U12-type or minor introns are found in most multicellular eukaryotes and constitute ∼0.5% of all introns in species with a minor spliceosome. Although the biological significance for the evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome, predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA binding motif protein 48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5′ end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation of RBM48 in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron-containing genes have significant U12-type intron retention in RBM48 mutants. Comparing these results to maize rbm48 mutants defined a subset of minor intron-containing genes disrupted in both species. Mutations in the majority of these orthologous minor intron-containing genes have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of human RBM48 mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.
Centriole duplication occurs once in each cell cycle to maintain centrosome number. A previous genome-wide screen revealed that depletion of 14 RNA splicing factors leads to a specific defect in centriole duplication, but the cause of this deficit remains unknown. Here, we identified an additional pre-mRNA splicing factor, WBP11, as a novel protein required for centriole duplication. Loss of WBP11 results in the retention of ∼200 introns, including multiple introns in TUBGCP6, a central component of the γ-TuRC. WBP11 depletion causes centriole duplication defects, in part by causing a rapid decline in the level of TUBGCP6. Several additional splicing factors that are required for centriole duplication interact with WBP11 and are required for TUBGCP6 expression. These findings provide insight into how the loss of a subset of splicing factors leads to a failure of centriole duplication. This may have clinical implications because mutations in some spliceosome proteins cause microcephaly and/or growth retardation, phenotypes that are strongly linked to centriole defects.
more » « less- NSF-PAR ID:
- 10128447
- Publisher / Repository:
- DOI PREFIX: 10.1083
- Date Published:
- Journal Name:
- The Journal of Cell Biology
- Volume:
- 219
- Issue:
- 1
- ISSN:
- 0021-9525
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Setaria viridis and maize that an uncharacterized member of this family, CRM Family Member1 (CFM1), promotes the splicing of most of the introns that had not previously been shown to require a CRM domain protein. ASetaria mutant expressing mutated CFM1 was strongly disrupted in the splicing of three chloroplast tRNAs:trnI ,trnV andtrnA . Analyses by RNA gel blot and polysome association suggest that the tRNA deficiencies lead to compromised chloroplast protein synthesis and the observed whole‐plant chlorotic phenotypes. Co‐immunoprecipitation data demonstrate that the maize CFM1 ortholog is bound to introns whose splicing is disrupted in thecfm1 mutant. With these results, CRM domain proteins have been shown to promote the splicing of all but two of the introns found in angiosperm chloroplast genomes.
