Role of SSD1 in Phenotypic Variation of Saccharomyces cerevisiae Strains Lacking DEG1-Dependent Pseudouridylation.

Yeast phenotypes associated with the lack of wobble uridine (U34) modifications in tRNA were shown to be modulated by an allelic variation of SSD1, a gene encoding an mRNA-binding protein. We demonstrate that phenotypes caused by the loss of Deg1-dependent tRNA pseudouridylation are similarly affected by SSD1 allelic status. Temperature sensitivity and protein aggregation are elevated in deg1 mutants and further increased in the presence of the ssd1-d allele, which encodes a truncated form of Ssd1. In addition, chronological lifespan is reduced in a deg1 ssd1-d mutant, and the negative genetic interactions of the U34 modifier genes ELP3 and URM1 with DEG1 are aggravated by ssd1-d. A loss of function mutation in SSD1, ELP3, and DEG1 induces pleiotropic and overlapping phenotypes, including sensitivity against target of rapamycin (TOR) inhibitor drug and cell wall stress by calcofluor white. Additivity in ssd1 deg1 double mutant phenotypes suggests independent roles of Ssd1 and tRNA modifications in TOR signaling and cell wall integrity. However, other tRNA modification defects cause growth and drug sensitivity phenotypes, which are not further intensified in tandem with ssd1-d. Thus, we observed a modification-specific rather than general effect of SSD1 status on phenotypic variation in tRNA modification mutants. Our results highlight how the cellular consequences of tRNA modification loss can be influenced by protein targeting specific mRNAs.

Oxidosqualene Cyclase Knock-Down in Latex of Taraxacum koksaghyz Reduces Triterpenes in Roots and Separated Natural Rubber.

In addition to natural rubber (NR), several triterpenes are synthesized in laticifers of the Russian dandelion (Taraxacum koksaghyz). Detailed analysis of NR and resin contents revealed different concentrations of various pentacyclic triterpenes such as α-, ß-amyrin and taraxasterol, which strongly affect the mechanical properties of the resulting rubber material. Therefore, the reduction of triterpene content would certainly improve the industrial applications of dandelion NR. We developed T. koksaghyz plants with reduced triterpene contents by tissue-specific downregulation of major laticifer-specific oxidosqualene cyclases (OSCs) by RNA interference, resulting in an almost 67% reduction in the triterpene content of NR. Plants of the T1 generation showed no obvious phenotypic changes and the rubber yield also remained unaffected. Hence, this study will provide a solid basis for subsequent modern breeding programs to develop Russian dandelion plants with low and stable triterpene levels.

Enhanced tolerance to a combination of heat stress and drought in Arabidopsis plants deficient in ICS1 is associated with modulation of photosynthetic reaction center proteins.

Plants are exposed to multiple abiotic stresses that simultaneously occur under natural environmental conditions. Studies deciphering acclimation of plants to stress combinations are, however, still scarce. ISOCHORISMATE SYNTHASE 1 (ICS1) is known as a crucial enzyme required for synthesis of salicylic acid and phylloquinone, one of the components of the photosystem I complex. Although the significance of ICS1 in the regulation of abiotic stress response and pathogen defense in plants has been evidenced in previous studies, the role of this enzyme in the acclimation of plants to stress combinations is still largely unknown. In this study, we demonstrated the enhanced tolerance of Arabidopsis salicylic acid induction deficient 2-1(sid2-1) mutant deficient in ICS1 to a combination of heat stress and drought. H2 O2 -dependent stomatal closure and accumulation of total soluble sugars are associated with the enhanced tolerance of sid2-1 plants to this stress combination. In addition, sid2-1 plants showed higher accumulation of reaction center proteins (D1 and D2) in photosystem II accompanied by enhanced expression of transcripts involved in repair of these reaction center proteins. Furthermore, investigation of chlorophyll fluorescence indicated that mechanisms for dissipating the excess energy might be activated in sid2-1 plants specifically under a combination of heat stress and drought. Taken together, our findings suggest that maintenance of photosynthetic apparatus as well as prevention of excess water loss might enhance the tolerance of sid2-1 plants deficient in ICS1 to a combination of heat stress and drought.

Pseudouridine-Free Escherichia coli Ribosomes.

Pseudouridine (Ψ) is present at conserved, functionally important regions in the ribosomal RNAs (rRNAs) from all three domains of life. Little, however, is known about the functions of Ψ modifications in bacterial ribosomes. An Escherichia coli strain has been constructed in which all seven rRNA Ψ synthases have been inactivated and whose ribosomes are devoid of all Ψs. Surprisingly, this strain displays only minor defects in ribosome biogenesis and function, and cell growth is only modestly affected. This is in contrast to a strong requirement for Ψ in eukaryotic ribosomes and suggests divergent roles for rRNA Ψ modifications in these two domains.IMPORTANCE Pseudouridine (Ψ) is the most abundant posttranscriptional modification in RNAs. In the ribosome, Ψ modifications are typically located at conserved, critical regions, suggesting they play an important functional role. In eukarya and archaea, rRNAs are modified by a single pseudouridine synthase (PUS) enzyme, targeted to rRNA via a snoRNA-dependent mechanism, while bacteria use multiple stand-alone PUS enzymes. Disruption of Ψ modification of rRNA in eukarya seriously impairs ribosome function and cell growth. We have constructed an E. coli multiple deletion strain lacking all Ψ modifications in rRNA. In contrast to the equivalent eukaryotic mutants, the E. coli strain is only modestly affected in growth, decoding, and ribosome biogenesis, indicating a differential requirement for Ψ modifications in these two domains.

Targeted gene deletion of Leishmania major UDP-galactopyranose mutase leads to attenuated virulence.

Considering the high incidence of galactofuranose (Gal(f)) in pathogens and its absence from higher eukaryotes, the enzymes involved in the biosynthesis of this unusual monosaccharide appear as attractive drug targets. However, although the importance of Gal(f) in bacterial survival or pathogenesis is established, its role in eukaryotic pathogens is still undefined. Recently, we reported the identification and characterization of the first eukaryotic UDP-galactopyranose mutases. This enzyme holds a central role in Gal(f) metabolism by providing UDP-Gal(f) to all galactofuranosyltransferases. In this work, the therapeutical potential of Gal(f) metabolism in Leishmania major was hence evaluated by targeted replacement of the GLF gene encoding UDP-galactopyranose mutase. In L. major, Gal(f) is present in the membrane anchor of the lipophosphoglycan (LPG) and in glycoinositolphospholipids. Accordingly, the generated glf(-) mutant is deficient in LPG backbone and expresses truncated glycoinositolphospholipids. These structural changes do not influence the in vitro growth of the parasite but lead to an attenuation of virulence comparable with that observed with a mutant exclusively deficient in LPG.

Prolonged exclusive breast-feeding from vegan mother causing an acute onset of isolated methylmalonic aciduria due to a mild mutase deficiency.

We describe a case of methylmalonic aciduria (MMA) occurred in a 22-month-old boy whose diet was exclusively based upon breast-feeding from a mother following a long-lasting strict vegetarian diet. Clinical picture showed a dramatic onset, with a profound drowsiness associated with a severe metabolic acidosis, hyperammonemia, macrocytic anemia, ketonuria, and massive methylmalonic aciduria without homocystinuria. Both symptoms and biochemical findings quickly improved thanks to prompt vitamin B(12)parenteral therapy. Biochemical and enzymatic findings allowed a diagnosis of mild mutase deficiency, which only and inadequate dietary B(12)contribution might have revealed. Our case highlights the risk of a prolonged strictly vegetarian diet of lactating mother for providing inadequate amounts of some nutrients to the breast-fed baby. Moreover, such a dietary behaviour could dramatically unmask otherwise clinically unapparent metabolic defects in the baby.