Biology subjects

Rio, D. C.

Publications and source records attributed to Rio, D. C..

4 recordsLinked to original sources

bioRxiv2023

Splicing accuracy varies across human introns, tissues and age

Alternative splicing impacts most multi-exonic human genes. Inaccuracies during this process may have an important role in ageing and disease. Here, we investigated mis-splicing using RNA-sequencing data from ~14K control samples and 42 human body sites, focusing on split reads partially mapping to known transcripts in annotation. We show that mis-splicing occurs at different rates across introns and tissues and that these splicing inaccuracies are primarily affected by the abundance of core components of the spliceosome assembly and its regulators. Using publicly available data on short-hairpin RNA-knockdowns of numerous spliceosomal components and related regulators, we found support for the importance of RNA-binding proteins in mis-splicing. We also demonstrated that age is positively correlated with mis-splicing, and it affects genes implicated in neurodegenerative diseases. This in-depth characterisation of mis-splicing can have important implications for our understanding of the role of splicing inaccuracies in human disease and the interpretation of long-read RNA-sequencing data.

genetics↗

bioRxiv2022

Highly efficient generation of isogenic pluripotent stem cell models using prime editing

The recent development of prime editing (PE) genome engineering technologies has the potential to significantly simplify the generation of human pluripotent stem cell (hPSC)-based disease models. PE is a multi-component editing system that uses a Cas9-nickase fused to a reverse transcriptase (nCas9-RT) and an extended PE guide RNA (pegRNA). Once reverse transcribed, the pegRNA extension functions as a repair template to introduce precise designer mutations at the target site. Here, we systematically compared the editing efficiencies of PE to conventional gene editing methods in hPSCs. This analysis revealed that PE is overall more efficient and precise than homology-directed repair (HDR) of site-specific nuclease-induced double-strand breaks (DSBs). Specifically, PE is more effective in generating heterozygous editing events to create autosomal dominant disease-associated mutations. By stably integrating the nCas9-RT into hPSCs we achieved editing efficiencies equal to those reported for cancer cells, suggesting that the expression of the PE components, rather than cell-intrinsic features, limit PE in hPSCs. To improve the efficiency of PE in hPSCs, we optimized the delivery modalities for the PE components. Delivery of the nCas9-RT as mRNA combined with synthetically generated chemically-modified pegRNAs and nicking guide RNAs (ngRNAs) improved editing efficiencies up to 13-fold compared to transfecting the prime editing components as plasmids or ribonucleoprotein particles (RNPs). Finally, we demonstrated that this mRNA-based delivery approach can be used repeatedly to yield editing efficiencies exceeding 60% and to correct or introduce familial mutations causing Parkinsons disease in hPSCs.

molecular biology↗

bioRxiv2022

Analysis of altered pre-mRNA splicing patterns caused by a mutation in the RNA binding protein hnRNPA1 linked to amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disease characterized by loss of motor neurons. Human genetic studies have linked mutations in RNA binding proteins as causative for this disease. The hnRNPA1 protein, a known pre-mRNA splicing factor, is mutated in a number of ALS patients. Here, we generate two cell models to investigate how a mutation in the C-terminal low complexity domain of hnRNPA1 affects global pre-mRNA splicing patterns and RNA binding. We show that a single amino acid change in the C-terminal low complexity domain (D262V) leads to changes in splicing of thousands of transcripts whose genes are linked to the DNA damage response, cilia organization and translation. We also show that there are changes in RNA binding of the mutant hnRNPA1 protein to transcripts whose splicing patterns change. Finally, we show that cells expressing the hnRNPA1 D262V mutation exhibit an aggregation phenotype, markedly reduced growth rates and changes in stress granules. This study shows that global changes in pre-mRNA splicing patterns caused by a single mutation in the hnRNPA1 protein lead to phenotypes related to ALS and that specific cellular pathways are affected.

biochemistry↗

bioRxiv2020

Structural evolution of the tissue-specific U2AF2 paralog and alternative splicing factor LS2

The Drosophila melanogaster LS2 protein is a tissue-specific paralog of U2AF2 that mediates testis-specific alternative splicing. In order to understand the structural mechanisms underlying the distinct RNA binding specificity we determined the solution structures of the LS2 RNA recognition motif (RRM) domains and characterized their interaction with cis-regulatory guanosine-rich RNA motifs found in intron regions upstream of alternatively spliced exons. We show that the guanosine-rich RNA adopts a G quadruplex (G4) fold in vitro. The LS2 tandem RRMs adopt canonical RRM folds that are connected by a 38-residue linker that harbors a small helical motif 0. The LS2 RRM2 domain and the 0 helix in the interdomain linker mediate interactions with the G4 RNA. The functional importance of these unique molecular features in LS2 is validated by mutational analysis in vitro and RNA splicing assays in vivo. RNA sequencing data confirm the enrichment of G4-forming LS2 target motifs near LS2-affected exons. Our data indicate a role of G quadruplex structures as cis-regulatory motifs in introns for the regulation of alternative splicing, that engage non-canonical interactions with a tandem RRM protein. These results highlight the intriguing molecular evolution of a tissue-specific splicing factor from its conserved U2AF2 paralog as a result of (retro-) gene duplication in D. melanogaster.

biochemistry↗