Biology subjects

Beusch, I.

Publications and source records attributed to Beusch, I..

4 recordsLinked to original sources

bioRxiv2022

Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles

The spliceosome is a staggeringly complex machine comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and interrogated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A-complex but also in components that act as late as the second chemical step after SF3b is dissociated. Viable resistance substitutions map not only to the pladienolide B binding site but also to the G-patch (ATPase activator) domain of SUGP1, which lacks orthologs in yeast. We used these mutants and biochemical approaches to identify the spliceosomal disassemblase DHX15/hPrp43 as the ATPase ligand for SUGP1. These and other data support a model in which SUGP1 promotes splicing fidelity by triggering early spliceosome disassembly in response to kinetic blocks. Our approach provides a template for the analysis of essential cellular machines in humans.

molecular biology↗

bioRxiv2022

The structure of yeast Npl3 bound to RNA reveals a cooperative sequence-specific recognition and an RNA chaperone role in splicing

The conserved SR-like protein Npl3 promotes splicing of diverse pre-mRNAs. However, the RNA sequence(s) recognized by the RNA Recognition Motifs (RRM1 & RRM2) of Npl3 during the splicing reaction remain elusive. Here, we developed a split-iCRAC approach in yeast to uncover the consensus sequence bound to each RRM. High-resolution NMR structures show that RRM2 recognizes a 5-GNGG-3 motif leading to an unusual mille-feuille topology. These structures also reveal how RRM1 preferentially interacts with a CC-dinucleotide upstream of this motif, and how the inter-RRM linker and the region C-terminal to RRM2 contributes to cooperative RNA-binding. Structure-guided functional studies show that Npl3 genetically interacts with U2 snRNP specific factors and we provide evidence that Npl3 melts U2 snRNA stem-loop I, a prerequisite for U2/U6 duplex formation within the catalytic center of the Bact spliceosomal complex. Thus, our findings provide insights into an unanticipated RNA chaperoning role for Npl3 during spliceosome active site formation.

biophysics↗

bioRxiv2022

Sensitive detection and structural characterisation of UV-induced cross-links in protein-RNA complexes using CLIR-MS

Protein-RNA interactions underpin many critical biological processes, demanding the development of technologies to precisely characterize their nature and functions. Many such technologies depend upon cross-linking under mild irradiation conditions to stabilize contacts between amino acids and nucleobases; for example, the cross-linking of stable isotope labelled RNA coupled to mass spectrometry (CLIR-MS) method. A deeper understanding of the CLIR-MS workflow is required to maximize its impact for structural biology, particularly addressing the low abundance of cross-linking products and the information content of spatial/geometric restraints reflected by a cross-link. Here, we present a vastly improved CLIR-MS pipeline that features enhanced sample preparation, data acquisition and interpretation. These advances significantly increase the number of detected cross-link products per sample. We demonstrate that the procedure is robust against variation of key experimental parameters, including irradiation energy and temperature. Using this improved protocol on four protein-RNA complexes representing canonical and non-canonical RNA-binding domains, we propose for the first time the distances encoded by protein-RNA cross-links, enabling their use as structural restraints. We also compared the cross-linking of canonical RNA with 4-thiouracil-labeled counterparts, showing slight, but noticeable differences. The improved understanding of protein-RNA cross-links refines the structural interpretation of complexes and facilitates the adoption of the method in integrative/hybrid structural biology.

biochemistry↗

bioRxiv2021

Coupling of spliceosome complexity to intron diversity

We determined that over 40 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution of S. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeast Cryptococcus neoformans. We found they govern splicing efficiency of introns with divergent spacing between intron elements. Importantly, most of these factors also suppress usage of weak nearby cryptic/alternative splice sites. Among these, orthologs of GPATCH1 and the helicase DHX35 display correlated functional signatures and copurify with each other as well as components of catalytically active spliceosomes, identifying a conserved G-patch/helicase pair that promotes splicing fidelity. We propose that a significant fraction of spliceosomal proteins in humans and most eukaryotes are involved in limiting splicing errors, potentially through kinetic proofreading mechanisms, thereby enabling greater intron diversity.

molecular biology↗