Spliceosome

{{see also|Splicing (genetics)}}

In 1977, work by the Sharp and Roberts labs revealed that genes of higher organisms are "split" or present in several distinct segments along the DNA molecule.{{cite journal | vauthors = Berget SM, Moore C, Sharp PA | title = Spliced segments at the 5' terminus of adenovirus 2 late mRNA | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 74 | issue = 8 | pages = 3171–5 | date = August 1977 | pmid = 269380 | pmc = 431482 | doi = 10.1073/pnas.74.8.3171 | bibcode = 1977PNAS...74.3171B | doi-access = free }}{{cite journal | vauthors = Chow LT, Roberts JM, Lewis JB, Broker TR | title = A map of cytoplasmic RNA transcripts from lytic adenovirus type 2, determined by electron microscopy of RNA:DNA hybrids | journal = Cell | volume = 11 | issue = 4 | pages = 819–36 | date = August 1977 | pmid = 890740 | doi = 10.1016/0092-8674(77)90294-X | s2cid = 37967144 }} The coding regions of the gene are separated by non-coding DNA that is not involved in protein expression. The split gene structure was found when adenoviral mRNAs were hybridized to endonuclease cleavage fragments of single stranded viral DNA. It was observed that the mRNAs of the mRNA-DNA hybrids contained 5' and 3' tails of non-hydrogen bonded regions. When larger fragments of viral DNAs were used, forked structures of looped out DNA were observed when hybridized to the viral mRNAs. It was realised that the looped out regions, the introns, are excised from the precursor mRNAs in a process Sharp named "splicing". The split gene structure was subsequently found to be common to most eukaryotic genes. Phillip Sharp and Richard J. Roberts were awarded the [https://www.nobelprize.org/prizes/medicine/1993/summary/ Nobel Prize in Medicine 1993] for the discovery of introns and the splicing process.

Each spliceosome is composed of five small nuclear RNAs (snRNA) and a range of associated protein factors. When these small RNAs are combined with the protein factors, they make RNA-protein complexes called snRNPs (small nuclear ribonucleoproteins, pronounced "snurps").

The snRNAs that make up the major spliceosome are named U1, U2, U4, U5, and U6, so-called because they are rich in uridine, and participate in several RNA-RNA and RNA-protein interactions.

The assembly of the spliceosome occurs on each pre-mRNA (also known as heterogeneous nuclear RNA, hn-RNA) at each exon:intron junction. The pre-mRNA introns contains specific sequence elements that are recognized and utilized during spliceosome assembly. These include the 5' end splice site, the branch point sequence, the polypyrimidine tract, and the 3' end splice site. The spliceosome catalyzes the removal of introns, and the ligation of the flanking exons.{{cn|date=July 2024}}

Introns typically have a GU nucleotide sequence at the 5' end splice site, and an AG at the 3' end splice site. The 3' splice site can be further defined by a variable length of polypyrimidines, called the polypyrimidine tract (PPT), which serves the dual function of recruiting factors to the 3' splice site and possibly recruiting factors to the branch point sequence (BPS). The BPS contains the conserved adenosine required for the first step of splicing.{{cn|date=July 2024}}

Many proteins exhibit a zinc-binding motif, which underscores the importance of zinc in the splicing mechanism.{{cite journal | vauthors = Agafonov DE, Deckert J, Wolf E, Odenwälder P, Bessonov S, Will CL, Urlaub H, Lührmann R | title = Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method | journal = Molecular and Cellular Biology | volume = 31 | issue = 13 | pages = 2667–82 | date = July 2011 | pmid = 21536652 | pmc = 3133382 | doi = 10.1128/mcb.05266-11 }}{{cite journal | vauthors = Kuhn AN, van Santen MA, Schwienhorst A, Urlaub H, Lührmann R | title = Stalling of spliceosome assembly at distinct stages by small-molecule inhibitors of protein acetylation and deacetylation | journal = RNA | volume = 15 | issue = 1 | pages = 153–75 | date = January 2009 | pmid = 19029308 | pmc = 2612777 | doi = 10.1261/rna.1332609 }}{{cite journal | vauthors = Patil V, Canzoneri JC, Samatov TR, Lührmann R, Oyelere AK | title = Molecular architecture of zinc chelating small molecules that inhibit spliceosome assembly at an early stage | journal = RNA | volume = 18 | issue = 9 | pages = 1605–11 | date = September 2012 | pmid = 22832025 | pmc = 3425776 | doi = 10.1261/rna.034819.112 }} The first molecular-resolution reconstruction of U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) complex was reported in 2016.{{cite journal | vauthors = Cate JH | title = STRUCTURE. A Big Bang in spliceosome structural biology | journal = Science | volume = 351 | issue = 6280 | pages = 1390–2 | date = March 2016 | pmid = 27013712 | doi = 10.1126/science.aaf4465 | s2cid = 206648185 }}

Image:Yeast tri-snRNP.jpg{{cite journal | vauthors = Häcker I, Sander B, Golas MM, Wolf E, Karagöz E, Kastner B, Stark H, Fabrizio P, Lührmann R | title = Localization of Prp8, Brr2, Snu114 and U4/U6 proteins in the yeast tri-snRNP by electron microscopy | journal = Nature Structural & Molecular Biology | volume = 15 | issue = 11 | pages = 1206–12 | date = November 2008 | pmid = 18953335 | doi = 10.1038/nsmb.1506 | s2cid = 22982227 }} fields of negatively stained yeast (Saccharomyces cerevisiae) tri-snRNPs. Below left is a schematic illustration of the interaction of tri-snRNP proteins with the U4/U6 snRNA duplex. Below right is a cartoon model of the yeast tri-snRNP with shaded areas corresponding to U5 (gray), U4/U6 (orange) and the linker region (yellow).]]

Cryo-EM has been applied extensively by Shi et al. to elucidate the near-/atomic structure of spliceosome in both yeast{{cite journal | vauthors = Yan C, Hang J, Wan R, Huang M, Wong CC, Shi Y | title = Structure of a yeast spliceosome at 3.6-angstrom resolution | journal = Science | volume = 349 | issue = 6253 | pages = 1182–91 | date = September 2015 | pmid = 26292707 | doi = 10.1126/science.aac7629 | bibcode = 2015Sci...349.1182Y | s2cid = 22194712 }} and humans.{{cite journal | vauthors = Zhang X, Yan C, Hang J, Finci LI, Lei J, Shi Y | title = An Atomic Structure of the Human Spliceosome | journal = Cell | volume = 169 | issue = 5 | pages = 918–929.e14 | date = May 2017 | pmid = 28502770 | doi = 10.1016/j.cell.2017.04.033 | doi-access = free }} The molecular framework of spliceosome at near-atomic-resolution demonstrates Spp42 component of U5 snRNP forms a central scaffold and anchors the catalytic center in yeast. The atomic structure of the human spliceosome illustrates the step II component Slu7 adopts an extended structure, poised for selection of the 3'-splice site. All five metals (assigned as Mg2+) in the yeast complex are preserved in the human complex.{{cn|date=July 2024}}

{{main|Alternative splicing}}

Alternative splicing (the re-combination of different exons) is a major source of genetic diversity in eukaryotes. Splice variants have been used to account for the relatively small number of protein coding genes in the human genome, currently estimated at around 20,000. One particular Drosophila gene, Dscam, has been speculated to be alternatively spliced into 38,000 different mRNAs, assuming all of its exons can splice independently of each other.{{cite journal | vauthors = Schmucker D, Clemens JC, Shu H, Worby CA, Xiao J, Muda M, Dixon JE, Zipursky SL | title = Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity | journal = Cell | volume = 101 | issue = 6 | pages = 671–84 | date = June 2000 | pmid = 10892653 | doi = 10.1016/S0092-8674(00)80878-8 | doi-access = free }}

Pre-mRNA splicing factors were originally found to be concentrated in nuclear bodies known as nuclear speckles.

{{cite journal | vauthors = Spector DL, Lamond AI | title = Nuclear speckles | journal = Cold Spring Harbor Perspectives in Biology | volume = 3 | issue = 2 | pages = a000646 | date = Feb 2011 | pmid = 20926517 | pmc = 3039535 | doi = 10.1101/cshperspect.a000646 | department = Review }} It was originally postulated that nuclear speckles are either sites of mRNA splicing or storage sites of mRNA splicing factors. It is now understood that nuclear speckles help concentrate splicing factors near genes that are physically located close to them. Genes located farther from speckles can still be transcribed and spliced, but their splicing is less efficient compared to those closer to speckles.{{cite journal | vauthors = Bhat P, Chow A, Emert B et al | title = Genome organization around nuclear speckles drives mRNA splicing efficiency. | journal = Nature | volume = 629 | issue = 5 | pages = 1165–1173 | date = May 2024 | pmid = 38720076 | pmc = 11164319 | doi = 10.1038/s41586-024-07429-6 | bibcode = 2024Natur.629.1165B }} RNA splicing is a biochemical reaction, and like all biochemical reactions, its rate depends on the concentration of enzymes and substrates. In this case, the enzymes are the spliceosomes, and the substrates are the pre-mRNAs. By varying the concentration of spliceosomes and pre-mRNAs based on their proximity to nuclear speckles, cells could potentially regulate the efficiency of splicing.

The model for formation of the spliceosome active site involves an ordered, stepwise assembly of discrete snRNP particles on the pre-mRNA substrate. The first recognition of pre-mRNAs involves U1 snRNP binding to the 5' end splice site of the pre-mRNA and other non-snRNP associated factors to form the commitment complex, or early (E) complex in mammals.{{cite journal | vauthors = Jamison SF, Crow A, Garcia-Blanco MA | title = The spliceosome assembly pathway in mammalian extracts | journal = Molecular and Cellular Biology | volume = 12 | issue = 10 | pages = 4279–87 | date = October 1992 | pmid = 1383687 | pmc = 360351 | doi = 10.1128/MCB.12.10.4279 }}{{cite journal | vauthors = Seraphin B, Rosbash M | title = Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing | journal = Cell | volume = 59 | issue = 2 | pages = 349–58 | date = October 1989 | pmid = 2529976 | doi = 10.1016/0092-8674(89)90296-1 | s2cid = 18553973 }} The commitment complex is an ATP-independent complex that commits the pre-mRNA to the splicing pathway.{{cite journal | vauthors = Legrain P, Seraphin B, Rosbash M | title = Early commitment of yeast pre-mRNA to the spliceosome pathway | journal = Molecular and Cellular Biology | volume = 8 | issue = 9 | pages = 3755–60 | date = September 1988 | pmid = 3065622 | pmc = 365433 | doi = 10.1128/MCB.8.9.3755 | url = }} U2 snRNP is recruited to the branch region through interactions with the E complex component U2AF (U2 snRNP auxiliary factor) and possibly U1 snRNP. In an ATP-dependent reaction, U2 snRNP becomes tightly associated with the branch point sequence (BPS) to form complex A. A duplex formed between U2 snRNP and the pre-mRNA branch region bulges out the branch adenosine specifying it as the nucleophile for the first transesterification.{{cite journal | vauthors = Query CC, Moore MJ, Sharp PA | title = Branch nucleophile selection in pre-mRNA splicing: evidence for the bulged duplex model | journal = Genes & Development | volume = 8 | issue = 5 | pages = 587–97 | date = March 1994 | pmid = 7926752 | doi = 10.1101/gad.8.5.587 | doi-access = free }}

The presence of a pseudouridine residue in U2 snRNA, nearly opposite of the branch site, results in an altered conformation of the RNA-RNA duplex upon the U2 snRNP binding. Specifically, the altered structure of the duplex induced by the pseudouridine places the 2' OH of the bulged adenosine in a favorable position for the first step of splicing.{{cite journal | vauthors = Newby MI, Greenbaum NL | title = Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine | journal = Nature Structural Biology | volume = 9 | issue = 12 | pages = 958–65 | date = December 2002 | pmid = 12426583 | doi = 10.1038/nsb873 | s2cid = 39628664 }} The U4/U5/U6 tri-snRNP (see Figure 1) is recruited to the assembling spliceosome to form complex B, and following several rearrangements, complex C is activated for catalysis.{{Cite book| vauthors = Burge CB, Tuschl T, Sharp PA |year = 1999 |chapter= Splicing precursors to mRNAs by the spliceosomes | veditors = Gesteland RF, Cech TR, Atkins JF |title=The RNA World |publisher=Cold Spring Harbor Lab. Press |pages=525–60 |isbn=978-0-87969-380-0 }}{{cite journal | vauthors = Staley JP, Guthrie C | title = Mechanical devices of the spliceosome: motors, clocks, springs, and things | journal = Cell | volume = 92 | issue = 3 | pages = 315–26 | date = February 1998 | pmid = 9476892 | doi = 10.1016/S0092-8674(00)80925-3 | doi-access = free }} It is unclear how the tri-snRNP is recruited to complex A, but this process may be mediated through protein-protein interactions and/or base pairing interactions between U2 snRNA and U6 snRNA.{{cn|date=July 2024}}

The U5 snRNP interacts with sequences at the 5' and 3' splice sites via the invariant loop of U5 snRNA{{cite journal | vauthors = Newman AJ, Teigelkamp S, Beggs JD | title = snRNA interactions at 5' and 3' splice sites monitored by photoactivated crosslinking in yeast spliceosomes | journal = RNA | volume = 1 | issue = 9 | pages = 968–80 | date = November 1995 | pmid = 8548661 | pmc = 1369345 | url = http://www.rnajournal.org/cgi/reprint/1/9/968 | access-date = 2008-03-07 | archive-date = 2005-02-23 | archive-url = https://web.archive.org/web/20050223232712/http://www.rnajournal.org/cgi/reprint/1/9/968 | url-status = live }} and U5 protein components interact with the 3' splice site region.{{cite journal | vauthors = Chiara MD, Palandjian L, Feld Kramer R, Reed R | title = Evidence that U5 snRNP recognizes the 3' splice site for catalytic step II in mammals | journal = The EMBO Journal | volume = 16 | issue = 15 | pages = 4746–59 | date = August 1997 | pmid = 9303319 | pmc = 1170101 | doi = 10.1093/emboj/16.15.4746 }}

Upon recruitment of the tri-snRNP, several RNA-RNA rearrangements precede the first catalytic step and further rearrangements occur in the catalytically active spliceosome. Several of the RNA-RNA interactions are mutually exclusive; however, it is not known what triggers these interactions, nor the order of these rearrangements. The first rearrangement is probably the displacement of U1 snRNP from the 5' splice site and formation of a U6 snRNA interaction. It is known that U1 snRNP is only weakly associated with fully formed spliceosomes,{{cite journal | vauthors = Moore MJ, Sharp PA | title = Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing | journal = Nature | volume = 365 | issue = 6444 | pages = 364–8 | date = September 1993 | pmid = 8397340 | doi = 10.1038/365364a0 | bibcode = 1993Natur.365..364M | s2cid = 4361512 }} and U1 snRNP is inhibitory to the formation of a U6-5' splice site interaction on a model of substrate oligonucleotide containing a short 5' exon and 5' splice site.{{cite journal | vauthors = Konforti BB, Koziolkiewicz MJ, Konarska MM | title = Disruption of base pairing between the 5' splice site and the 5' end of U1 snRNA is required for spliceosome assembly | journal = Cell | volume = 75 | issue = 5 | pages = 863–73 | date = December 1993 | pmid = 8252623 | doi = 10.1016/0092-8674(93)90531-T | doi-access =free }} Binding of U2 snRNP to the branch point sequence (BPS) is one example of an RNA-RNA interaction displacing a protein-RNA interaction. Upon recruitment of U2 snRNP, the branch binding protein SF1 in the commitment complex is displaced since the binding site of U2 snRNA and SF1 are mutually exclusive events.{{cn|date=July 2024}}

Within the U2 snRNA, there are other mutually exclusive rearrangements that occur between competing conformations. For example, in the active form, stem loop IIa is favored; in the inactive form a mutually exclusive interaction between the loop and a downstream sequence predominates. It is unclear how U4 is displaced from U6 snRNA, although RNA has been implicated in spliceosome assembly, and may function to unwind U4/U6 and promote the formation of a U2/U6 snRNA interaction. The interactions of U4/U6 stem loops I and II dissociate and the freed stem loop II region of U6 folds on itself to form an intramolecular stem loop and U4 is no longer required in further spliceosome assembly. The freed stem loop I region of U6 base pairs with U2 snRNA forming the U2/U6 helix I. However, the helix I structure is mutually exclusive with the 3' half of an internal 5' stem loop region of U2 snRNA.{{cn|date=July 2024}}

{{see also|Minor spliceosome}}

Some eukaryotes have a second spliceosome, the so-called minor spliceosome.{{cite journal | vauthors = Patel AA, Steitz JA | title = Splicing double: insights from the second spliceosome | journal = Nature Reviews. Molecular Cell Biology | volume = 4 | issue = 12 | pages = 960–70 | date = December 2003 | pmid = 14685174 | doi = 10.1038/nrm1259 | s2cid = 21816910 | authorlink2 = Joan A. Steitz }}

A group of less abundant snRNAs, U11, U12, U4atac, and U6atac, together with U5, are subunits of the minor spliceosome that splices a rare class of pre-mRNA introns, denoted U12-type. The minor spliceosome is located in the nucleus like its major counterpart,{{cite journal | vauthors = Pessa HK, Will CL, Meng X, Schneider C, Watkins NJ, Perälä N, Nymark M, Turunen JJ, Lührmann R, Frilander MJ | title = Minor spliceosome components are predominantly localized in the nucleus | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 105 | issue = 25 | pages = 8655–60 | date = June 2008 | pmid = 18559850 | pmc = 2438382 | doi = 10.1073/pnas.0803646105 | doi-access = free }} though there are exceptions in some specialised cells including anucleate platelets{{cite journal | vauthors = Denis MM, Tolley ND, Bunting M, Schwertz H, Jiang H, Lindemann S, Yost CC, Rubner FJ, Albertine KH, Swoboda KJ, Fratto CM, Tolley E, Kraiss LW, McIntyre TM, Zimmerman GA, Weyrich AS | title = Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets | journal = Cell | volume = 122 | issue = 3 | pages = 379–91 | date = August 2005 | pmid = 16096058 | pmc = 4401993 | doi = 10.1016/j.cell.2005.06.015 }} and the dendroplasm (dendrite cytoplasm) of neuronal cells.{{cite journal | vauthors = Glanzer J, Miyashiro KY, Sul JY, Barrett L, Belt B, Haydon P, Eberwine J | title = RNA splicing capability of live neuronal dendrites | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 102 | issue = 46 | pages = 16859–64 | date = November 2005 | pmid = 16275927 | pmc = 1277967 | doi = 10.1073/pnas.0503783102 | bibcode = 2005PNAS..10216859G | doi-access = free }}

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{{refbegin}}

• {{cite book| vauthors = Butcher SE | veditors = Sigel A, Sigel H, Sigel RK |title=Structural and catalytic roles of metal ions in RNA|series=Metal Ions in Life Sciences|volume=9|year=2011|publisher=RSC Publishing|doi=10.1039/9781849732512-00235 |pages=235–51|chapter=Chapter 8. The Spliceosome and Its Metal Ions | pmid = 22010274 |isbn=978-1-84973-094-5 }}
• {{cite journal | vauthors = Nilsen TW | title = The spliceosome: the most complex macromolecular machine in the cell? | journal = BioEssays | volume = 25 | issue = 12 | pages = 1147–9 | date = December 2003 | pmid = 14635248 | doi = 10.1002/bies.10394 | doi-access = }}

{{refend}}

{{Commons category| Spliceosomes}}

• [http://www.pdbe.org/emsearch/spliceosom* 3D macromolecular structures of Spliceosomes from the EM Data Bank(EMDB)]
• {{MeshName|Spliceosomes}}

{{Post transcriptional modification}}

Category:Organelles

Category:Gene expression

Category:Protein complexes

Category:RNA splicing