Biology subjects

Hunter, O.

Publications and source records attributed to Hunter, O..

3 recordsLinked to original sources

bioRxiv2023

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

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 yeast Saccharomyces cerevisiae by 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. Splicing is inhibited within minutes after addition of Plad-B, and different introns appear inhibited to different extents. Intron-specific inhibition differences are also observed during 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. Introns with variant BP sequences seem particularly sensitive, echoing observations from mammalian cells, where monitoring individual introns is complicated by multi-intron gene architecture and alternative splicing. The compact yeast system may hasten characterization of splicing inhibitors, accelerating improvements in selectivity and therapeutic efficacy.

molecular biology↗

bioRxiv2022

RNA structure landscape of S. cerevisiae introns

Pre-mRNA secondary structures are hypothesized to play widespread roles in regulating RNA processing pathways, but these structures have been difficult to visualize in vivo. Here, we characterize S. cerevisiae pre-mRNA structures through transcriptome-wide dimethyl sulfate (DMS) probing, enriching for low-abundance pre-mRNA through splicing inhibition. We cross-validate structures found from phylogenetic and mutational studies and identify new structures within the majority of probed introns (102 of 161). We find widespread formation of "zipper stems" between the 5 splice site and branch point, "downstream stems" between the branch point and the 3 splice site, and previously uncharacterized long stems that distinguish pre-mRNA from spliced mRNA. Multi-dimensional chemical mapping reveals examples where intron structures can form in vitro without the presence of binding partners, and structure ensemble prediction suggests that such structures appear in introns across the Saccharomyces genus. We develop a high-throughput functional assay to characterize variants of RNA structure (VARS-seq) and we apply the method on 135 sets of stems across 7 introns, identifying structured elements that alter retained intron levels at a distance from canonical splice sites. This transcriptome-wide inference of intron RNA structures suggests new ideas and model systems for understanding how pre-mRNA folding influences gene expression.

biochemistry↗

bioRxiv2019

Cus2 enforces the first ATP-dependent step of splicing by binding to yeast SF3b1 through a UHM-ULM interaction

Stable recognition of the intron branchpoint by the U2 snRNP to form the pre-spliceosome is the first ATP-dependent step of splicing. Genetic and biochemical data from yeast indicate that Cus2 aids U2 snRNA folding into the stem IIa conformation prior to pre-spliceosome formation. Cus2 must then be removed by an ATP-dependent function of Prp5 before assembly can progress. However, the location from which Cus2 is displaced and the nature of its binding to the snRNP are unknown. Here, we show that Cus2 contains a conserved UHM (U2AF homology motif) that binds Hsh155, the yeast homolog of human SF3b1, through a conserved ULM (U2AF ligand motif). Mutations in either motif block binding and allow pre-spliceosome formation without ATP. A 2.0 [A] resolution structure of the Hsh155 ULM in complex with the UHM of Tat-SF1, the human homolog of Cus2, and complementary binding assays show that the interaction is highly similar between yeast and humans. Furthermore, Tat-SF1 enforces ATP-dependence of pre-spliceosome formation in yeast extracts, showing that it can replace Cus2 function. Cus2 is removed before pre-spliceosome formation, and both Cus2 and its Hsh155 ULM binding site are absent from available cryo-EM structure models. However, our data are consistent with the apparent location of the disordered Hsh155 ULM between the U2 stem-loop IIa and the HEAT-repeats of Hsh155 that interact with Prp5. We suggest a model in which Prp5 uses ATP to remove Cus2 from Hsh155 such that extended base pairing between U2 snRNA and the intron branchpoint can occur.

molecular biology↗