Biology subjects

Carrocci, T. J.

Publications and source records attributed to Carrocci, T. J..

3 recordsLinked to original sources

bioRxiv2025

A Yeast-Based High-Throughput Screening Platform for the Discovery of Novel pre-mRNA Splicing Modulators

Pre-mRNA splicing is a crucial process in eukaryotic gene expression, and splicing dysregulation has been linked to various diseases. However, very few small molecules have been discovered that can modulate spliced mRNA formation or inhibit the splicing machinery itself. This study presents a novel high-throughput screening (HTS) platform for identifying compounds that modulate splicing. Our platform comprises a two-tiered screening approach: a primary screen measuring growth inhibition in sensitized in S. cerevisiae (yeast) strains and a secondary screen that relies on production of a fluorescent protein as a readout for splicing inhibition. Using this approach, we identified 4 small molecules that cause accumulation of unspliced pre-mRNA in vivo in yeast. In addition, cancer cells expressing a myelodysplastic syndrome-associated splicing factor mutation (SRSF2P95H) are more sensitive to one of these compounds than those expressing the wild-type version of the protein. Transcriptome analyses showed that this compound causes widespread changes in gene expression in sensitive SRSF2P95H-expressing cells. Our results demonstrate the utility of using a yeast-based HTS to identify compounds capable of changing pre-mRNA splicing outcomes.

biochemistry↗

bioRxiv2025

CAKUT variants in PRPF8, DYRK2, and CEP78: implications for splicing and ciliogenesis

IntroductionCongenital anomalies of the kidney and urinary tract (CAKUT) are the leading cause of chronic kidney disease in children and young adults. Although over 50 monogenic causes have been identified, many remain unresolved. PRPF8 is a core spliceosome component, essential for pre-mRNA splicing, and further localizes to the distal mother centriole to promote ciliogenesis. MethodsWe performed trio exome sequencing in 208 CAKUT families and identified strong variants in PRPF8 and the EDD-DYRK2-DDB1VprBP complex. Functional validation included splicing assays in yeast (Saccharomyces cerevisiae), Sonic hedgehog (Shh) signaling in RPE-1 cells, co-immunoprecipitation for protein complex assembly, and in situ hybridization in mouse embryos. Protein interactions were modeled using AlphaFold. ResultsWe identified heterozygous de novo or inherited variants in PRPF8, DYRK2, DDB1, EDD and CEP78. Yeast assays revealed that while most PRPF8 variants preserved growth and splicing at consensus splice sites, the de novo PRPF8R1681W variant impaired splicing of non-consensus splice sites and was inviable at elevated temperature. CAKUT variants failed to rescue prp28-1 and U4-cs1 alleles but showed variant-specific synthetic interactions with brr2-1, including weak suppression or synthetic sickness at elevated temperatures. Shh signaling was reduced in [~]50% of PRPF8 variants expressed in RPE-1 cells. CEP78 truncating variants abrogated binding to CEP350 and VPRBP. Two DYRK2 variants disrupted EDD-DYRK2-DDB1VprBP complex formation without affecting kinase activity. In situ hybridization revealed strong Prpf8 expression in the developing collecting duct and urothelium. ConclusionVariants in PRPF8 and components of the EDD-DYRK2-DDB1VprBP complex may contribute to CAKUT through impaired pre-mRNA splicing and defective ciliogenesis. These findings uncover an entirely new functional network of candidate genes for CAKUT and ciliopathies, significantly broadening our understanding of disease mechanisms and offering novel entry points for mechanistic studies. Translational StatementOur study identifies a previously unrecognized molecular network involving PRPF8 and the EDD-DYRK2-DDB1VprBP complex, revealing a novel pathogenic mechanism in CAKUT. These results introduce a new class of candidate genes and pathways essential for kidney development. As the genetic etiology of CAKUT remains unknown in most patients, our findings underscore the need for targeted genetic testing and functional studies to enhance diagnosis, advance mechanistic insight, and enable more personalized clinical management.

genetics↗

bioRxiv2024

Functional Analysis of the Zinc Finger Modules of the S. cerevisiae Splicing Factor Luc7

Identification of splice sites is a critical step in pre-mRNA splicing since definition of the exon/intron boundaries controls what nucleotides are incorporated into mature mRNAs. The intron boundary with the upstream exon is initially identified through interactions with the U1 snRNP. This involves both base pairing between the U1 snRNA and the pre-mRNA as well as snRNP proteins interacting with the 5 splice site/snRNA duplex. In yeast, this duplex is buttressed by two conserved protein factors, Yhc1 and Luc7. Luc7 has three human paralogs (LUC7L, LUC7L2, and LUC7L3) which play roles in alternative splicing. What domains of these paralogs promote splicing at particular sites is not yet clear. Here, we humanized the zinc finger domains of the yeast Luc7 protein in order to understand their roles in splice site selection using reporter assays, transcriptome analysis, and genetic interactions. While we were unable to determine a function for the first zinc finger domain, humanization of the second zinc finger domain to mirror that found in LUC7L or LUC7L2 resulted in altered usage of nonconsensus 5 splice sites. In contrast, the corresponding zinc finger domain of LUC7L3 could not support yeast viability. Further, humanization of Luc7 can suppress mutation of the ATPase Prp28, which is involved in U1 release and exchange for U6 at the 5 splice site. Our work reveals a role for the second zinc finger of Luc7 in splice site selection and suggests that different zinc finger domains may have different ATPase requirements for release by Prp28.

biochemistry↗