Biology subjects

Igel, H.

Publications and source records attributed to Igel, H..

4 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↗

bioRxiv2021

Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling

Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18S rRNA and 73 sites in 25S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct, undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and acute environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function. HighlightsO_LImethod enabling single-molecule profiling of RNA modifications is developed and reveals heterogeneous classes of modified ribosomes. C_LIO_LIrRNA 2O methylation and pseudouridylation modifications are independent of each other. C_LIO_LIin functional centers of the ribosome are modified in a concerted fashion. C_LIO_LIof function for RNA helicases Dbp3 and Prp43 produce discrete overlapping subpopulations of incompletely modified ribosomes. C_LIO_LImodification profiles are resilient to rapidly changing nutrient conditions and perturbation of translation C_LI

molecular biology↗

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↗

bioRxiv2019

Rapidly evolving protointrons in Saccharomyces genomes revealed by a hungry spliceosome

Introns are a prevalent feature of eukaryotic genomes, yet their origins and contributions to genome function and evolution remain mysterious. In budding yeast, repression of the highly transcribed intron-containing ribosomal protein genes (RPGs) globally increases splicing of non-RPG transcripts through reduced competition for the spliceosome. We show that under these \"hungry spliceosome\" conditions, splicing occurs at more than 150 previously unannotated locations we call protointrons that do not overlap known introns. Protointrons use a less constrained set of splice sites and branchpoints than standard introns, including in one case AT-AC in place of GT-AG. Protointrons are not conserved in all closely related species, suggesting that most are not under selection. Some are found in non-coding RNAs (e. g. CUTs and SUTs), where they may contribute to the creation of new genes. Others are found across boundaries between noncoding and coding sequences, or within coding sequences, where they offer pathways to the creation of new protein variants, or new regulatory controls for existing genes. We define protointrons as (1) nonconserved intron-like sequences that are (2) infrequently spliced, and importantly (3) are not currently understood to contribute to gene expression or regulation in the way that standard introns function. A very few protointrons in S. cerevisiae challenge this classification by their increased splicing frequency and potential function, consistent with the proposed evolutionary process of \"intronization\", whereby new standard introns are created. This snapshot of intron evolution highlights the important role of the spliceosome in the expansion of transcribed genomic sequence space, providing a pathway for the rare events that may lead to the birth of new eukaryotic genes and the refinement of existing gene function.\n\nAuthor SummaryThe protein coding information in eukaryotic genes is broken by intervening sequences called introns that are removed from RNA during transcription by a large protein-RNA complex called the spliceosome. Where introns come from and how the spliceosome contributes to genome evolution are open questions. In this study, we find more than 150 new places in the yeast genome that are recognized by the spliceosome and spliced out as introns. Since they appear to have arisen very recently in evolution by sequence drift and do not appear to contribute to gene expression or its regulation, we call these protointrons. Protointrons are found in both protein-coding and non-coding RNAs and are not efficiently removed by the splicing machinery. Although most protointrons are not conserved, a few are spliced more efficiently, and are located where they might begin to play functional roles in gene expression, as predicted by the proposed process of intronization. The challenge now is to understand how spontaneously appearing splicing events like protointrons might contribute to the creation of new genes, new genetic controls, and new protein isoforms as genomes evolve.

genomics↗