Editing Spliceosome (section)

==Assembly==
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.<ref>{{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 }}</ref><ref>{{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 }}</ref>  The commitment complex is an ATP-independent complex that commits the pre-mRNA to the splicing pathway.<ref name="pmid3065622">{{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 = }}</ref>  U2 snRNP is recruited to the branch region through interactions with the E complex component [[U2AF2|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.<ref>{{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 }}</ref>

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.<ref>{{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 }}</ref>  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.<ref>{{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 }}</ref><ref name="pmid9476892">{{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 }}</ref> 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<ref name=Newman95>{{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 }}</ref> and U5 protein components interact with the 3' splice site region.<ref name="pmid9303319">{{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 }}</ref>

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,<ref>{{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 }}</ref> 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.<ref name="pmid8252623">{{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 =  }}</ref>  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.<ref name="pmid9476892" /> 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}}