Minor spliceosome inactivation causes microcephaly due to cell cycle defects and death of self-amplifying radial glial cells (original) (raw)
Related papers
2017
Inactivation of the minor spliceosome has been linked to microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). To interrogate how minor intron splicing regulates cortical development, we employed Emx1-Cre to ablate Rnu11, which encodes the minor spliceosome-specific U11 small nuclear RNA (snRNA), in the developing cortex (pallium). Rnu11 cKO mice were born with microcephaly, caused by death of self-amplifying radial glial cells (RGCs). However, both intermediate progenitor cells (IPCs) and neurons were produced in the U11-null pallium. RNAseq of the pallium revealed elevated minor intron retention in the mutant, particularly in genes regulating cell cycle. Moreover, the only downregulated minor intron-containing gene (MIG) was Spc24, which regulates kinetochore assembly. These findings were consistent with the observation of fewer RGCs entering cytokinesis prior to RGC loss, underscoring the requirement of minor splicing for cell cycle progression in RGCs. Overall, we pr...
Trp53 ablation fails to prevent microcephaly in mouse pallium with impaired minor intron splicing
2021
Mutations in minor spliceosome component RNU4ATAC, a small nuclear RNA (snRNA), are linked to primary microcephaly. We have reported that in the conditional knockout (cKO) mice for Rnu11, another minor spliceosome snRNA, minor intron splicing defect in minor intron-containing genes (MIGs) regulating cell cycle resulted in cell cycle defects, with a concomitant increase in γH2aX+ cells and p53-mediated apoptosis. Trp53 ablation in the Rnu11 cKO mice did not prevent microcephaly. However, RNAseq analysis of the double knockout (dKO) pallium reflected transcriptomic shift towards the control from the Rnu11 cKO. We found elevated minor intron retention and alternative splicing across minor introns in the dKO. Disruption of these MIGs resulted in cell cycle defects that were more severe and detected earlier in the dKO, but with delayed detection of γH2aX+ DNA damage. Thus, p53 might also play a role in causing DNA damage in the developing pallium. In all, our findings further refine our ...
PLOS Genetics
Malfunction of pre-mRNA processing factors are linked to several human diseases including cancer and neurodegeneration. Here we report the identification of a de novo heterozygous missense mutation in the SNRPE gene (c.65T>C (p.Phe22Ser)) in a patient with non-syndromal primary (congenital) microcephaly and intellectual disability. SNRPE encodes SmE, a basal component of pre-mRNA processing U snRNPs. We show that the microcephalylinked SmE variant is unable to interact with the SMN complex and as a consequence fails to assemble into U snRNPs. This results in widespread mRNA splicing alterations in fibroblast cells derived from this patient. Similar alterations were observed in HEK293 cells upon SmE depletion that could be rescued by the expression of wild type but not mutant SmE. Importantly, the depletion of SmE in zebrafish causes aberrant mRNA splicing alterations and reduced brain size, reminiscent of the patient microcephaly phenotype. We identify the EMX2 mRNA, which encodes a protein required for proper brain development, as a major mis-spliced down stream target. Together, our study links defects in the SNRPE gene to microcephaly and suggests that alterations of cellular splicing of specific mRNAs such as EMX2 results in the neurological phenotype of the disease.
Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex
Cell, 2016
Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dyn...
The minor and major spliceosome interact to regulate alternative splicing around minor introns
2020
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, whic...
Loss of U11 small nuclear RNA in the developing mouse limb results in micromelia
Development, 2020
Disruption of the minor spliceosome due to mutations in RNU4ATAC is linked to primordial dwarfism in microcephalic osteodysplastic primordial dwarfism type 1, Roifman syndrome, and Lowry-Wood syndrome. Similarly, primordial dwarfism in domesticated animals is linked to positive selection in minor spliceosome components. Despite being vital for limb development and size regulation, its role remains unexplored. Here we disrupt minor spliceosome function in the developing mouse limb by ablating one of its essential components, U11 small nuclear RNA, which resulted in micromelia. Notably, earlier loss of U11 corresponded to increased severity. We find that limb size is reduced due to elevated minor intron retention in minor intron-containing genes that regulate cell cycle. As a result, limb progenitor cells experience delayed prometaphase to metaphase transition and prolonged S-phase. Moreover, we observed death of rapidly dividing, distally located progenitors. Despite cell cycle defec...
Minor spliceosome disruption causes limb growth defects without altering patterning
2020
Disruption of the minor spliceosome causes primordial dwarfism in microcephalic osteodysplastic primordial dwarfism type 1. Similarly, primordial dwarfism in domesticated animals is linked to positive selection in minor spliceosome components. Despite the importance of minor intron splicing in limb size regulation, its role in limb development remains unexplored. Here we show that loss of U11 small nuclear RNA, an essential minor spliceosome component, results in stunted limbs that maintain patterning. Notably, earlier loss of U11 corresponded to increased severity. We find that limb size is reduced due to elevated minor intron retention in minor intron-containing genes that regulate cell cycle. Limb progenitor cells experience delayed prometaphase to metaphase transition and prolonged S-phase, resulting in death of rapidly dividing, distally located progenitors. Consequently, crucial limb patterning genes are upregulated and their expression is maintained spatially to achieve basic...
Neuropathology, 2014
To explore the molecular pathogenesis of amyotrophic lateral sclerosis (ALS), the nuclear function of TAR-DNA binding protein 43 kDa (TDP-43) must be elucidated. TDP-43 is a nuclear protein that colocalizes with Cajal body or Gem in cultured cells. Several recent studies have reported that the decreasing number of Gems accompanied the depletion of the causative genes for ALS, TDP-43 and FUS. Gems play an important role in the pathogenesis of spinal muscular atrophy. Gems are the sites of the maturation of spliceosomes, which are composed of uridylaterich (U) snRNAs (small nuclear RNAs) and protein complex, small nuclear ribonuclearprotein (snRNP). Spliceosomes regulate the splicing of pre-mRNA and are classified into the major or minor classes, according to the consensus sequence of acceptor and donor sites of pre-mRNA splicing. Although the major class of spliceosomes regulates most pre-mRNA splicing, minor spliceosomes also play an important role in regulating the splicing or global speed of pre-mRNA processing. A mouse model of spinal muscular atrophy, in which the number of Gems is decreased, shows fewer subsets U snRNAs. Interestingly, in the central nervous system, U snRNAs belonging to the minor spliceosomes are markedly reduced. In ALS, the U12 snRNA is decreased only in the tissue affected by ALS and not in other tissues. Although the molecular mechanisms underlying the decreased U12 snRNA resulting in cell dysfunction and cell death in motor neuron diseases remain unclear, these findings suggest that the disturbance of nuclear bodies and minor splicing may underlie the common molecular pathogenesis of motor neuron diseases.
Splicing Segregation: The Minor Spliceosome Acts outside the Nucleus and Controls Cell Proliferation
Cell, 2007
The functional relevance and the evolution of two parallel mRNA splicing systems in eukaryotes-a major and minor spliceosome that differ in abundance and splicing rate-are poorly understood. We report here that partially spliced pre-mRNAs containing minor-class introns undergo nuclear export and that minor-class snRNAs are predominantly cytoplasmic in vertebrates. Cytoplasmic interference with the minor spliceosome further indicated its functional segregation from the nucleus. In keeping with this, minor splicing was only weakly affected during mitosis. By selectively interfering with snRNA function in zebrafish development and in mammalian cells, we revealed a conserved role for minor splicing in cell-cycle progression. We argue that the segregation of the splicing systems allows for processing of partially unspliced cytoplasmic transcripts, emerging as a result of different splicing rates. The segregation offers a mechanism accounting for spliceosome evolution in a single lineage and provides a means for nucleus-independent control of gene expression.
Primary microcephaly (MCPH) is characterized by reduced brain size and intellectual disability. The exact pathophysiological mechanism underlying MCPH remains to be elucidated, but dysfunction of neuronal progenitors in the developing neocortex plays a major role. Using homozygosity mapping and whole exome sequencing, we identified a homozygous missense mutation (p.W155C) in Ribosomal RNA Processing 7 Homolog A, RRP7A, which segregated with MCPH in a consanguineous family with 10 affected individuals. RRP7A is expressed in neural stem cells/radial glial cells of the developing human forebrain, and targeted mutation of Rrp7a leads to defects in both neurogenesis and proliferation in a mouse stem cell model. RRP7A localizes to centrosomes, cilia and nucleoli, and patient-derived fibroblasts display defects in processing of ribosomal RNA, resorption of primary cilia and cell cycle progression. Finally, analysis of zebrafish embryos with loss-of-function mutation in rrp7a confirmed that...