Alternative Splicing Regulates Targeting of Malate Dehydrogenase in Yarrowia lipolytica

Abstract

Alternative pre-mRNA splicing is a major mechanism contributing to the proteome complexity of most eukaryotes, especially mammals. In less complex organisms, such as yeasts, the numbers of genes that contain introns are low and cases of alternative splicing (AS) with functional implications are rare . We report the first case of AS with functional consequences in the yeast Yarrowia lipolytica . The splicing pattern was found to govern the cellular localization of malate dehydrogenase, an enzyme of the central carbon metabolism. This ubiquitous enzyme is involved in the tricarboxylic acid cycle in mitochondria and in the glyoxylate cycle, which takes place in peroxisomes and the cytosol. In Saccharomyces cerevisiae , three genes encode three compartment-specific enzymes. In contrast, only two genes exist in Y. lipolytica . One gene ( YlMDH1 , YALI0D16753g) encodes a predicted mitochondrial protein, whereas the second gene ( YlMDH2 , YALI0E14190g) generates the cytosolic and peroxisomal forms through the alternative use of two 3′-splice sites in the second intron. Both splicing variants were detected in cDNA libraries obtained from cells grown under different conditions. Mutants expressing the individual YlMdh2p isoforms tagged with fluorescent proteins confirmed that they localized to either the cytosolic or the peroxisomal compartment.

Origin of Spliceosomal Introns and Alternative Splicing

“…This may be achieved by on/off protein switches (Bingham et al 1988), or by coupling missense AS to NMD (Lewis et al 2003;Lareau et al 2007;Yap et al 2012), leading to transcript degradation before translation. Third, inclusion of alternative sequences may lead to differences in intracellular transport, either at the mRNA (Buckley et al 2011) or protein level (Freitag et al 2012;Kabran et al 2012). Nevertheless, despite a plethora of described examples of each kind, a major unanswered question is still to what extent AS is functional or simply splicing "noise" (Sorek et al 2004;Irimia et al 2008;Roy and Irimia 2008).…”

“…First, significant amounts of AS (usually intron retention) have now been described in nearly all well-studied species. These include representatives of all eukaryotic supergroups (McGuire et al 2008;Labadorf et al 2010;Otto et al 2010;Rhind et al 2011;Shen et al 2011;Curtis et al 2012;Sebé-Pedró s et al 2013), and even some intron-poor species (Pleiss et al 2007;Kabran et al 2012). Second, LECA's intron -exon structures seem to have met all classic requirements for AS to occur Koonin et al 2013): It was intron-rich (Irimia et al 2007b) and had heterogeneous splice signals, which are associated with splicing variation within and across genomes (Stamm et al 2000;Ast 2004;Baek and Green 2005).…”

A molecular genetic toolbox for Yarrowia lipolytica

“…However, previously studied proteins are involved in beta-oxidation (Mfe1p), or are not a constant peroxisome resident (Pex3p) [106]. Malate synthase has been studied for alternative splicing, with one form localized by an N-terminal GFP to the peroxisome matrix by plasmid-based expression [107]. Peroxisome maturation has been studied in detail in Y. lipolytica , following a series of microbody structures which contain peroxisome associated proteins (Pex6p and Pex2p) and enzyme functions such as beta-oxidation and hydrogen peroxide detoxification [13].…”

Role of Pex11p in Lipid Homeostasis in Yarrowia lipolytica

“…In S. cerevisiae, plants, and mammals, Pex11 is involved in peroxisome fission, and giant peroxisomes are observed in ⌬pex11 mutants (3)(4)(5)(6)(7)(8). In order to determine if this protein has the same function in Y. lipolytica, we generated mutants of the wild type and ⌬Ylpex11 that constitutively expressed a peroxisome-targeted RedStar2 fluorescent protein: JMY3175 (the wild-type JMY2900 with RedStar2SKL) and JMY3170 (the ⌬Ylpex11 strain expressing RedStar2SKL; RedStar2SKLp) (25). Experiments were performed in minimum media.…”