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

Boesler, Carsten; Rigo, Norbert; Anokhina, Maria M.; Tauchert, M.J.; Agafonov, Dmitry E.; Kastner, Berthold; Urlaub, Henning; Ficner, Ralf; Will, Cindy L.; Lührmann, Reinhard

doi:10.1038/ncomms11997

Cited by 66 publications

(109 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Like the DEAD-box proteins Sub2 and hPrp5, hPrp28 may not require rapid ATP hydrolysis for its function during splicing. However, in the presence of an ATPase deficient hPrp28 mutant, 37S human pre-B complexes accumulate (Boesler et al 2016). The latter contain the U4/U6.U5 trisnRNP, in addition to U1 and U2, but in contrast to B complexes, the tri-snRNP is not stably associated.…”

Section: Structural Characterization Of the B Atpγs Complex By Emmentioning

confidence: 99%

ATPγS stalls splicing after B complex formation but prior to spliceosome activation

Agafonov

Santen

Kastner

et al. 2016

RNA

Self Cite

View full text Add to dashboard Cite

The ATP analog ATPγS inhibits pre-mRNA splicing in vitro, but there have been conflicting reports as to which step of splicing is inhibited by this small molecule and its inhibitory mechanism remains unclear. Here we have dissected the effect of ATPγS on pre-mRNA splicing in vitro. Addition of ATPγS to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATPγS concentrations ≥0.5 mM, precatalytic B complexes accumulate, demonstrating a block prior to or during the spliceosome activation stage. Affinity purification of the ATPγS-stalled B complexes (B ATPγS ) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that B ATPγS complexes are compositionally more homogeneous than B complexes previously isolated in the presence of ATP. In particular, they contain little or no Prp19/CDC5L complex proteins, indicating that these proteins are recruited after assembly of the precatalytic spliceosome. Under the electron microscope, B ATPγS complexes exhibit a morphology highly similar to B complexes, indicating that the ATPγS-induced block in the transformation of the B to B act complex is not due to a major structural defect. Likely mechanisms whereby ATPγS blocks spliceosome assembly at the activation stage, including inhibition of the RNA helicase Brr2, are discussed. Given their more homogeneous composition, B complexes stalled by ATPγS may prove highly useful for both functional and structural analyses of the precatalytic spliceosome and its conversion into an activated B act spliceosomal complex.

show abstract

Section: Structural Characterization Of the B Atpγs Complex By Emmentioning

confidence: 99%

ATPγS stalls splicing after B complex formation but prior to spliceosome activation

Agafonov

Santen

Kastner

et al. 2016

RNA

Self Cite

View full text Add to dashboard Cite

show abstract

“…Interestingly, the FgSad1 D76N ‐GFP displayed much higher affinity to the U2 snRNP proteins than FgSad1‐GFP in both pull‐down and Y2H experiments, indicating that D76N increased the association of FgSad1 with U2 snRNP. In this study, both FgSad1‐GFP and FgSad1 D76N ‐GFP precipitated the U1 snRNP protein FgPrp39 at similar intensities, indicating both proteins are associated with the pre‐B complex in which the tri‐snRNP is loosely attached (Boesler et al ., ). Sad1 was reported to play a role in tri‐snRNP docking (Makarova et al ., ) which involves U2/U6 helix II formation (Schneider et al ., ,b).…”

Section: Discussionmentioning

confidence: 97%

“…In addition to acting as a clamp between U4/U6 and U5 snRNPs, the hSad1 also interacts with the PWI domain of Brr2 to keep it separated from its substrate U4/U6 di‐snRNP in the human tri‐snRNP (Agafonov et al ., ). hSad1 is abundant in human pre‐B‐complex (Boesler et al ., ) but displaced in B complex, which triggers tri‐snRNP rearrangement that would allow the movement of Brr2 towards its RNA substrate (Bertram et al ., ).…”

Section: Introductionmentioning

confidence: 99%

Spontaneous mutations in FgSAD1 suppress the growth defect of the Fgprp4 mutant by affecting tri‐snRNP stability and its docking in Fusarium graminearum

Fan

Yan

et al. 2019

Environmental Microbiology

View full text Add to dashboard Cite

Summary FgPrp4, the only kinase in the spliceosome, is not essential for viability, but is important for splicing efficiency in Fusarium graminearum. The Fgprp4 deletion mutant had severe growth defects but often produced spontaneous suppressors with faster growth rate. To better understand the suppression mechanism, we identified and characterized spontaneous mutations in the tri‐snRNP‐specific protein, FgSad1, which suppressed the growth defects of Fgprp4. The L512P mutation was verified for its suppressive effects on Fgprp4, suggesting that mutations in FgSad1 may have effects involving FgPrp4 phosphorylation on FgSad1. Phosphoproteomics analysis showed that FgSad1 may not be the direct substrate of FgPrp4 kinase. Furthermore, truncation analysis showed that the N‐terminal, extra RS‐rich region of FgSad1 is critical for its function and is post‐translationally modified. The P258S or S269P mutations in FgSad1 increased its interactions with the U5 protein FgPrp8 and the U4/U6 protein FgPrp31, which may result in tri‐snRNP stabilization. Additionally, the D76N mutation increased the association of FgSad1 with the U2 snRNP. These data indicate that suppressor mutations in FgSad1 increase the stability of the tri‐snRNP and/or the affinity of FgSad1 with U2 snRNP and therefore potentially facilitate the docking of tri‐snRNP into the spliceosome.

show abstract

“…[20][21][22] SF2 helicases can be grouped into 5 families, 3 of which are represented among the spliceosomal remodeling enzymes: 3 DEAD box proteins (Prp5, Sup2/UAP56, Prp28) act during initial spliceosome assembly and activation, a single Ski2-like helicase (Brr2) is involved in spliceosome activation and 4 DEAH/RHA enzymes (Prp2, Prp16, Prp22, Prp43) are required during spliceosome activation, catalysis and disassembly. 20 The most dramatic rearrangements occur during spliceosome activation, where the Prp28 helicase aids in the displacement of U1 snRNA from the 5SS, 8,23,24 followed by Brr2 unwinding the U4 and U6 snRNAs [25][26][27] and leading to displacement of U4 snRNA and U4/U6-bound proteins. 28,29 These processes allow U6 to engage in alternative interactions with the 5SS and U2 snRNA, as well as to form a catalytically important internal stem-loop, thereby building up the spliceosome's active site.…”

Section: Introductionmentioning

confidence: 99%

“…Subsequently, the pre-formed U4/U6 U5 tri-snRNP loosely associates with the spliceosome, giving rise to the catalytically inactive pre-B complex. 8 Within the tri-snRNP, the U4 and U6 snRNAs are extensively base-paired via 2 regions (stems I and II) [9][10][11][12][13][14] and decorated by several proteins. [12][13][14] Association of the U4/U6 di-snRNP with the U5 snRNP is mainly accomplished by protein-protein interactions.…”

Section: Introductionmentioning

confidence: 99%

Functions and regulation of the Brr2 RNA helicase during splicing

2016

View full text Add to dashboard Cite

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

show abstract

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

Cited by 66 publications

References 49 publications

ATPγS stalls splicing after B complex formation but prior to spliceosome activation

ATPγS stalls splicing after B complex formation but prior to spliceosome activation

Spontaneous mutations in FgSAD1 suppress the growth defect of the Fgprp4 mutant by affecting tri‐snRNP stability and its docking in Fusarium graminearum

Functions and regulation of the Brr2 RNA helicase during splicing

Contact Info

Product

Resources

About