Targeted high throughput mutagenesis of the human spliceosome reveals its<i>in vivo</i>operating principles

Beusch, Irene; Rao, Beiduo; Studer, Michael; Luhovska, Tetiana; Šukytė, Viktorija; Lei, Susan; Oses-Prieto, Juan; SeGraves, Em; Burlingame, Alma L.; Jonas, Steven; Madhani, Hiten D.

doi:10.1101/2022.11.13.516350

Cited by 2 publications

(1 citation statement)

References 72 publications

(119 reference statements)

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“…Thus mammalian introns that may be intrinsically more susceptible to disassembly by SUG1P and DHX15 in the absence of inhibitors or mutations could also be more sensitive to inhibitors that disrupt U2-BP helix loading. Relevant to this model is the finding that certain mutations in SUG1P produce a subtle Plad-B resistance in human cells, possibly by delaying discard of a Plad-B inhibited complex to provide time for escape of the block (Beusch et al 2023). Although we currently have only partial understanding of the factors involved in recognition and discard of complexes poised to use an incorrect BP in either system, this class of inhibitors is positioned to take advantage of BP fidelity mechanisms to create the observed intron-specific blocks to splicing.…”

Section: Discussionmentioning

confidence: 99%

Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain

Hunter,

Talkish,

Quick-Cleveland

et al. 2023

Preprint

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Intron branch point (BP) recognition by the U2 snRNP is a critical step of splicing, vulnerable to recurrent cancer mutations and bacterial natural product inhibitors. The BP binds a conserved pocket in the SF3B1 (human) or Hsh155 (yeast) U2 snRNP protein. Amino acids that line this pocket affect binding of splicing inhibitors like Pladienolide-B (Plad-B), such that organisms differ in their sensitivity. To study the mechanism of splicing inhibitor action in a simplified system, we modified the naturally Plad-B resistant yeastSaccharomyces cerevisiaeby changing 14 amino acids in the Hsh155 BP pocket to those from human. This humanized yeast grows normally, and splicing is largely unaffected by the mutation. Minutes after addition of Plad-B splicing is inhibited, however different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed when we measure co-transcriptional splicing in Plad-B using single-molecule intron tracking (SMIT) to minimize gene-specific transcription and decay rates that cloud estimates of inhibition by standard RNA-seq. Comparison of Plad-B intron sensitivities to those of the structurally distinct inhibitor Thailanstatin-A reveals intron-specific differences in sensitivity to different compounds. This work exposes a complex relationship between binding of different members of this class of inhibitors to the spliceosome and intron-specific rates of BP recognition and catalysis. The results echo and extend observations from mammalian cells, where inhibitor studies are complicated by multi-intron gene architecture and alternative splicing. The more compact yeast system may hasten characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.

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Section: Discussionmentioning

confidence: 99%

Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain

Hunter,

Talkish,

Quick-Cleveland

et al. 2023

Preprint

View full text Add to dashboard Cite

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Structural basis of catalytic activation in human splicing

Schmitzová¹,

Cretu

Dienemann³

et al. 2023

Nature

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Pre-mRNA splicing follows a pathway driven by ATP-dependent RNA helicases. A crucial event of the splicing pathway is the catalytic activation, which takes place at the transition between the activated Bact and the branching-competent B* spliceosomes. Catalytic activation occurs through an ATP-dependent remodelling mediated by the helicase PRP2 (also known as DHX16)1–3. However, because PRP2 is observed only at the periphery of spliceosomes3–5, its function has remained elusive. Here we show that catalytic activation occurs in two ATP-dependent stages driven by two helicases: PRP2 and Aquarius. The role of Aquarius in splicing has been enigmatic6,7. Here the inactivation of Aquarius leads to the stalling of a spliceosome intermediate—the BAQR complex—found halfway through the catalytic activation process. The cryogenic electron microscopy structure of BAQR reveals how PRP2 and Aquarius remodel Bact and BAQR, respectively. Notably, PRP2 translocates along the intron while it strips away the RES complex, opens the SF3B1 clamp and unfastens the branch helix. Translocation terminates six nucleotides downstream of the branch site through an assembly of PPIL4, SKIP and the amino-terminal domain of PRP2. Finally, Aquarius enables the dissociation of PRP2, plus the SF3A and SF3B complexes, which promotes the relocation of the branch duplex for catalysis. This work elucidates catalytic activation in human splicing, reveals how a DEAH helicase operates and provides a paradigm for how helicases can coordinate their activities.

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Targeted high throughput mutagenesis of the human spliceosome reveals itsin vivooperating principles

Cited by 2 publications

References 72 publications

Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain

Broad variation in response of individual introns to splicing inhibitors in a humanized yeast strain

Structural basis of catalytic activation in human splicing

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