Abstract
Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla. At its catalytic core resides the activated splicing complex Bact consisting of the three small nuclear ribonucleoprotein complexes (snRNPs) U2, U5 and U6 and the so-called NineTeen complex (NTC) which is important for spliceosomal activation. CWC15 is an integral part of the NTC in humans and it is associated with the NTC in other species. Here we show the ubiquitous expression and developmental importance of the Arabidopsis ortholog of yeast CWC15. CWC15 associates with core components of the Arabidopsis NTC and its loss leads to inefficient splicing. Consistent with the central role of CWC15 in RNA splicing, cwc15 mutants are embryo lethal and additionally display strong defects in the female haploid phase. Interestingly, the haploid male gametophyte or pollen in Arabidopsis, on the other hand, can cope without functional CWC15, suggesting that developing pollen might be more tolerant to CWC15-mediated defects in splicing than either embryo or female gametophyte.
Highlights
Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla
We address the importance of the predicted splicing factor CWC15 in the model plant Arabidopsis thaliana
Adding peptides with lower counts to our analysis, we detected a majority of all components of the U5 small nuclear ribonucleoprotein complexes (snRNPs), NTC, NTR, and associated splicing factors (Supplementary Table 2) that were recently described in a multitude of structural cryo-EM reports for yeast and human spliceosomes[15,28]. These results suggest that CWC15 is potentially part of the NineTeen complex, which has an important general role in splicing in Arabidopsis thaliana[26,33]
Summary
Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla. The Arabidopsis zygote initiates a precise developmental program, which results in a heart-shaped embryo already comprising all major seedling organs: two primary leaves or cotyledons, a shoot meristem, a hypocotyl, and a primary root with a root m eristem[4] This invariant embryo patterning and development is impaired in mutants defective for various cellular response pathways e.g. responses to phytohormones, small RNA pathways, vesicular trafficking, cytoskeletal structure, and cell cycle control[5,6,7,8,9]. Mutations in genes that are components of the RNA splicing machinery (spliceosome) affect gametophyte function and embryogenesis Mutations such as gfa1/clotho, rtf[2], sus2/prp[8], or bud[13] cause severely reduced transmission of the mutant alleles via the female gametophyte and cause embryo lethality in A rabidopsis[10,11,12,13,14]. Mutations in CLOTHO, which is a homolog of the yeast U5-associated Snu[114], and ATROPOS, whose homolog has a demonstrated role in U2 a ssembly[27], result in defective female gametophytes, whereas male transmission is less severely a ffected[11]
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