Mutation in minor spliceosome components is linked to the developmental disorder microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). Here, we inactivated the minor spliceosome in the developing mouse cortex (pallium) by ablating , which encodes the crucial minor spliceosome small nuclear RNA (snRNA) U11. conditional knockout mice were born with microcephaly, which was caused by the death of self-amplifying radial glial cells (RGCs), while intermediate progenitor cells and neurons were produced. RNA sequencing suggested that this cell death was mediated by upregulation of p53 (Trp53 - Mouse Genome Informatics) and DNA damage, which were both observed specifically in U11-null RGCs. Moreover, U11 loss caused elevated minor intron retention in genes regulating the cell cycle, which was consistent with fewer RGCs in S-phase and cytokinesis, alongside prolonged metaphase in RGCs. In all, we found that self-amplifying RGCs are the cell type most sensitive to loss of minor splicing. Together, these findings provide a potential explanation of how disruption of minor splicing might cause microcephaly in MOPD1.
In the mammalian genome, each histone family contains multiple replication-dependent paralogs, which are found in clusters where their transcription is thought to be coupled to the cell cycle. Here, we wanted to interrogate the transcriptional regulation of these paralogs during retinal development and aging. We employed deep sequencing, quantitative PCR, in situ hybridization (ISH), and microarray analysis, which revealed that replication-dependent histone genes were not only transcribed in progenitor cells but also in differentiating neurons. Specifically, by ISH analysis we found that different histone genes were actively transcribed in a subset of neurons between postnatal day 7 and 14. Interestingly, within a histone family, not all paralogs were transcribed at the same level during retinal development. For example, expression of Hist1h1b was higher embryonically, while that of Hist1h1c was higher postnatally. Finally, expression of replication-dependent histone genes was also observed in the aging retina. Moreover, transcription of replication-dependent histones was independent of rapamycin-mediated mTOR pathway inactivation. Overall, our data suggest the existence of variant nucleosomes produced by the differential expression of the replication-dependent histone genes across retinal development. Also, the expression of a subset of replication-dependent histone isotypes in senescent neurons warrants re-examining these genes as "replication-dependent." Thus, our findings underscore the importance of understanding the transcriptional regulation of replication-dependent histone genes in the maintenance and functioning of neurons.
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 owing 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 defects and cell death, the spatial expression of key limb patterning genes was maintained. Overall, we show that the minor spliceosome is required for limb development via size control potentially shared in disease and domestication.
In eukaryotes, gene expression requires splicing, which starts with the identification of exon-intron boundaries by the small, nuclear RNA (snRNAs) of the spliceosome, aided by associated proteins. In the mammalian genome, <1% of introns lack canonical exon-intron boundary sequences and cannot be spliced by the canonical splicing machinery. These introns are spliced by the minor spliceosome, consisting of unique snRNAs (U11, U12, U4atac, and U6atac). The importance of the minor spliceosome is underscored by the disease microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1), which is caused by mutation in U4atac. Thus, it is important to understand the expression and function of the minor spliceosome and its targets in mammalian development, for which we used the mouse as our model. Here, we report enrichment of the minor snRNAs in the developing head/central nervous system (CNS) between E9.5 and E12.5, along with enrichment of these snRNAs in differentiating retinal neurons. Moreover, dynamic expression kinetics of minor intron-containing genes (MIGs) was observed across retinal development. DAVID analysis of MIGs that were cotranscriptionally upregulated embryonically revealed enrichment for RNA metabolism and cell cycle regulation. In contrast, MIGs that were cotranscriptionally upregulated postnatally revealed enrichment for protein localization/transport, vesicle-mediated transport, and calcium transport. Finally, we used U12 morpholino to inactivate the minor spliceosome in the postnatal retina, which resulted in apoptosis of differentiating retinal neurons. Taken together, our data suggest that the minor spliceosome may have distinct functions in embryonic versus postnatal development. Importantly, we show that the minor spliceosome is crucial for the survival of terminally differentiating retinal neurons.
Our study showed that CitK is not required for a subset of RPCs before E14, but is necessary for RPC survival post E14. This in turn results in normal early embryonic neurogenesis, but severely compromised later embryonic and postnatal neurogenesis.
BackgroundThe retina as a model system with extensive information on genes involved in development/maintenance is of great value for investigations employing deep sequencing to capture transcriptome change over time. This in turn could enable us to find patterns in gene expression across time to reveal transition in biological processes.MethodsWe developed a bioinformatics pipeline to categorize genes based on their differential expression and their alternative splicing status across time by binning genes based on their transcriptional kinetics. Genes within same bins were then leveraged to query gene annotation databases to discover molecular programs employed by the developing retina.ResultsUsing our pipeline on RNA-Seq data obtained from fractionated (nucleus/cytoplasm) developing retina at embryonic day (E) 16 and postnatal day (P) 0, we captured high-resolution as in the difference between the cytoplasm and the nucleus at the same developmental time. We found de novo transcription of genes whose transcripts were exclusively found in the nuclear transcriptome at P0. Further analysis showed that these genes enriched for functions that are known to be executed during postnatal development, thus showing that the P0 nuclear transcriptome is temporally ahead of that of its cytoplasm. We extended our strategy to perform temporal analysis comparing P0 data to either P21-Nrl-wildtype (WT) or P21-Nrl-knockout (KO) retinae, which predicted that the KO retina would have compromised vasculature. Indeed, histological manifestation of vasodilation has been reported at a later time point (P60).ConclusionsThus, our approach was predictive of a phenotype before it presented histologically. Our strategy can be extended to investigating the development and/or disease progression of other tissue types.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2822-z) contains supplementary material, which is available to authorized users.
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 patterning. Overall, these findings reveal a potential mechanism shared in disease and domestication.The minor spliceosome, which consists of five small nuclear RNAs (snRNAs) U11, U12, U4atac, U5, and U6atac and associated proteins, splices <0.5% of introns, termed minor introns, found in <2% of genes, termed minor intron-containing genes (MIGs) 1-3 . In metazoans, the minor spliceosome, MIGs, and the position of minor introns within MIGs are all highly conserved 4-6 . This conservation could be explained by the enrichment of MIGs in essential functions required for cell survival 7 , which is consistent with embryonic lethality observed in multiple animal models with constitutive loss of minor spliceosome function [8][9][10] . Consequently, it is unsurprising that human diseases with minor spliceosome loss-of-function mutations have not been discovered. However, mutations that result in partial loss of minor spliceosome function have been reported [11][12][13][14][15][16] .For example, microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1), Roifman syndrome (RS), and Lowry-Wood syndrome (LWS) are linked to partial inhibition of the minor spliceosome 11,12,14,16 . Here, individuals harbor disparate mutations in RNU4ATAC, which encodes the U4atac snRNA, and display symptoms including microcephaly, micrognathia, vertebral deficits, and primordial dwarfism 16,17 . The severity of primordial dwarfism observed in MOPD1, RS, and LWS is observed along a gradation, with MOPD1 being the most severe, RS moderate, and LWS mild 16,17 . Nonetheless, in all cases, the basic patterning of the limb skeletal elements, including the presence of a stylopod (humerus; femur), zeugopod (radius/ulna; tibia/fibula), and autopod (hand; foot), is maintained 16,17 .Since unique mutations in RNU4ATAC have been linked to MOPD1, RS, and LWS, the gradation of primordial dwarfism might be due to differential inhibition of the minor spliceosome 16,17 . This suggests that the activity of the minor spliceosome might serve as a rheostat for tissue size cont...
Processing of mRNAs including, alternative splicing (AS), mRNA transport and translation regulation are crucial to eukaryotic gene expression. For example, >90% of the gene in the human genome are known to undergo alternative splicing thereby expanding the proteome production capacity of a limited number of genes. Similarly, mRNA export and translation regulation plays a vital role in regulating protein production. Thus, it is important to understand how these RNA binding proteins including alternative splicing factors (ASFs) and mRNA transport and translation factors regulate these processes. Here we report the expression of an ASF, Serine-arginine rich splicing factor 10 (Sfrs10) and a mRNA translation regulation factor, CUGBP, elav like family member 4 (Celf4) in the developing mouse retina. Sfrs10 was expressed throughout postnatal (P) retinal development and was observed progressively in newly differentiating neurons. Immunofluorescence (IF) showed Sfrs10 in retinal ganglion cells (RGCs) at P0, followed by amacrine and bipolar cells, and at P8 it was enriched in red/green cone photoreceptor cells. By P22, Sfrs10 was observed in rod photoreceptors in a peri-nuclear pattern. Like Sfrs10, Celf4 was also observed in the developing retina, but with two distinct retinal isoforms. In situ hybridization (ISH) showed progressive expression of Celf4 in differentiating neurons, which was confirmed by IF that showed a dynamic shift in Celf4 localization. Early in development Celf4 expression was restricted to the nuclei of newly differentiating RGCs and later (E16 onwards) it was observed in the initial segments of RGC axons. Later, during postnatal development, Celf4 was observed in amacrine and bipolar cells, but here it was predominantly cytoplasmic and enriched in the two synaptic layers. Specifically, at P14, Celf4 was observed in the synaptic boutons of rod bipolar cells marked by Pkc-α. Thus, Celf4 might be regulating AS early in development besides its known role of regulating mRNA localization/translation. In all, our data suggests an important role for AS and mRNA localization/translation in retinal neuron differentiation.
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