Abstract
Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two IFT genes, IFT81 (fla9) and IFT121 (ift121-2), which lead to flagellar assembly defects in the unicellular green alga Chlamydomonas reinhardtii. The splicing defects in these ift mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a dgr14 deletion mutant, which suppresses the 3′ splice site mutation in IFT81, and a frameshift mutant of FRA10, which suppresses the 5′ splice site mutation in IFT121. Surprisingly, we found dgr14-1 and fra10 mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in SMG1, which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the ift mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the ift mutant phenotype, make Chlamydomonas an attractive organism to identify new proteins involved in splicing through suppressor screening.
Highlights
Pre-messenger RNA splicing, which removes noncoding introns from nascent RNAs to produce functional mRNAs, is an important and precisely controlled process
Splicing of most introns found in eukaryotic nuclei is facilitated by the spliceosome, which is a dynamic complex that contains multiple uridine-rich small nuclear ribonucleoproteins and proteins associated with these snRNPs [1,2]
Our study provides the first functional study of the involvement of FRA10AC1 in 2 pre-mRNA splicing and suggests that, like C. elegans, the Chlamydomonas DGR14 protein is involved in pre-mRNA splicing regulation
Summary
Pre-messenger RNA splicing, which removes noncoding introns from nascent RNAs to produce functional mRNAs, is an important and precisely controlled process. The major spliceosome, which is composed of U1, U2, U4/U6 and U5 snRNPs and associated proteins, recognizes conserved nucleotide sequences at the 50 splice donor site (GT), the 30 splice acceptor site (AG) and the branch site Mutations in these splice sites usually cause aberrant transcripts and it is estimated that splice site mutations cause approximately 15% of human genetic diseases [3]. The minor spliceosome, which is composed of U11, U12, U4atac, U6atac and U5 and many of the associated proteins found in the major spliceosome, is responsible for the removal of only approximately 0.3% of introns It recognizes different conserved nucleotide sequences at the 50 (AT) and 30 (AC) splice sites. In our study described here, we focus on two peripheral proteins, DGCR14 and FRA10AC1 [5]
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