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P53 ablation fails to completely rescue microcephaly caused by impaired minor intron splicing

ABSTRACT: Mutations in the minor spliceosome components such as RNU4atac, a small nuclear RNA (snRNA), are linked to primary microcephaly. We have reported that in the conditional knockout (cKO) mice for Rnu11, a minor spliceosome snRNA, minor intron splicing defect in minor intron-containing genes (MIGs) regulating cell cycle resulted in cell cycle defect with a concomitant increase in yH2aX+ cells and P53-mediated apoptosis. Trp53 ablation in the Rnu11 cKO mice did not prevent microcephaly. Although RNAseq analysis of the double knockout (dKO) pallium reflected transcriptomic shift towards the control from the cKO. We found elevated minor intron retention and alternative splicing across minor introns in the dKO. Disruption of these minor intron-containing genes (MIGs) resulted in cell cycle defect that was detected earlier and was more severe in the dKO, but with delayed detection of yH2aX+ DNA damage. Thus, P53 might also play a role in causing DNA damage in the developing pallium. In all, our findings further refine our understanding of the role of the minor spliceosome in cortical development and identify MIGs underpinning microcephaly in minor spliceosome-related disease.

ORGANISM(S): Mus musculus

PROVIDER: GSE168366 | GEO | 2021/09/30

REPOSITORIES: GEO

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Minor spliceosome inactivation in the developing mouse cortex causes self-amplifying radial glial cell death and microcephaly.

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Minor intron splicing efficiency increases with the development of lethal prostate cancer

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2023-06-08 | PXD026949 | Pride

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Project description:Mutations in minor spliceosome components are linked to diseases such as Roifman syndrome, Lowry-Wood syndrome, and early-onset cerebellar ataxia (EOCA). Here we report that besides increased minor intron retention, Roifman syndrome and EOCA can also be characterized by elevated alternative splicing (AS) around minor introns. Consistent with the idea that the assembly/activity of the minor spliceosome informs AS in minor intron-containing genes (MIGs), inhibition of all minor spliceosome snRNAs led to upregulated AS. Notably, alternatively spliced MIG isoforms were bound to polysomes in the U11-null dorsal telencephalon, which suggested that aberrant MIG protein expression could contribute to disease pathogenesis. In agreement, expression of an aberrant isoform of the MIG Dctn3 by in utero electroporation, affected radial glial cell divisions. Finally, we show that AS around minor introns is executed by the major spliceosome and is regulated by U11-59K of the minor spliceosome, which forms exon-bridging interactions with proteins of the major spliceosome. Overall, we extend the exon-definition model to MIGs and postulate that disruptions of exon-bridging interactions might contribute to disease severity and pathogenesis.

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CRISPR-Cas9 knockdown of RBM48 in K562 cells

Project description:U12-type or minor (U12) introns are spliced by a distinct minor spliceosome and are found in the vast majority of multicellular eukaryotes, including plants and animals. Although U12 introns constitute less than 0.5% of all introns in many species, minor intron containing genes (MIGs) are important for organismal growth, development and pathology. We recently reported that maize RNA Binding Motif Protein 48 (RBM48) is required for U12-type intron splicing. Maize rbm48 mutants have genome-wide defects of U12 intron splicing, leading to abnormal endosperm cell differentiation and proliferation. To investigate whether RBM48 mediated U12 splicing is conserved between maize and humans, we generated a CRISPR/Cas9-mediated RBM48 functional knockout of the human RBM48 ortholog (RBM48 FunKO) in human K-562 cells.

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A splicing-dependent transcriptional checkpoint associated with pre-spliceosome formation

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2020-11-26 | GSE162136 | GEO

Minor intron retention drives clonal hematopoietic disorders and diverse cancer predisposition

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2021-02-09 | GSE149455 | GEO

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Project description:We identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of the U11/U12-65K protein, a known unique component of the minor spliceosome that functions during the early steps of minor intron recognition as a component of the U11/U12 di-snRNP. We show that both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with the U11/U12-65K protein, which uses the N-terminal region to interact with U11 snRNP during the intron recognition step. Finally, we show that while RBM41 knockout cells are viable, they show alterations in the splicing of U12-type introns, particularly differential U12-type 3' splice site usage. Together, our results highlight the role 3’-terminal stem-loop of U12 snRNA as a dynamic binding platform for the paralogous U11/U12-65K and RBM41 proteins, which function at distinct stages of minor spliceosome assembly/disassembly cycle.

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