Talkish, J. | Search bioRxiv

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↗