The target of the DEAH-box NTP triphosphatase Prp43 in Saccharomyces cerevisiae spliceosomes is the U2 snRNP-intron interaction

Fourmann, Jean-Baptiste; Dybkov, Olexandr; Agafonov, Dmitry E.; Tauchert, M.J.; Urlaub, Henning; Ficner, Ralf; Fabrizio, Patrizia; Lührmann, Reinhard

doi:10.7554/elife.15564

Cited by 51 publications

(87 citation statements)

References 48 publications

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“…For the first three nucleotides at the 5’ end (U1–U3) two alternative conformations are observed (Figure 1—figure supplement 2). The structure reveals the basis for the sequence-independent RNA binding of Prp43 which is in line with previous biochemical data (Fourmann et al, 2016b; Bohnsack et al, 2009; Tanaka and Schwer, 2006). …”

Section: Resultssupporting

confidence: 90%

“…To dismantle purified ILSs (Fourmann et al, 2016b), samples were incubated with distinct combinations of a 10-fold molar excess over the spliceosome of recombinant scPrp43, ctPrp43, ctPrp43-IDSB, ctPrp43-HT, ctPrp43-HL, ctPrp43-HT&HL and scNtr(1•2). An additional step of incubation was performed for the variant ctPrp43-IDSB in the presence of 0.5 mM DTT for 5 min at 23°C.…”

Section: Methodsmentioning

confidence: 99%

“…In the course of this process, Prp43 acts at the latest stage of the splicing cycle and it is required to dismantle the intron-lariat spliceosome into the excised lariat and the U2•U5•U6 snRNPs (Arenas and Abelson, 1997; Fourmann et al, 2013, 2016a). Recently, the target substrate of Prp43 during this process was revealed which is the RNA network between the U2 snRNP and the branch site of the intron (Fourmann et al, 2016b). Indeed, in the spliceosome, Prp43 was crosslinked exclusively to the pre-mRNA and not to any snRNAs.…”

Section: Introductionmentioning

confidence: 99%

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Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

Tauchert

Fourmann

Lührmann

et al. 2017

eLife

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216

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The DEAH-box helicase Prp43 is a key player in pre-mRNA splicing as well as the maturation of rRNAs. The exact modus operandi of Prp43 and of all other spliceosomal DEAH-box RNA helicases is still elusive. Here, we report crystal structures of Prp43 complexes in different functional states and the analysis of structure-based mutants providing insights into the unwinding and loading mechanism of RNAs. The Prp43•ATP-analog•RNA complex shows the localization of the RNA inside a tunnel formed by the two RecA-like and C-terminal domains. In the ATP-bound state this tunnel can be transformed into a groove prone for RNA binding by large rearrangements of the C-terminal domains. Several conformational changes between the ATP- and ADP-bound states explain the coupling of ATP hydrolysis to RNA translocation, mainly mediated by a β-turn of the RecA1 domain containing the newly identified RF motif. This mechanism is clearly different to those of other RNA helicases.DOI: http://dx.doi.org/10.7554/eLife.21510.001

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Section: Resultssupporting

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

Tauchert

Fourmann

Lührmann

et al. 2017

eLife

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216

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“…We have recently shown that the spliceosomal target of Prp43 is the pre-mRNA branchsite/U2 snRNP interaction (42). Our data revealed that Prp43 fused to the GP motif of Ntr1 is a minimal discard enzyme leading to spliceosome disassembly, while Ntr2 is not strictly required for disassembly (42).…”

Section: Introductionmentioning

confidence: 99%

Regulation of Prp43-mediated disassembly of spliceosomes by its cofactors Ntr1 and Ntr2

Fourmann

Tauchert

Ficner

et al. 2016

Nucleic Acids Research

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The DEAH-box NTPase Prp43 disassembles spliceosomes in co-operation with the cofactors Ntr1/Spp382 and Ntr2, forming the NTR complex. How Prp43 is regulated by its cofactors to discard selectively only intron-lariat spliceosomes (ILS) and defective spliceosomes and to prevent disassembly of earlier and properly assembled/wild-type spliceosomes remains unclear. First, we show that Ntr1΄s G-patch motif (Ntr1GP) can be replaced by the GP motif of Pfa1/Sqs1, a Prp43΄s cofactor in ribosome biogenesis, demonstrating that the specific function of Ntr1GP is to activate Prp43 for spliceosome disassembly and not to guide Prp43 to its binding site in the spliceosome. Furthermore, we show that Ntr1΄s C-terminal domain (CTD) plays a safeguarding role by preventing Prp43 from disrupting wild-type spliceosomes other than the ILS. Ntr1 and Ntr2 can also discriminate between wild-type and defective spliceosomes. In both type of spliceosomes, Ntr1-CTD impedes Prp43-mediated disassembly while the Ntr1GP promotes disassembly. Intriguingly, Ntr2 plays a specific role in defective spliceosomes, likely by stabilizing Ntr1 and allowing Prp43 to enter a productive interaction with the GP motif of Ntr1. Our data indicate that Ntr1 and Ntr2 act as ‘doorkeepers’ and suggest that both cofactors inspect the RNP structure of spliceosomal complexes thereby targeting suboptimal spliceosomes for Prp43-mediated disassembly.

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“…However, Ntr2 is not essential for association of Prp43 with the spliceosome, yet its presence is required to prevent Prp43-mediated disruption of intact spliceosomal intermediates other than the intron-lariat spliceosome. 118 Thus, after accommodation of Prp43, Brr2 might further modulate Prp43 activities based on its intractions with Ntr2. Similar to the structure of a post-step 1 spliceosome lacking Prp16, 103 Brr2 is also flexibly tethered in a post-catalytic intron-lariat spliceosome and Prp43 has not been located in that structure, 81 presently leaving the structural basis for such putative regulatory interactions unresolved.…”

Section: Brr2 Regulation During Splicingmentioning

confidence: 99%

Functions and regulation of the Brr2 RNA helicase during splicing

2016

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Pre-mRNA splicing entails the stepwise assembly of an inactive spliceosome, its catalytic activation, splicing catalysis and spliceosome disassembly. Transitions in this reaction cycle are accompanied by compositional and conformational rearrangements of the underlying RNA-protein interaction networks, which are driven and controlled by 8 conserved superfamily 2 RNA helicases. The Ski2-like helicase, Brr2, provides the key remodeling activity during spliceosome activation and is additionally implicated in the catalytic and disassembly phases of splicing, indicating that Brr2 needs to be tightly regulated during splicing. Recent structural and functional analyses have begun to unravel how Brr2 regulation is established via multiple layers of intra-and inter-molecular mechanisms. Brr2 has an unusual structure, including a long N-terminal region and a catalytically inactive C-terminal helicase cassette, which can auto-inhibit and auto-activate the enzyme, respectively. Both elements are essential, also serve as protein-protein interaction devices and the N-terminal region is required for stable Brr2 association with the tri-snRNP, tri-snRNP stability and retention of U5 and U6 snRNAs during spliceosome activation in vivo. Furthermore, a C-terminal region of the Prp8 protein, comprising consecutive RNase H-like and Jab1/MPN-like domains, can both up-and downregulate Brr2 activity. Biochemical studies revealed an intricate cross-talk among the various cis-and transregulatory mechanisms. Comparison of isolated Brr2 to electron cryo-microscopic structures of yeast and human U4/U6 U5 tri-snRNPs and spliceosomes indicates how some of the regulatory elements exert their functions during splicing. The various modulatory mechanisms acting on Brr2 might be exploited to enhance splicing fidelity and to regulate alternative splicing. KEYWORDSPre-mRNA splicing; regulation of pre-mRNA splicing; remodeling of RNAprotein complexes; RNA helicase structure, function and regulation; spliceosome catalytic activation

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The target of the DEAH-box NTP triphosphatase Prp43 in Saccharomyces cerevisiae spliceosomes is the U2 snRNP-intron interaction

Cited by 51 publications

References 48 publications

Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

Structural insights into the mechanism of the DEAH-box RNA helicase Prp43

Regulation of Prp43-mediated disassembly of spliceosomes by its cofactors Ntr1 and Ntr2

Functions and regulation of the Brr2 RNA helicase during splicing

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