Search for: All records

Abstract PremiseThe extraction of high‐quality RNA is the critical first step for the analysis of gene expression and gene space. This remains particularly challenging in plants, and especially in ferns, where the disruption of the cell wall and separation of organic compounds from nucleic acids is not trivial. MethodsWe developed a cetyltrimethylammonium bromide (CTAB)‐based RNA extraction protocol that consistently performs well across a large phylogenetic breadth of ferns—a lineage of plants high in secondary compounds—and in an array of tissue types. Two alternative options (precipitation vs. clean‐up without intermediate precipitation) are presented, both of which yield high‐quality RNA extracts with optical density (OD) ratios of OD 260/280 = 1.9–2.1 and OD 260/230 > 1.6, and RNA integrity numbers >7. ConclusionsThis study presents an efficient protocol for the extraction of high‐quality RNA from multiple tissues and across the fern phylogeny, a clade of plants that still lags behind other major lineages in the development of genomic resources. We hope that this method can be used to help facilitate the closing of this gap. 

Abstract Historically, xenia effects were hypothesized to be unique genetic contributions of pollen to seed phenotype, but most examples represent standard complementation of Mendelian traits. We identified the imprinteddosage-effect defective1(ded1) locus in maize (Zea mays) as a paternal regulator of seed size and development. Hypomorphic alleles show a 5–10% seed weight reduction whended1is transmitted through the male, while homozygous mutants are defective with a 70–90% seed weight reduction.Ded1encodes an R2R3-MYB transcription factor expressed specifically during early endosperm development with paternal allele bias. DED1 directly activates early endosperm genes and endosperm adjacent to scutellum cell layer genes, while directly repressing late grain-fill genes. These results demonstrate xenia as originally defined: Imprinting ofDed1causes the paternal allele to set the pace of endosperm development thereby influencing grain set and size. 

Abstract 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.