Minor splicing snRNAs are enriched in the developing mouse CNS and are crucial for survival of differentiating retinal neurons

Abstract

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.

Minor intron splicing is regulated by FUS and affected by ALS ‐associated FUS mutants

“…The minor spliceosome is essential for development (Otake et al , ; Baumgartner et al , ), and dysfunctional minor splicing has been associated with several developmental and neurological disorders. In humans, mutations in the U4atac snRNA lead to the developmental disorder Taby‐Linder syndrome (TALS), which is characterized by the growth retardation as well as central nervous system malformations (Pierce & Morse, ; Jafarifar et al , ).…”

Minor spliceosome inactivation causes microcephaly due to cell cycle defects and death of self-amplifying radial glial cells

“…Cryosections (16 µm) of mouse heads (for E10-E12 and P0), telencephalons (E13-E14) and whole brains (for P21) (n=3 for each timepoint and genotype) were used for section ISH. Section ISH was performed using an antisense, digoxigenin-labeled U11 RNA probe, which was detected using either alkaline phosphatase or fluorescent labeling (FISH), as described in Baumgartner et al (2014). The U11 probe was generated using the U11 expression primers in Table S7.…”

“…RNA was extracted using the DirectZOL RNA MiniPrep kit (Zymo Research, R2050), according to the manufacturer's instructions. For total RNA samples, 500 ng RNA was used for cDNA synthesis, which was performed as previously described (Baumgartner et al, 2014).…”

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

“…16 μm cryosections of mouse heads (for E10–E12 and P0), telencephalons (E13–E14), and whole brains (for P21), N =3 for each time-point and genotype, were used for section in situ hybridization (SISH). SISH was performed using antisense, digoxigenin-labeled U11 RNA probe, which was detected using either alkaline phosphatase (AP) or fluorescent labeling (FISH), as described (Baumgartner et al, 2014). The U11 probe was generated using the U11 expression primers in Table S5.…”