 | doi-access = free }}</ref> If exon 6 is skipped, the resulting mRNA encodes a soluble Fas protein that does not promote apoptosis. The inclusion or skipping of the exon depends on two antagonistic proteins, [[TIA1|TIA-1]] and polypyrimidine tract-binding protein (PTB).
* The 5' donor site in the intron downstream from exon 6 in the pre-mRNA has a weak agreement with the consensus sequence, and is not bound usually by the U1 snRNP. If U1 does not bind, the exon is skipped (see "a" in accompanying figure).
* Binding of TIA-1 protein to an intronic splicing enhancer site stabilizes binding of the U1 snRNP.<ref name=Matlin/> The resulting 5' donor site complex assists in binding of the splicing factor U2AF to the 3' splice site upstream of the exon, through a mechanism that is not yet known (see b).<ref name=Izquierdo2005>{{cite journal | vauthors = Izquierdo JM, Majós N, Bonnal S, Martínez C, Castelo R, Guigó R, Bilbao D, Valcárcel J | display-authors = 6 | title = Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition | journal = Molecular Cell | volume = 19 | issue = 4 | pages = 475–84 | date = August 2005 | pmid = 16109372 | doi = 10.1016/j.molcel.2005.06.015 | doi-access = free }}
</ref>
* Exon 6 contains a pyrimidine-rich exonic splicing silencer, ''ure6'', where PTB can bind. If PTB binds, it inhibits the effect of the 5' donor complex on the binding of U2AF to the acceptor site, resulting in exon skipping (see c).

This mechanism is an example of exon definition in splicing. A spliceosome assembles on an intron, and the snRNP subunits fold the RNA so that the 5' and 3' ends of the intron are joined. However, recently studied examples such as this one show that there are also interactions between the ends of the exon. In this particular case, these exon definition interactions are necessary to allow the binding of core splicing factors prior to assembly of the spliceosomes on the two flanking introns.<ref name=Izquierdo2005/>

====Repressor-activator competition: HIV-1 ''tat'' exon 2====

[[File:Tat exon2 splicing.jpg|thumb|left|Alternative splicing of HIV-1 tat exon 2]]
[[HIV]], the [[retrovirus]] that causes [[AIDS]] in humans, produces a single primary RNA transcript, which is alternatively spliced in multiple ways to produce over 40 different mRNAs.<ref name=Zahler2004>{{cite journal | vauthors = Zahler AM, Damgaard CK, Kjems J, Caputi M | title = SC35 and heterogeneous nuclear ribonucleoprotein A/B proteins bind to a juxtaposed exonic splicing enhancer/exonic splicing silencer element to regulate HIV-1 tat exon 2 splicing | journal = The Journal of Biological Chemistry | volume = 279 | issue = 11 | pages = 10077–84 | date = March 2004 | pmid = 14703516 | doi = 10.1074/jbc.M312743200 | doi-access = free }}</ref> Equilibrium among differentially spliced transcripts provides multiple mRNAs encoding different products that are required for viral multiplication.<ref name=Jaquenet2001>{{cite journal | vauthors = Jacquenet S, Méreau A, Bilodeau PS, Damier L, Stoltzfus CM, Branlant C | title = A second exon splicing silencer within human immunodeficiency virus type 1 tat exon 2 represses splicing of Tat mRNA and binds protein hnRNP H | journal = The Journal of Biological Chemistry | volume = 276 | issue = 44 | pages = 40464–75 | date = November 2001 | pmid = 11526107 | doi = 10.1074/jbc.M104070200 | doi-access = free }}</ref> One of the differentially spliced transcripts contains the ''tat'' gene, in which exon 2 is a cassette exon that may be skipped or included. The inclusion of tat exon 2 in the RNA is regulated by competition between the splicing repressor hnRNP A1 and the SR protein SC35. Within exon 2 an exonic splicing silencer sequence (ESS) and an exonic splicing enhancer sequence (ESE) overlap. If A1 repressor protein binds to the ESS, it initiates [[cooperative binding]] of multiple A1 molecules, extending into the 5' donor site upstream of exon 2 and preventing the binding of the core splicing factor U2AF35 to the polypyrimidine tract. If SC35 binds to the ESE, it prevents A1 binding and maintains the 5' donor site in an accessible state for assembly of the spliceosome. Competition between the activator and repressor ensures that both mRNA types (with and without exon 2) are produced.<ref name=Zahler2004/>