It is revealed that mutations in U4atac affect the splicing of a large set of transcripts involved in innate immunity, hematopoiesis, and intestinal cell functions, including the Drosophila Janus kinase (JAK) homolog hopscotch (hop).
Abstract
The small nuclear RNA U4atac is a core component of the minor spliceosome. In humans, homozygous or compound heterozygous point mutations in U4atac cause rare developmental disorders, such as Roifman syndrome, characterized by growth restriction, brain anomalies, and immune deficiency. To better define the pathophysiological role of U4atac mutations, we here establish a model of minor spliceosome dysfunction by generating a Drosophila melanogaster CRISPR/Cas9-induced U4atac mutant in the highly conserved stem II region. U4atac homozygous mutants exhibit growth and neurodevelopmental defects, immunodeficiency, and gastrointestinal symptoms. Using bulk RNA-sequencing and functional assays, we reveal that mutations in U4atac affect the splicing of a large set of transcripts involved in innate immunity, hematopoiesis, and intestinal cell functions, including the Drosophila Janus kinase (JAK) homolog hopscotch (hop). Importantly, U4atac deficiency reduces Hop expression and causes Hop-related hematopoietic defects at the embryonic and larval stages. Notably, we also observe reduced expression of Jak1 and attenuated activation of downstream signaling in patients with Roifman syndrome. Thus, our work identifies alterations of Jak signaling as part of the pathogenesis of RNU4atac-opathy.
Myotonic dystrophy type 1 (DM1) is caused by (CUG)n-expanded DMPK transcripts that sequester the splicing factor MBNL1 in the nucleus, resulting in widespread splicing abnormalities. Although significant progress has been made in understanding DM1 pathogenesis, the contribution of DMPK transcript levels to disease severity, and the variability of these levels across cell types, tissues, and patients, remains poorly understood. To investigate this in a quantitative manner, we developed isogenic human immortalized myoblast models with inducible modulation of DMPK RNA levels using CRISPR activation (CRISPRa) and interference (CRISPRi) guided by synthetic sgRNAs. CRISPRa elevated DMPK RNA levels by more than three-fold, intensifying MBNL1-dependent splicing defects. In contrast, CRISPRi reduced DMPK RNA expression by approximately 80%, partially rescuing splicing abnormalities. These changes were validated by visualizing (CUG)n foci using RNA FISH. Lowering DMPK transcript levels increased the availability of free nucleoplasmic MBNL1, whereas upregulation further depleted MBNL1, reinforcing the central role of MBNL1 sequestration in repeat RNA toxicity. Our findings demonstrate that expanded DMPK transcript levels modulate free MBNL1 concentration and alternative splicing in a dose-dependent manner, underscoring the central role of repeat RNA expression in DM1 pathogenesis. These models provide a powerful platform for dissecting variability in DMPK expression and for defining the therapeutic thresholds required for effective DMPK knockdown, thereby offering critical insights for the design and evaluation of DMPK and MBNL1-directed therapeutic strategies.
Lise Ripken, Thomas D. Hoekman, M. Willemse et al.· Human Molecular Genetics· 0 citations
Heterochromatin Protein 1γ, encoded by the Cbx3 gene, is a crucial epigenetic regulator that plays an essential role in mammalian meiotic progression. However, the functional divergence and conservation of this protein in teleosts—organisms possessing duplicated Cbx3 paralogs due to whole-genome duplication—remain to be elucidated. Building on previous research, we focused on cbx3a in Nile tilapia (Oreochromis niloticus), a significant aquaculture species and an excellent model for teleost reproductive studies, emphasizing its role in spermatogenesis. Expression analysis revealed that Cbx3a is localized to primordial germ cells and is sustained in spermatogonia, spermatocytes, and spermatids during spermatogenesis. CRISPR/Cas9-mediated knockout of cbx3a demonstrated that Cbx3a deficiency induces germ cell apoptosis, meiotic arrest, and sperm defects, including shortened tails and impaired motility, resulting in profound defects in sperm quantity and quality, strongly implying compromised male fertility. Transcriptomic analysis further identified dysregulated molecular pathways, including cytokine signaling and neuroactive ligand–receptor interactions. This provides novel mechanistic insights into HP1γ-mediated epigenetic regulation of meiosis. Notably, cbx3a mutants exhibited phenotypic bifurcation: a subset showed meiotic defects accompanied by sporadic germ cell apoptosis, whereas others underwent full meiotic arrest with pervasive germ cell apoptosis in adult gonads. Collectively, these findings clarify the essential and conserved role of Cbx3a/HP1γ in Nile tilapia spermatogenesis, thereby advancing the field of vertebrate reproductive epigenetics and providing a valuable theoretical basis for potential applications in reproductive management, such as improving sperm quality.
It is concluded that PC2C331S may perturb protein stability and/or polycystin complex formation prior to ciliary/EV trafficking and a C. elegans pipeline for mechanistic classification of conserved ADPKD-associated missense variants is established.
Juan Wang, Carlos Nava Cruz, Jonathon D. Walsh et al.· Genetics· 0 citations
ELAV/Hu RNA-binding proteins (RBPs) are key regulators of neuronal alternative splicing and polyadenylation programs across animals. How ELAV/Hu RBPs achieve gene-specific regulation by recognizing spaced U-rich motifs through multimerization, remains uncertain. We determined X-ray crystal structures of ELAV RNA recognition motif 3 (RRM3) to reveal that multimerization is mediated by two evolutionarily conserved interfaces in non-RNA-binding parts of the RRM to form a tetramer and RNA binding is not required for multimerization. Mutational probing of these two interfaces in Drosophila photoreceptor neurons shows that both interfaces contribute to ELAV function in development. Notably, multimerization defective Drosophila elav mutants are embryonic lethal. Genomic profiling demonstrates that multimerization is required to direct neuronal alternative splicing and polyadenylation programs of some, but not all ELAV target genes. Our study provides a structural basis for a mechanistic understanding how ELAV/Hu proteins can extract gene-specific regulation from a landscape of redundant sequence motifs.
Thomas C. Dix, David W. J. Mcquarrie, Ulrike Bräuer et al.· Genes & Development· 0 citations