Jesús de la Cruz

Unwinding RNA in Saccharomyces cerevisiae: DEAD-box proteins and related families

Members of the RNA-helicase family are defined by several evolutionary conserved motifs. They are found in all organisms - from bacteria to humans - and many viruses. The minimum number of RNA helicases present within a eukaryotic cell can be predicted from the complete sequence of the Saccharomyces cerevisiae genome. Recent progress in the functional analysis of various family members has given new insights into, and confirmed the significance of these proteins for, most cellular RNA metabolic processes.

Protein trans-Acting Factors Involved in Ribosome Biogenesis in Saccharomyces cerevisiae

The synthesis of ribosomes is one of the major cellular activities, and in eukaryotes, it takes place primarily, although not exclusively, in a specialized subnuclear compartment termed the nucleolus (125, 155). There, the rRNA genes are transcribed as precursors (pre-rRNAs), which undergo processing and covalent modification. Maturation of pre-rRNAs is intimately linked to their assembly with the ribosomal proteins (r-proteins). These processes depend on various cis-acting elements (6, 188), and they require a large number of nonribosomal protein trans-acting factors (97, 174, 193). Experimental evidence suggests that the basic outline of ribosome synthesis is conserved throughout eukaryotes. However, most of our knowledge comes from the combination of molecular genetic and biochemical approaches in the yeast Saccharomyces cerevisiae. This minireview is aimed at giving an insight into the functions of the many protein trans-acting factors involved in ribosome biogenesis in S. cerevisiae.

Cloning and characterization of a chitinase (CHIT42) cDNA from the mycoparasitic fungus Trichoderma harzianum

A cDNA of Trichoderma harzianum (chit42), coding for an endochitinase of 42 kDa, has been cloned using synthetic oligonucleotides corresponding to aminoacid sequences of the purified chitinase. The cDNA codes for a protein of 423 amino acids. Analysis of the N-terminal amino-acid sequence of the chitinase, and comparison with that deduced from the nucleotide sequence, revealed post-translational processing of a putative signal peptide of 22 amino acids and a second peptide of 12 amino acids. The chit42 sequence presents overall similarities with filamentous fungal and bacterial chitinases and to a lesser extent with yeast and plant chitinases. The deduced aminoacid sequence has putative catalytic, phosphorylation and glycosylation domains. Expression of chit42 mRNA is strongly induced by chitin and chitin-containing cell walls and is subjected to catabolite repression. Southern analysis shows that it is present as a single-copy gene in T. harzianum. chit42 is also detected in several tested mycoparasitic and non-mycoparasitic fungal strains.

Primary structure and expression pattern of the 33-kDa chitinase gene from the mycoparasitic fungus Trichoderma harzianum

A gene (chit33) from the mycoparasitic fungus Trichoderma harzianum, coding for a chitinase of 33 kDa, has been isolated and characterized. Partial amino-acid sequences from the purified 33-kDa chitinase were obtained. The amino-terminal peptide sequence was employed to design an oligonucleotide probe and was used as a primer to isolate a 1.2-kb cDNA. The cDNA codes for a protein of 321 amino acids, which includes a putative signal peptide of 19 amino acids. All microsequenced peptides found in this sequence, indicate that this cDNA codes for the 33-kDa chitinase. A high homology (approximately 43% identity) was found with fungal and plant chitinases, including yeast chitinases. However enzyme characteristics suggest a nutritional (saprophytic or mycoparasitic), rather than a morphogenetic, role for this chitinase. The chit33 gene appears as a single copy in the T. harzianum genome, is strongly suppressed by glucose, and de-repressed under starvation conditions as well as in the presence of autoclaved mycelia and/or fungal cell walls. The 33-kDa chitinase seems to be very stable except under starvation conditions. The independent regulation of each of the chitinases in T. harzianum indicates different specific roles.

An Antifungal Exo-α-1,3-Glucanase (AGN13.1) from the Biocontrol Fungus Trichoderma harzianum

ABSTRACT Trichoderma harzianum secretes α-1,3-glucanases when it is grown on polysaccharides, fungal cell walls, or autoclaved mycelium as a carbon source (simulated antagonistic conditions). We have purified and characterized one of these enzymes, named AGN13.1. The enzyme was monomeric and slightly basic. AGN13.1 was an exo-type α-1,3-glucanase and showed lytic and antifungal activity against fungal plant pathogens. Northern and Western analyses indicated that AGN13.1 is induced by conditions that simulated antagonism. We propose that AGN13.1 contributes to the antagonistic response of T. harzianum.

Dbp6p Is an Essential Putative ATP-Dependent RNA Helicase Required for 60S-Ribosomal-Subunit Assembly in Saccharomyces cerevisiae

ABSTRACT A previously uncharacterized Saccharomyces cerevisiaeopen reading frame, YNR038W, was analyzed in the context of the European Functional Analysis Network. YNR038W encodes a putative ATP-dependent RNA helicase of the DEAD-box protein family and was therefore named DBP6 (DEAD-box protein 6). Dbp6p is essential for cell viability. In vivo depletion of Dbp6p results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase labeling of pre-rRNA and steady-state analysis of pre-rRNA and mature rRNA by Northern hybridization and primer extension show that Dbp6p depletion leads to decreased production of the 27S and 7S precursors, resulting in a depletion of the mature 25S and 5.8S rRNAs. Furthermore, hemagglutinin epitope-tagged Dbp6p is detected exclusively within the nucleolus. We propose that Dbp6p is required for the proper assembly of preribosomal particles during the biogenesis of 60S ribosomal subunits, probably by acting as an rRNA helicase.

Functions of Ribosomal Proteins in Assembly of Eukaryotic Ribosomes In Vivo

The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.

Molecular characterization and heterologous expression of an endo-β-1,6-glucanase gene from the mycoparasitic fungus Trichoderma harzianum

Hydrolytic enzymes from the filamentous fungus Trichoderma harzianum have been described as critical elements of the mycoparasitic action of Trichoderma against fungal plant pathogens. In this report we describe the first genomic and cE)NA clones encoding a β-1,6-endoglucanase gene. The deduced protein sequence has limited homology with other β-glucanases. Northern experiments show a marked repression of mRNA accumulation by glucose. The protein has been successfully produced in Saccharomyces cerevisiae upon construction of a transcriptional fusion of the cDNA with a yeast promoter. This S. cerevisiae recombinant strain shows a strong lytic action on agar plates containing β-1,6-glucan.

Spb4p, an essential putative RNA helicase, is required for a late step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae.

Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, Al, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.

Has1p, a member of the DEAD‐box family, is required for 40S ribosomal subunit biogenesis in Saccharomyces cerevisiae†

The Has1 protein, a member of the DEAD‐box family of ATP‐dependent RNA helicases in Saccharomyces cerevisiae, has been found by different proteomic approaches to be associated with 90S and several pre‐60S ribosomal complexes. Here, we show that Has1p is an essential trans‐acting factor involved in 40S ribosomal subunit biogenesis. Polysome analyses of strains genetically depleted of Has1p or carrying a temperature‐sensitive has1‐1 mutation show a clear deficit in 40S ribosomal subunits. Analyses of pre‐rRNA processing by pulse‐chase labelling, Northern hybridization and primer extension indicate that these strains form less 18S rRNA because of inhibition of processing of the 35S pre‐rRNA at the early cleavage sites A0, A1 and A2. Moreover, processing of the 27SA3 and 27SB pre‐rRNAs is delayed in these strains. Therefore, in addition to its role in the biogenesis of 40S ribosomal subunits, Has1p is required for the optimal synthesis of 60S ribosomal subunits. Consistent with a role in ribosome biogenesis, Has1p is localized to the nucleolus. On sucrose gradients, Has1p is associated with a high‐molecular‐weight complex sedimenting at positions equivalent to 60S and pre‐60S ribosomal particles. A mutation in the ATP‐binding motif of Has1p does not support growth of a has1 null strain, suggesting that the enzymatic activity of Has1p is required in ribosome biogenesis. Finally, sequence comparisons suggest that Has1p homologues exist in all eukaryotes, and we show that a has1 null strain can be fully complemented by the Candida albicans homologue.