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Conserved role for spliceosomal component PRPF40A in microexon splicing
Bikash Choudhary1, Adam Norris2
1Department of Biochemistry, University of California, Riverside, California 92521, USA.
Abstract:
Microexons (exons ≤30 nt) are important features of neuronal transcriptomes, but pose mechanistic challenges to the splicing machinery. We previously showed that PRP-40, a component of the U1 spliceosome, is globally required for microexon splicing in Caenorhabditis elegans Here we show that the homologous PRPF40A is also globally required for microexon splicing in mouse neuroblastoma cells. We find that PRPF40A coregulates microexons along with SRRM4, a neuron-specific regulator of microexon splicing. The relationship between exon size and dependence on PRPF40A/SRRM4 is distinct, with SRRM4-dependence exhibiting a size threshold (∼30 nt) and PRPF40A-dependence exhibiting a graded decrease as exon size increases. Finally, we show that PRPF40A knockdown causes an increase in productive splicing of its spliceosomal binding partner Luc7l by the skipping of a small "poison exon." Similar homeostatic cross-regulation is often observed across paralogous RNA-binding proteins. Here we find this concept likewise applies across evolutionarily unrelated but functionally and physically coupled spliceosomal components.
Insights
PRPF40A is essential for splicing microexons in mouse cells, working with SRRM4. This splicing factor also regulates its own gene expression, revealing cross-regulation within the spliceosome.
Area of Science:
- Molecular Biology
- Genetics
- Neuroscience
Background:
- Microexons are crucial for neuronal gene expression but present splicing challenges.
- Previous work identified PRP-40 as vital for microexon splicing in C. elegans.
Purpose of the Study:
- To investigate the role of the homologous PRPF40A in microexon splicing in mammalian cells.
- To elucidate the relationship between PRPF40A, SRRM4, and microexon size.
- To explore potential cross-regulatory mechanisms involving PRPF40A.
Main Methods:
- Utilized mouse neuroblastoma cells to study microexon splicing.
- Performed knockdown experiments to assess the function of PRPF40A.
- Analyzed the interplay between PRPF40A and SRRM4 in regulating microexons of varying sizes.
- Investigated the impact of PRPF40A knockdown on the splicing of its binding partner, Luc7l.
Main Results:
- PRPF40A is globally required for microexon splicing in mouse neuroblastoma cells.
- PRPF40A and SRRM4 coregulate microexons, with distinct size-dependent relationships.
- SRRM4-dependence shows a threshold at ~30 nt, while PRPF40A-dependence decreases gradually with increasing exon size.
- PRPF40A knockdown enhances productive splicing of Luc7l by promoting the skipping of a poison exon.
Conclusions:
- PRPF40A is a key regulator of microexon splicing in mammals, analogous to its C. elegans counterpart.
- The findings reveal a novel size-dependent regulatory mechanism for microexons involving PRPF40A and SRRM4.
- Demonstrated homeostatic cross-regulation between spliceosomal components, extending this concept to evolutionarily distinct proteins.
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