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The spliceosome removes introns from messenger RNA precursors (pre-mRNA). Decades of biochemistry and genetics combined with recent structural studies of the spliceosome have produced a detailed view of the mechanism of splicing. In this review, we aim to make this mechanism understandable and provide several videos of the spliceosome in action to illustrate the intricate choreography of splicing. The U1 and U2 small nuclear ribonucleoproteins (snRNPs) mark an intron and recruit the U4/U6.U5 tri-snRNP. Transfer of the 5′ splice site (5′SS) from U1 to U6 snRNA triggers unwinding of U6 snRNA from U4 snRNA. U6 folds with U2 snRNA into an RNA-based active site that positions the 5′SS at two catalytic metal ions. The branch point (BP) adenosine attacks the 5′SS, producing a free 5′ exon. Removal of the BP adenosine from the active site allows the 3′SS to bind, so that the 5′ exon attacks the 3′SS to produce mature mRNA and an excised lariat intron.
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Supplemental Video 1: An overview of pre-mRNA splicing. Structures derive from PDB codes 6G90 for yeast A complex (31), 5GAN for yeast tri-snRNP (53), 5ZWM for yeast pre-B complex (38), 5NRL for yeast B complex (51), 5GM6 for yeast Bact complex (82), 5LJ5 for yeast C complex (29), 5MQ0 for yeast C* complex (85), and 6EXN for yeast P complex (87). The structure of the full intron RNA is built freely to connect ordered parts of the structures, and is for illustrative purposes only. Video used with permission from the MRC Laboratory of Molecular Biology, Cambridge, UK.
Supplemental Video 2: Assembly of the A complex. Structures derive from PDB codes 6G90 for yeast A complex (31) and 6QW6 for human tri-snRNP (56). Initial U1 binding is shown using the core of yeast U1 snRNP which corresponds to the human U1 snRNP structure (34, 35). Data not shown from other important structures (37, 38, 53). Transcription of narration: “An intron is marked by conserved motifs at the 5′ splice site, branch point, and 3′ splice site. The U1 snRNP binds to the 5′ splice site, and U2 snRNP binds to the branch point, forming the prespliceosome, or A complex. The remaining 3 snRNPs are preassembled into the tri-snRNP.” Video used with permission from the MRC Laboratory of Molecular Biology, Cambridge, UK.
Supplemental Video 3: Assembly of pre-B complex and its transition to B complex. Structures derive from PDB codes 6QX9 for human pre-B complex (56) and 6AHD for human B complex (79). One possible pathway for the transition is shown. Prp28 is shown leaving with U1 snRNP, and 5′SS annealing to U6 snRNA is shown before Brr2 loading on U4 snRNA, but an alternative order of events is possible. The U6 snRNA ACAGAGA box is a flexible loop not modeled in the structure, but for visualization is shown here in one possible conformation. Transcription of narration: “Here is the human tri-snRNP. Pre-B complex is formed when U1 and U2 snRNPs bind. U2 in green binds stably, while U1 in purple is more transient and docks between the RecA domains of Prp28 helicase. Upon binding, RecA2 clamps around U1 snRNA, promoting release of the 5′ splice site as U1 and Prp28 dissociate. The 5′ splice site anneals to the U6 ACAGAGA box, shown in red. This exposes the Brr2 binding site on U4, in yellow. A concerted set of conformational rearrangements result in Brr2 binding U4 snRNA to form the B-complex spliceosome, primed for catalytic activation.” Video adapted from Charenton et al. (56).
Supplemental Video 4: Spliceosome activation, showing transition from B complex to Bact complex and formation of the active site. Structures derive from PDB codes 6AHD for human B complex (79) and 5Z56 for human Bact complex (94a), with other structures informing the transition (148, 94b). U4/U6 di-snRNP proteins are shown departing before Brr2 action on the U4/U6 duplex but these events may coincide. Transcription of narration: “This is human B complex. The 5′SS is properly positioned while the branch point adenosine is distant and the active site is not formed. At some unknown time, the B-complex proteins and other factors dissociate. This probably coincides with the activity of the Brr2 helicase, which unwinds the U4/U6 duplex. In the absence of its U4 chaperone, U6 can fold into the active site together with U2. The newly formed active site holds two catalytic magnesium ions close to the 5′SS. However, the branching reaction cannot occur because Cwc24 shields the 5′SS, and SF3b encapsulates the branch point. This new conformation of the spliceosome is stabilized by the NTC and NTR complexes, forming Bact complex.” Video used with permission from the MRC Laboratory of Molecular Biology, Cambridge, UK.
Supplemental Video 5: The branching reaction, showing the transition from Bact complex to C complex via B* complex. Structures derive from PDB codes 5GM6 for yeast Bact complex (82), 6J6N and 6J6Q for yeast B* complex (91), and 5LJ5 for yeast C complex (29). B* and C complex structures were modified according to a 2.8 Å resolution cryo-EM map of yeast C complex (M.E. Wilkinson, unpublished). Data not shown from other important structures (30, 81). The B* structures were used to animate docking of the branch helix and the effects of Cwc25. Transcription of narration: “In Bact complex, the branching reaction is inhibited because the 5′SS is protected by Cwc24, and the branch point is sequestered inside SF3b. To relieve this inhibition, the helicase Prp2 pulls the intron near the 3′SS. By an unknown mechanism, this triggers loss of the RES complex, SF3b, and Cwc24. The exposed branch helix can now start to dock into the active site. Docking is accompanied by a large conformational change to B* complex. For branching chemistry to occur, the step I factors Yju2 and Isy1 further dock the branch helix into the active site. Cwc25 then penetrates the branch helix to ultimately promote chemistry. The product of branching is cleavage at the 5′SS and formation of a 2′–5′ link between the 5′SS and branch point. The resultant spliceosome after branching is called C complex.” Video used with permission from the MRC Laboratory of Molecular Biology, Cambridge, UK.
Supplemental Video 6: The exon ligation reaction, showing the transition from C complex to P complex via C* complex. Structures derive from PDB codes 5LJ5 for yeast C complex (29) and 6EXN for yeast P complex (87). Data not shown from other important structures (30, 85, 86, 88, 89). To animate 3′SS docking a hairpin was modelled between the BP and 3′SS as suggested by Liu et al. (88). Transcription of narration: “C complex is formed immediately after the branching reaction. To enable exon ligation, the helicase Prp16 pulls the intron near the 3′SS. This triggers dissociation of the step I factors. The branch helix then undocks, accompanied by a change to the exon ligation conformation, forming C* complex. The step II factors Prp18 and Slu7 then bind along with the helicase Prp22. This promotes docking of the 3′SS into the active site. The 3′SS is recognized by pairing to the 5′SS and branch point adenosine. Upon exon ligation, the 5′ and 3′ exons are connected to form mRNA. The resultant spliceosome after exon ligation is called P complex.” Video used with permission from the MRC Laboratory of Molecular Biology, Cambridge, UK.
Supplemental Video 7: Release of mRNA and spliceosome disassembly, showing the transition from P complex to ILS. Structures derive from PDB codes 6EXN for yeast P complex (87) and 5Y88 for yeast ILS (90). Data not shown from other important structures (88, 89). There is a wider gap between the Prp8 Large domain and N-terminal domain in ILS complex compared with P complex. Therefore the mRNA is shown sliding out this gap, but this is not known to be the case. Transcription of narration: “P complex contains the ligated exons and lariat intron. To release mRNA, the helicase Prp22 pulls on the 3′ exon. This results in loss of exon ligation factors, and release of spliced mRNA. The resultant Intron Lariat Spliceosome, or ILS complex, is disassembled by the helicase Prp43, which binds Syf1 near U2/U6 helix II. Binding of the Ntr1 complex stimulates Prp43 activity, resulting in disassembly of the spliceosome into U6 snRNA, U2 snRNP, U5 snRNP, and NTC and NTR proteins; and release of the excised lariat intron, which is degraded.” Video used with permission from the MRC Laboratory of Molecular Biology, Cambridge, UK.