ABSTRACT
Mannoproteins are non-filamentous glycoproteins localized to the outermost layer of the yeast cell wall. The physiological roles of these structural components have not been completely elucidated due to the limited availability of appropriate tools. As the perturbation of mannoproteins may affect cell morphology, we investigated mannoprotein mutants in Saccharomyces cerevisiae via high-dimensional morphological phenotyping. The mannoprotein mutants were morphologically classified into seven groups using clustering analysis with Gaussian mixture modeling. The pleiotropic phenotypes of cluster I mutant cells (ccw12Δ) indicated that CCW12 plays major roles in cell wall organization. Cluster II (ccw14Δ, flo11Δ, srl1Δ, and tir3Δ) mutants exhibited altered mother cell size and shape. Mutants of cluster III and IV exhibited no or very small morphological defects. Cluster V (dse2Δ, egt2Δ, and sun4Δ) consisted of endoglucanase mutants with cell separation defects due to incomplete septum digestion. The cluster VI mutant cells (ecm33Δ) exhibited perturbation of apical bud growth. Cluster VII mutant cells (sag1Δ) exhibited differences in cell size and actin organization. Biochemical assays further confirmed the observed morphological defects. Further investigations based on various omics data indicated that morphological phenotyping is a complementary tool that can help with gaining a deeper understanding of the functions of mannoproteins.
ABSTRACT
Exon-intron architecture is one of the major features directing the splicing machinery to the short exons that are located within long flanking introns. However, the evolutionary dynamics of exon-intron architecture and its impact on splicing is largely unknown. Using a comparative genomic approach, we analyzed 17 vertebrate genomes and reconstructed the ancestral motifs of both 3' and 5' splice sites, as also the ancestral length of exons and introns. Our analyses suggest that vertebrate introns increased in length from the shortest ancestral introns to the longest primate introns. An evolutionary analysis of splice sites revealed that weak splice sites act as a restrictive force keeping introns short. In contrast, strong splice sites allow recognition of exons flanked by long introns. Reconstruction of the ancestral state suggests these phenomena were not prevalent in the vertebrate ancestor, but appeared during vertebrate evolution. By calculating evolutionary rate shifts in exons, we identified cis-acting regulatory sequences that became fixed during the transition from early vertebrates to mammals. Experimental validations performed on a selection of these hexamers confirmed their regulatory function. We additionally revealed many features of exons that can discriminate alternative from constitutive exons. These features were integrated into a machine-learning approach to predict whether an exon is alternative. Our algorithm obtains very high predictive power (AUC of 0.91), and using these predictions we have identified and successfully validated novel alternatively spliced exons. Overall, we provide novel insights regarding the evolutionary constraints acting upon exons and their recognition by the splicing machinery.
