Biology subjects

Fica, S. M.

Publications and source records attributed to Fica, S. M..

7 recordsLinked to original sources

bioRxiv2026

Somatic DDX41 mutations confer neomorphic splicing activity in the pathogenesis of myelodysplastic neoplasms

Germline mutations in the RNA helicase DDX41 are the most common genetic predisposition to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), representing 5-7% of AML. Over 70% of patients acquire a somatic mutation at specific hotspots (R525H/G530D) in the helicase domain of DDX41 in trans, which is linked to disease progression. DDX41 has diverse cellular functions, including RNA splicing, ribosome biogenesis, R-loop resolution and inflammation. However, the mechanisms by which somatic DDX41 mutations drive MDS/AML pathogenesis are yet to be determined. Here, we systematically evaluated the function of pathogenic, missense germline and somatic DDX41 mutations. We report that DDX41 somatic mutations are gain of function alleles and fail to rescue AML cell growth, distinct from germline DDX41 mutations. DDX41 somatic mutations R525H and G530D drive widespread splicing changes in isogenic AML cell lines, consistent in genetically engineered murine bone marrow HSPCs and CD34+ cells from DDX41-mutant MDS/AML patients. Mechanistically, DDX41-R525H exhibits increased RNA binding affinity compared to wildtype, with aberrantly spliced RNA targets overlapping with enriched binding. Strikingly, aberrant splicing of SEPTIN7 is enriched exclusively in CD34+ HSPCs of patients harboring both germline and somatic DDX41 mutations but absent in samples with germline mutation alone or lacking DDX41 mutations altogether. SEPTIN7 mis-splicing introduces a premature stop codon resulting in its downregulation at the protein level. SEPTIN7 is a known regulator of cytokinesis, HSC polarity and repopulation capacity. Our results identify a novel mis-spliced candidate, SEPTIN7, whose deregulation implicates a mechanism by which the DDX41 somatic mutations promote dysplastic hematopoiesis.

cancer biology↗

bioRxiv2026

Structural basis for alternative 3' splice site selection in the human spliceosome active center

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/741260v1_ufig1.gif" ALT="Figure 1"> View larger version (97K): org.highwire.dtl.DTLVardef@279388org.highwire.dtl.DTLVardef@46d636org.highwire.dtl.DTLVardef@997672org.highwire.dtl.DTLVardef@12f40ee_HPS_FORMAT_FIGEXP M_FIG C_FIG Key findingsO_LICryo-EM reveals SDE2 as a novel factor stabilizing the spliceosome active center C_LIO_LISDE2 {Delta}N structures visualize a stalled C* state with impaired docking-factor engagement. C_LIO_LISDE2, Prp18, and FAM32A read a cis-code to stabilize weaker proximal 3'-ss. C_LIO_LISDE2 {Delta}N destabilizes 3'-ss docking and rescues BRCA1 and CFTR mis-splicing in vivo. C_LI Accurate alternative splicing requires discrimination between adjacent 3' splice sites (3'-ss) during catalysis and is disrupted by pathogenic AG-gain mutations that create competing 3'-ss. Here, we present cryo-EM structures of human spliceosomes assembled on native-sequence pre-mRNAs, revealing how the catalytic core controls alternative 3'-ss selection. SDE2 is a previously unrecognized active-center component that promotes a docking-competent spliceosome conformation. Machine learning, in vivo transcriptomics, and in vitro biochemistry show how SDE2 cooperates with FAM32A and Prp18 to act as readers of a cis-regulatory code that governs 3'-ss selection during catalysis. These factors promote weaker, proximal site use by counteracting an intrinsic distal bias generated by active-site interactions with the distal-site -4 nucleotide. Structural or genetic perturbation of these exon-ligation factors destabilizes proximal 3'-ss docking and restores canonical splicing in disease-relevant CFTR and BRCA1 AG-gain alleles. Our work establishes the spliceosome active center as a tunable regulatory hub for alternative splicing.

molecular biology↗

bioRxiv2023

The evolution of splice site sequence preference is linked to the U6 snRNA m6A methyltransferase METTL16

Eukaryotic genes are interrupted by introns that are removed from transcribed RNAs by splicing. The extent of alternative splicing is the best genomic predictor of developmental complexity, yet it is unclear what mediates change in patterns of splicing complexity between species. Here we show that variation in 5 splice site sequence preferences correlate with the presence of the U6 snRNA methyltransferase METTL16 and the splicing factor SNRNP27K. We used inter-species association mapping with Saccharomycotina species to correlate splicing phenotypes with the presence or absence of splicing factors. The greatest variation in 5 splice site sequence occurred at the +4 position and involved a preference switch between adenosine and uridine. Loss of METTL16 and SNRNP27K orthologs, or a single SNRNP27K methionine residue, was associated with a preference for +4U. These findings are consistent with splicing analyses of mutants defective in either METTL16 or SNRNP27K orthologs and models derived from spliceosome structures, demonstrating that inter-species association mapping is a powerful orthogonal approach to molecular studies. We conclude that variation in concerted processes of 5 splice site selection by U6 snRNA is crucial to evolutionary change in splicing complexity.

bioinformatics↗

bioRxiv2022

m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5' splice site

Alternative splicing of messenger RNAs is associated with the evolution of developmentally complex eukaryotes. Splicing is mediated by the spliceosome, and docking of the pre-mRNA 5 splice site into the spliceosome active site depends upon pairing with the conserved ACAGA sequence of U6 snRNA. In some species, including humans, the central adenosine of the ACAGA box is modified by N6 methylation, but the role of this m6A modification is poorly understood. Here we show that m6A modified U6 snRNA determines the accuracy and efficiency of splicing. We reveal that the conserved methyltransferase, FIO1, is required for Arabidopsis U6 snRNA m6A modification. Arabidopsis fio1 mutants show disrupted patterns of splicing that can be explained by the sequence composition of 5 splice sites and cooperative roles for U5 and U6 snRNA in splice site selection. U6 snRNA m6A influences 3 splice site usage and reinforces splicing fidelity at elevated temperature. We generalise these findings to reveal two major classes of 5 splice site in diverse eukaryotes, which display anti-correlated interaction potential with U5 snRNA loop 1 and the U6 snRNA ACAGA box. We conclude that U6 snRNA m6A modification contributes to the selection of degenerate 5 splice sites crucial to alternative splicing.

molecular biology↗

bioRxiv2020

Structural basis for conformational equilibrium of the catalytic spliceosome

The catalytic spliceosome exists in equilibrium between the branching (B*/ C) and exon ligation (C*/ P) conformations. Here we present the electron cryo-microscopy reconstruction of the Saccharomyces cerevisiae C complex at 2.8 [A] resolution and identify a novel C-complex intermediate (Ci) that elucidates the molecular basis for this equilibrium. In the Ci conformation, the exon-ligation factors Prp18 and Slu7 are already bound before ATP hydrolysis by Prp16, which destabilises the branching conformation. Biochemical assays suggest these pre-bound factors prime C complex for conversion to C* by Prp16. A complete model of the Prp19-complex (NTC) shows how the NTC pre-recruits the branching factors Yju2 and Isy1 before branching. Prp16 remodels Yju2 binding after branching, allowing Yju2 to remain associated with the C* and P spliceosomes and promote exon ligation. Our results explain how Prp16 action modulates dynamic binding of step-specific factors to alternatively stabilise the C or C* conformation and establish equilibrium of the catalytic spliceosome.

biophysics↗

bioRxiv2020

psiCLIP reveals dynamic RNA binding by DEAH-box helicases before and after exon ligation

Eight RNA helicases remodel the spliceosome to effect pre-mRNA splicing but their mechanism of action remains poorly understood. We have developed "purified spliceosome iCLIP" (psiCLIP) to define helicase-RNA contacts in specific spliceosomal states. psiCLIP reveals previously unappreciated dynamics of spliceosomal helicases. The binding profile of the helicase Prp16 is influenced by the distance between the branch-point and 3 splice site, while Prp22 binds diffusely on the intron before exon ligation but switches to more narrow binding downstream of the exon junction after exon ligation. Notably, depletion of the exon-ligation factor Prp18 destabilizes Prp22 binding to the pre-mRNA, demonstrating that psiCLIP can be used to study the relationships between helicases and auxiliary splicing factors. Thus, psiCLIP is sensitive to spliceosome dynamics and complements the insights from structural and imaging studies by providing crucial positional information on helicase-RNA contacts during spliceosomal remodeling.

molecular biology↗

bioRxiv2017

Post-catalytic spliceosome structure reveals mechanism of 3'-splice site selection

Introns are removed from eukaryotic mRNA precursors by the spliceosome in two transesterification reactions - branching and exon ligation. Following branching, the 5'-exon remains paired to U5 snRNA loop 1, but the mechanism of 3'-splice site recognition during exon ligation has remained unclear. Here we present the 3.7[A] cryo-EM structure of the yeast P complex spliceosome immediately after exon ligation. The 3'-splice site AG dinucleotide is recognised through non-Watson-Crick pairing with the 5'-splice site and the branch point adenosine. A conserved loop of Prp18 together with the -finger and the RNaseH domain of Prp8 clamp the docked 3'-splice site and 3'-exon. The step 2 factors Prp18 and Slu7 and the C-terminal domain of Yju2 stabilise a conformation competent for 3'-splice site docking and exon ligation. The structure accounts for the strict conservation of the GU and AG dinucleotides of the introns and provides insight into the catalytic mechanism of exon ligation.

molecular biology↗