mRNA Splicing - Major Pathway

Stable Identifier
Homo sapiens
pre-mRNA splicing, U2 Dependent Splicing
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Eukaryotic genes are transcribed to yield pre-mRNAs that are processed to add methyl guanosine cap structures and polyadenylate tails and to splice together segments of a pre-mRNA termed exons, thereby removing segments termed introns. More than 90% of human genes contain introns, with an average of 4.0 introns per gene and 3413 nucleotides per intron compared with 5.0 exons per gene and 50.9 nucleotides per exon (Deutsch and Long 1999). (Notable exceptions are the histone genes, which are intronless.)
Pre-mRNA splicing is performed by a large ribonucleoprotein complex, the spliceosome, which contains 5 small nuclear RNAs (snRNAs) and more than 150 proteins (reviewed in Will and Luhrmann 2011, Kastner et al. 2019, Yan et al. 2019, Fica et al. 2020, Wan et al. 2020, Wilkinson et al. 2020). The catalyst in the spliceosome comprises magnesium ions coordinated by the U6 snRNA that catalyze transesterification reactions between hydroxyl groups and phosphate groups from the pre-mRNA. The role of the U6 snRNA demonstrates that the spliceosome is a ribozyme hints at the origin of the spliceosome as a self-splicing group II intron.
The spliceosome is initially assembled cotranscriptionally on the pre-mRNA as the Spliceosomal E (Early) complex and then remodelled sequentially by association and dissociation of proteins and snRNAs to catalyze of the two reactions of splicing. First, a nucleophilic attack by the 2' hydroxyl group of a conserved adenine residue, the branch point, within the intron on the phosphate group of the 5' residue of the intron yields a lariat (looped) structure in the intron joined to the downstream (3') exon and a free upstream exon with a 3' hydroxyl group. Second, a nucleophilic attack by the 3' hydroxyl group of the upstream exon on the phosphate of the 5' residue of the downstream exon yields a spliced mRNA containing the upstream exon ligated to the downstream exon and a free intron containing a lariat structure.
The Spliceosomal E complex contains the U1 snRNP bound to the 5' splice site, SF1 bound to the branch point, and the U2AF complex bound to the polypyrimidine tract of the intron and the 3' splice site of the pre-mRNA (Zhuang and Weiner 1986, Hong et al. 1997, Das et al. 2000, Hartmuth et al. 2002, Rappsilber et al. 2002, Hegele et al. 2012, Makarov et al. 2012, Crisci et al. 2015, Kondo et al. 2015, Tan et al. 2016). SF1 and U2AF are displaced on the pre-mRNA and the U2 snRNP binds the branch region to yield the Spliceosomal A complex (Wu and Manley 1989, Fleckner et al. 1997, Neubauer et al. 1998, Hartmuth et al. 2002, Rappsilber et al. 2002, Xu et al. 2004, Behzadnia et al. 2007, Shen et al. 2008, Chen et al. 2017, Zhang et al. 2020). The U4/U6.U5 tri-snRNP, containing the U4 snRNA base-paired with the U6 snRNA plus the U5 snRNP and accessory proteins, binds the Spliceosomal A complex to form the Spliceosomal Pre-B complex (Hausner et al. 1990, Kataoka and Dreyfuss 2004, Chi et al. 2013, Mohlmann et al. 2014, Boesler et al. 2016, Zhan et al. 2018, Charenton et al. 2019, Kastner et al. 2019, Townsend et al. 2020). The U1 snRNP is replaced at the 5' splice site by the U6 snRNA and the spliceosome is remodelled to yield the Spliceosomal B complex (Ismaïli et al. 2001, Deckert et al. 2006, Bessonov et al. 2008, Wolf et al. 2009, Bessonov et al. 2010, Schmidt et al. 2014, Boesler et al. 2016, Bertram et al. 2017, Zhang et al. 2018, Kastner et al. 2019). The Spliceosomal B complex is activated to form the Spliceosomal Bact complex by dissociation of the U4 snRNP and Lsm proteins from the U6 snRNA, freeing the U6 snRNA to form the active site of the spliceosome (Lamond et al. 1988, Laggerbauer et al. 1998, Ajuh et al. 2000, Bessonov et al. 2010, Agafonov et al. 2011, Haselbach et al. 2018, Zhang et al. 2018, Kastner et al. 2019, Busetto et al. 2020). Dissociation of the SF3A and SF3B subcomplexes of the U2 snRNP allows the intron branch point to dock near the 5' splice site, forming the B* Spliceosomal complex. Reaction of the branch point at the 5' splice site, yields the Spliceosomal C complex (Jurica et al. 2002, Makarov et al. 2002, Rappsilber et al. 2002, Reichert et al. 2002, Kataoka and Dreyfuss 2004, Bessonov et al. 2010, Gencheva et al. 2010, Agafonov et al. 2011, Alexandrov et al. 2012, Barbosa et al. 2012, Steckelberg et al. 2012, Schmidt et al. 2014, Zhan et al. 2018, Kastner et al. 2019, Busetto et al. 2020). The branch point is rotated to allow the 3' splice site to enter the active site, yielding the Spliceosomal C* complex (Ortlepp et al. 1998, Zhou and Reed 1998, Jurica et al. 2002, Makarov et al. 2002, Rappsilber et al. 2002, Ilagan et al. 2013, Bertram et al. 2017, Zhang et al. 2017, Kastner et al. 2019). Reaction of the 3' hydroxyl group of the upstream exon at the 3' splice site yields the Spliceosomal P (postcatalytic) complex (Zhou et al. 2000, Kataoka and Dreyfuss 2004, Tange et al. 2005, Zhang and Krainer 2007, Chi et al. 2013, Ilagan et al. 2013, Bertram et al. 2017, Zhang et al. 2017, Fica et al. 2019, Zhang et al. 2019). The Spliceosomal P complex then dissociates to yield an mRNP containing the spliced mRNA and associated proteins, including the exon junction complex (EJC) (Ohno and Shimura 1996, Merz et al. 2007, Yoshimoto et al. 2009, Zanini et al. 2017, Felisberto-Rodrigues et al. 2019, Zhang et al. 2019, EJC reviewed in Schlautmann and Gehring 2020), and the Intron Lariat Spliceosome (ILS), which contains the intron lariat. The ILS is then disassembled to free its components for further splicing reactions and the intron lariat is degraded (Wen et al. 2008, Yoshimoto et al. 2009, Yoshimoto et al. 2014, Zhang et al. 2019, Studer et al. 2020).
Literature References
PubMed ID Title Journal Year
11014198 The protein Aly links pre-messenger-RNA splicing to nuclear export in metazoans

Reed, R, Luo, MJ, Katahira, J, Hurt, E, Straesser, K, Zhou, Z

Nature 2000
20980672 Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis

Sander, B, Will, CL, Lührmann, R, Anokhina, M, Golas, MM, Krasauskas, A, Bessonov, S, Stark, H, Urlaub, H

RNA 2010
10882114 Functional association of U2 snRNP with the ATP-independent spliceosomal complex E

Reed, R, Das, R, Zhou, Z

Mol Cell 2000
12411573 Small nuclear ribonucleoprotein remodeling during catalytic activation of the spliceosome

Makarova, OV, Will, CL, Gentzel, M, Lührmann, R, Wilm, M, Makarov, EM, Urlaub, H

Science 2002
29361316 Structure and Conformational Dynamics of the Human Spliceosomal Bact Complex

Komarov, I, Lührmann, R, Graf, B, Dybkov, O, Agafonov, DE, Haselbach, D, Stark, H, Urlaub, H, Kastner, B, Hartmuth, K

Cell 2018
18322460 Isolation of an active step I spliceosome and composition of its RNP core

Will, CL, Lührmann, R, Anokhina, M, Bessonov, S, Urlaub, H

Nature 2008
30765414 Structural Insights into Nuclear pre-mRNA Splicing in Higher Eukaryotes

Will, CL, Lührmann, R, Stark, H, Kastner, B

Cold Spring Harb Perspect Biol 2019
32494006 Molecular architecture of the human 17S U2 snRNP

Bertram, K, Will, CL, Lührmann, R, Dybkov, O, Agafonov, DE, Stark, H, Urlaub, H, Kastner, B, Zhang, Z, Hofele, R, Hartmuth, K

Nature 2020
29360106 Structure of the human activated spliceosome in three conformational states

Shi, Y, Li, L, Yan, C, Zhang, X, Lei, J, Zhan, X

Cell Res 2018
23345524 Rearrangements within human spliceosomes captured after exon ligation

Ilagan, JO, Chalkley, RJ, Jurica, MS, Burlingame, AL

RNA 2013
21536652 Semiquantitative proteomic analysis of the human spliceosome via a novel two-dimensional gel electrophoresis method

Will, CL, Lührmann, R, Deckert, J, Odenwälder, P, Agafonov, DE, Bessonov, S, Urlaub, H, Wolf, E

Mol Cell Biol 2011
14625303 A simple whole cell lysate system for in vitro splicing reveals a stepwise assembly of the exon-exon junction complex

Kataoka, N, Dreyfuss, G

J Biol Chem 2004
17095540 Protein composition of human mRNPs spliced in vitro and differential requirements for mRNP protein recruitment

Will, CL, Lührmann, R, Urlaub, H, Merz, C

RNA 2007
17332742 Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes

Sander, B, Will, CL, Lührmann, R, Deckert, J, Golas, MM, Dube, P, Stark, H, Urlaub, H, Behzadnia, N, Kastner, B, Hartmuth, K

EMBO J. 2007
16809785 Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions

Will, CL, Lührmann, R, Deckert, J, Boehringer, D, Urlaub, H, Stark, H, Behzadnia, N, Kastner, B, Hartmuth, K

Mol. Cell. Biol. 2006
26420826 Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins

Bagdiul, I, Raabe, M, Rain, JC, Urlaub, H, Raleff, F, Krämer, A, Crisci, A

Nucleic Acids Res. 2015
23222130 Aly and THO are required for assembly of the human TREX complex and association of TREX components with the spliced mRNA

Chang, X, Shi, M, Wu, G, Chi, B, Wang, L, Tan, M, Wang, Q, Cheng, H

Nucleic Acids Res. 2013
14713954 Prp5 bridges U1 and U2 snRNPs and enables stable U2 snRNP association with intron RNA

Kameoka, S, Xu, YZ, Huang, T, Query, CC, Newnham, CM, Konarska, MM

EMBO J 2004
25555158 Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5' splice site recognition

Kondo, Y, van Roon, AM, Nagai, K, Oubridge, C

Elife 2015
3757028 A compensatory base change in U1 snRNA suppresses a 5' splice site mutation

Zhuang, Y, Weiner, AM

Cell 1986
18593880 Distinct activities of the DExD/H-box splicing factor hUAP56 facilitate stepwise assembly of the spliceosome

Zhang, L, Shen, J, Zheng, X, Shen, H, Zhao, R, Green, MR

Genes Dev 2008
2558966 Mammalian pre-mRNA branch site selection by U2 snRNP involves base pairing

Manley, JL, Wu, J

Genes Dev 1989
30705154 A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation

Wilkinson, ME, Fica, SM, Nagai, K, Newman, AJ, Oubridge, C

Science 2019
9016565 Association of U2 snRNP with the spliceosomal complex E

Bennett, M, Hong, W, Reed, R, Feld Kramer, R, Wang, C, Xiao, Y

Nucleic Acids Res 1997
9242493 U2AF65 recruits a novel human DEAD box protein required for the U2 snRNP-branchpoint interaction

Valcárcel, J, Green, MR, Fleckner, J, Zhang, M

Genes Dev 1997
22365833 Dynamic protein-protein interaction wiring of the human spliceosome

Pena, V, Will, CL, Lührmann, R, Weimann, M, Wowro, S, Sourlis, C, Stelzl, U, Hegele, A, Grossmann, A, Kamburov, A

Mol. Cell 2012
31409651 Structural and functional characterisation of human RNA helicase DHX8 provides insights into the mechanism of RNA-stimulated ADP release

Thomas, JC, Workman, P, Felisberto-Rodrigues, C, Le Bihan, YV, Burke, R, McAndrew, C, van Montfort, RLM

Biochem J 2019
9524131 Human homologs of yeast prp16 and prp17 reveal conservation of the mechanism for catalytic step II of pre-mRNA splicing

Reed, R, Zhou, Z

EMBO J 1998
24448447 Mass spectrometry-based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

Lührmann, R, Deckert, J, Schmidt, C, Bessonov, S, Urlaub, H, Conrad, T, Grønborg, M

RNA 2014
24914973 Structural and functional analysis of the human spliceosomal DEAD-box helicase Prp28

Neumann, P, Möhlmann, S, Lührmann, R, Schmitt, A, Ficner, R, Mathew, R

Acta Crystallogr D Biol Crystallogr 2014
9539711 The human U5-200kD DEXH-box protein unwinds U4/U6 RNA duplices in vitro

Achsel, T, Lührmann, R, Laggerbauer, B

Proc Natl Acad Sci U S A 1998
32717639 Cryo-EM snapshots of the human spliceosome reveal structural adaptions for splicing regulation

Fica, SM

Curr Opin Struct Biol 2020
8608946 A human RNA helicase-like protein, HRH1, facilitates nuclear export of spliced mRNA by releasing the RNA from the spliceosome

Ohno, M, Shimura, Y

Genes Dev 1996
9701291 The mammalian homologue of Prp16p is overexpressed in a cell line tolerant to Leflunomide, a new immunoregulatory drug effective against rheumatoid arthritis

Achsel, T, Lührmann, R, Ortlepp, D, Kirschbaum, B, Müllner, S, Laggerbauer, B

RNA 1998
11233976 The 100-kda U5 snRNP protein (hPrp28p) contacts the 5' splice site through its ATPase site

Sha, M, Konarska, MM, Gustafson, EH, Ismaïli, N

RNA 2001
29301961 Structure of a human catalytic step I spliceosome

Yan, C, Shi, Y, Zhang, X, Lei, J, Zhan, X

Science 2018
32329775 Structural and functional insights into CWC27/CWC22 heterodimer linking the exon junction complex to spliceosomes

Paternina, JA, Bensaude, O, Hocq, R, Marquenet, E, Hennion, M, Basquin, J, Le Hir, H, Conti, E, Namane, A, Barbosa, I, Busetto, V

Nucleic Acids Res 2020
27683217 Stoichiometries of U2AF35, U2AF65 and U2 snRNP reveal new early spliceosome assembly pathways

Vorechovsky, I, Hudson, AJ, Eperon, IC, Chen, L, Kralovicova, J, Weinmeister, R, Eperon, LP

Nucleic Acids Res 2017
21441581 Spliceosome structure and function

Will, CL, Lührmann, R

Cold Spring Harb Perspect Biol 2011
32517083 A Day in the Life of the Exon Junction Complex

Gehring, NH, Schlautmann, LP

Biomolecules 2020
30602541 Molecular Mechanisms of pre-mRNA Splicing through Structural Biology of the Spliceosome

Wan, R, Yan, C, Shi, Y

Cold Spring Harb Perspect Biol 2019
28502770 An Atomic Structure of the Human Spliceosome

Yan, C, Shi, Y, Zhang, X, Finci, LI, Lei, J, Hang, J

Cell 2017
27377154 A spliceosome intermediate with loosely associated tri-snRNP accumulates in the absence of Prp28 ATPase activity

Tauchert, MJ, Will, CL, Rigo, N, Anokhina, MM, Agafonov, DE, Luhrmann, R, Urlaub, H, Boesler, C, Ficner, R, Kastner, B

Nat Commun 2016
22959432 CWC22 connects pre-mRNA splicing and exon junction complex assembly

Gehring, NH, Steckelberg, AL, Boehm, V, Gromadzka, AM

Cell Rep 2012
30728453 Structures of the human spliceosomes before and after release of the ligated exon

Shi, Y, Liu, D, Yan, C, Zhang, W, Zhang, X, Lei, J, Zhan, X

Cell Res 2019
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Nucleic Acids Res 2016
2176635 Evidence for base-pairing between mammalian U2 and U6 small nuclear ribonucleoprotein particles

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Genes Dev 1990
31794245 RNA Splicing by the Spliceosome

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Annu Rev Biochem 2020
2963332 Spliceosome assembly involves the binding and release of U4 small nuclear ribonucleoprotein

Lamond, AI, Grabowski, PJ, Sharp, PA, Konarska, MM

Proc. Natl. Acad. Sci. U.S.A. 1988
31815536 How Is Precursor Messenger RNA Spliced by the Spliceosome?

Wan, R, Shi, Y, Bai, R, Zhan, X

Annu Rev Biochem 2020
24304693 Identification of a novel component C2ORF3 in the lariat-intron complex: lack of C2ORF3 interferes with pre-mRNA splicing via intron turnover pathway

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Genes Cells 2014
19536130 Exon, intron and splice site locations in the spliceosomal B complex

Lührmann, R, Deckert, J, Stark, H, Merz, C, Kastner, B, Wolf, E

EMBO J 2009
9731529 Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex

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Nat. Genet. 1998
33243851 Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation

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Science 2020
23236153 Human spliceosomal protein CWC22 plays a role in coupling splicing to exon junction complex deposition and nonsense-mediated decay

Shu, MD, Alexandrov, A, Colognori, D, Steitz, JA

Proc Natl Acad Sci U S A 2012
12477934 Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method.

Hartmuth, K, Urlaub, H, Lührmann, R, Gentzel, M, Wilm, M, Vornlocher, HP, Will, CL

Proc Natl Acad Sci U S A 2002
30315277 Structures of the human pre-catalytic spliceosome and its precursor spliceosome

Shi, Y, Yan, C, Zhang, X, Lei, J, Zhan, X

Cell Res 2018
32179686 Structural basis for DEAH-helicase activation by G-patch proteins

Weber, ME, Ivanović, L, Jonas, S, Studer, MK, Marti, S

Proc Natl Acad Sci U S A 2020
22961380 Human CWC22 escorts the helicase eIF4AIII to spliceosomes and promotes exon junction complex assembly

Barrandon, C, Haque, N, Le Hir, H, Barbosa, I, Blanchette, M, Fiorini, F, Tomasetto, C

Nat Struct Mol Biol 2012
19165350 TFIP11 interacts with mDEAH9, an RNA helicase involved in spliceosome disassembly

Paine, ML, Wen, X, Tannukit, S

Int J Mol Sci 2008
30975767 Mechanism of 5' splice site transfer for human spliceosome activation

Charenton, C, Wilkinson, ME, Nagai, K

Science 2019
28062851 Human cactin interacts with DHX8 and SRRM2 to assure efficient pre-mRNA splicing and sister chromatid cohesion

Zanini, IM, Lorenzi, LE, Azzalin, CM, Soneson, C

J Cell Sci 2017
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Gencheva, M, Kato, M, Newo, AN, Lin, RJ

Biochem. J. 2010
12176931 Large-scale proteomic analysis of the human spliceosome.

Lamond, AI, Mann, M, Rappsilber, J, Ryder, U

Genome Res 2002
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EMBO J. 2000
10454621 Intron-exon structures of eukaryotic model organisms

Deutsch, M, Long, M

Nucleic Acids Res 1999
22110043 Functional mammalian spliceosomal complex E contains SMN complex proteins in addition to U1 and U2 snRNPs

Makarova, OV, Makarov, EM, Bottrill, A, Owen, N

Nucleic Acids Res. 2012
28781166 Cryo-EM Structure of a Pre-catalytic Human Spliceosome Primed for Activation

Bertram, K, Will, CL, Lührmann, R, Leelaram, MN, Dybkov, O, Haselbach, D, Agafonov, DE, Stark, H, Urlaub, H, Kastner, B

Cell 2017
11991638 Purification and characterization of native spliceosomes suitable for three-dimensional structural analysis.

Grigorieff, N, Moore, MJ, Licklider, LJ, Gygi, SR, Jurica, MS

RNA 2002
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Genes Dev 2002
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Nucleic Acids Res. 2009
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