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

@article{Agafonov2016ATPSSS,
  title={ATP$\gamma$S stalls splicing after B complex formation but prior to spliceosome activation},
  author={Dmitry E. Agafonov and Maria A van Santen and Berthold Kastner and Prakash Dube and Cindy L Will and Henning Urlaub and Reinhard L{\"u}hrmann},
  journal={RNA},
  year={2016},
  volume={22},
  pages={1329 - 1337}
}
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… 

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References

SHOWING 1-10 OF 51 REFERENCES

Spliceosome activation by PRP2 ATPase prior to the first transesterification reaction of pre-mRNA splicing

  • S. KimR. Lin
  • Biology, Chemistry
    Molecular and cellular biology
  • 1996
It is hypothesized that PRP2 functions as a molecular motor, similar to some DExH ATPases in transcription, in the activation of the precatalytic spliceosome for the transesterification reaction.

An ATP-independent complex commits pre-mRNA to the mammalian spliceosome assembly pathway.

A systematic analysis of the temporal and functional relationships among the H, E, A, and B splicing complexes concluded that E complex commits pre-mRNA to the splicing pathway and that this step is critical in determining the efficiency of mammalian spliceosome assembly.

Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism.

It appears that pre‐mRNA splicing, in common with other biological processes, can be regulated both positively and negatively by reversible protein phosphorylation.

Both phosphorylation and dephosphorylation of ASF/SF2 are required for pre-mRNA splicing in vitro.

Data is presented that suggests for the first time a requirement for SR protein dephosphorylation in pre-messenger RNA splicing in vitro and shows that deph phosphorylation of ASF/SF2 is required for thefirst transesterification reaction once the spliceosome has assembled.

Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing.

It is shown that the first step of splicing requires a novel ATP-independent conformational change and a model in which SF3 prevents premature nucleophilic attack of the chemically reactive hydroxyl of the branchpoint adenosine prior to the first transesterification.

Stalling of spliceosome assembly at distinct stages by small-molecule inhibitors of protein acetylation and deacetylation.

Mass-spectrometric analysis of affinity-purified stalled spliceosomes indicated that the intermediates differ in protein composition both from each other and from previously characterized native A and B splicing complexes, which suggests that the stalled complexes represent hitherto unobserved intermediates of splicingosome assembly.

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

The overall architecture of the human and S. cerevisiae B(act) complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved between lower and higher eukaryotes.

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

It is shown that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity, suggesting hPr p28 has an additional function at this stage of splicing.

Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components

The data suggest that Prp2 facilitates catalytic activation by remodeling the spliceosome, including destabilizing the SF3a and SF3b proteins, likely exposing the branch site before step 1.
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