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Control of gene expression at the post-transcriptional level is essential in all organisms, and RNA-binding proteins play critical roles from mRNA synthesis to decay. To fully understand this process, it is necessary to identify the complete set of RNA-binding proteins and the functional consequences of the protein-mRNA interactions. Here, we provide an overview of the proteins that bind to mRNAs and their functions in the pathogenic bloodstream form of Trypanosoma brucei. We describe the production of a small collection of open-reading frames encoding proteins potentially involved in mRNA metabolism. With this ORFeome collection, we used tethering to screen for proteins that play a role in post-transcriptional control. A yeast two-hybrid screen showed that several of the discovered repressors interact with components of the CAF1/NOT1 deadenylation complex. To identify the RNA-binding proteins, we obtained the mRNA-bound proteome. We identified 155 high-confidence candidates, including many not previously annotated as RNA-binding proteins. Twenty seven of these proteins affected reporter expression in the tethering screen. Our study provides novel insights into the potential trypanosome mRNPs composition, architecture and function.

Control of gene expression at the post-transcriptional level is essential in all organisms, and RNA-binding proteins play critical roles from mRNA synthesis to decay. To fully understand this process, it is necessary to identify the complete set of RNA-binding proteins and the functional consequences of the protein-mRNA interactions. Here, we provide an overview of the proteins that bind to mRNAs and their functions in the pathogenic bloodstream form of Trypanosoma brucei. We describe the production of a small collection of open-reading frames encoding proteins potentially involved in mRNA metabolism. With this ORFeome collection, we used tethering to screen for proteins that play a role in post-transcriptional control. A yeast two-hybrid screen showed that several of the discovered repressors interact with components of the CAF1/NOT1 deadenylation complex. To identify the RNA-binding proteins, we obtained the mRNA-bound proteome. We identified 155 high-confidence candidates, including many not previously annotated as RNA-binding proteins. Twenty seven of these proteins affected reporter expression in the tethering screen. Our study provides novel insights into the potential trypanosome mRNPs composition, architecture and function.

The covalently linked cell wall protein Ccw12p of Saccharomyces cerevisiae is a GPI-anchored protein (V. Mrsa et al., 1999, J Bacteriol 181: 3076-3086). Although only 121 amino acids long, the haemagglutinin-tagged protein released by laminarinase from the cell wall possesses an apparent molecular mass of > 300 kDa. A membrane-bound form with an apparent molecular mass of 58 kDa is highly O- and N-glycosylated and contains the GPI anchor. With a half-life of 2 min, the membrane form is transformed to the > 300 kDa form. The deletion mutant ccw12Delta grows slower than the wild type, is highly sensitive to Calcofluor white and contains 2.5 times more chitin. Further, compared with wild-type yeast, significantly more proteins are released from intact cells when treated with dithiothreitol. Interestingly, these defects become less pronounced when further GPI-anchored cell wall proteins are deleted. Mutant DeltaGPI (simultaneous deletion of CCW12, CCW13/DAN1, CCW14, TIP1 and CWP1) is similar in many respects to wild-type yeast. To find out how the cell wall is stabilized in mutant DeltaGPI, a genome-wide transcription analysis was performed. Of 159 significantly regulated genes, 14 encode either known or suspected cell wall-associated proteins. Analysis of genes affected in transcription revealed that SED1 and SRL1 in particular are required to reconstruct cell wall stability in the absence of multiple GPI-anchored mannoproteins.

Summary Glucose exerts profound effects upon yeast physiol- ogy. In general, the effects of high glucose concen- trations (>1%) upon Saccharomyces cerevisiae have been studied. In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by tran- script profiling. We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals. Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of >0.1%. We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post-transcriptional mecha- nisms combine to accelerate the accumulation of ribosomal protein mRNAs. Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose. However, our data indicate that yeast activates ribo- some biogenesis only when sufficient glucose is available to make this metabolic investment worth- while. In contrast, the regulation of metabolic func- tions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient.

Summary The transcriptome of Saccharomyces cerevisiae was screened using the high-density membrane hybridization method, under aerobic and hypoxic conditions, in wild-type and mutant backgrounds obtained by the disruption of the genes encoding the regulatory proteins Hap1, Rox1 and the Srb10 and Rox3 subunits of RNA polymerase II holoenzyme. None of the mutations studied was able to fully overcome the wild-type hypoxic response. Deletion of the hap1 gene changed the expression profiles of individual open reading frames (ORFs) under both aerobic and hypoxic conditions. Major changes associated with rox3 deletion were related to the hypoxic activation. Rox3 also caused a repressor effect (oxygen-independent) on a subset of genes related to subtelomeric proteins. With regard to the effect brought about by the deletion of rox1 and srb10, correspondence cluster analysis revealed that the transcriptome profile in aerobic conditions is very similar in the wild-type and both deletion strains. In contrast, however, differences were found during hypoxia between the subgroup formed by wild-type and the Δrox1 deletant compared with the Δsrb10 deletant. An analysis of selected ORFs responding to hypoxia, in association with a dependence on the regulatory factors studied, made it possible to identify the clusters that are related to different regulatory circuits.

The covalently linked cell wall protein Ccw12p of Saccharomyces cerevisiae is a GPI-anchored protein (V. Mrsa et al., 1999, J Bacteriol 181: 3076-3086). Although only 121 amino acids long, the haemagglutinin-tagged protein released by laminarinase from the cell wall possesses an apparent molecular mass of300 kDa. A membrane-bound form with an apparent molecular mass of 58 kDa is highly O- and N-glycosylated and contains the GPI anchor. With a half-life of 2 min, the membrane form is transformed to the300 kDa form. The deletion mutant ccw12Delta grows slower than the wild type, is highly sensitive to Calcofluor white and contains 2.5 times more chitin. Further, compared with wild-type yeast, significantly more proteins are released from intact cells when treated with dithiothreitol. Interestingly, these defects become less pronounced when further GPI-anchored cell wall proteins are deleted. Mutant DeltaGPI (simultaneous deletion of CCW12, CCW13/DAN1, CCW14, TIP1 and CWP1) is similar in many respects to wild-type yeast. To find out how the cell wall is stabilized in mutant DeltaGPI, a genome-wide transcription analysis was performed. Of 159 significantly regulated genes, 14 encode either known or suspected cell wall-associated proteins. Analysis of genes affected in transcription revealed that SED1 and SRL1 in particular are required to reconstruct cell wall stability in the absence of multiple GPI-anchored mannoproteins.

Glucose exerts profound effects upon yeast physiology. In general, the effects of high glucose concentrations (1%) upon Saccharomyces cerevisiae have been studied. In this paper, we have characterized the global responses of yeast cells to very low (0.01%), low (0.1%) and high glucose signals (1.0%) by transcript profiling. We show that yeast is more sensitive to very low glucose signals than was previously thought, and that yeast displays different responses to these different glucose signals. Genes involved in central metabolic pathways respond rapidly to very low glucose signals, whereas genes involved in the biogenesis of cytoplasmic ribosomes generally respond only to glucose concentrations of0.1%. We also show that cytoplasmic ribosomal protein mRNAs are transiently stabilized by glucose, indicating that both transcriptional and post-transcriptional mechanisms combine to accelerate the accumulation of ribosomal protein mRNAs. Presumably, this facilitates rapid ribosome biogenesis after exposure to glucose. However, our data indicate that yeast activates ribosome biogenesis only when sufficient glucose is available to make this metabolic investment worthwhile. In contrast, the regulation of metabolic functions in response to very low glucose signals presumably ensures that yeast can exploit even minute amounts of this preferred nutrient.

The transcriptome of Saccharomyces cerevisiae was screened using the high-density membrane hybridization method, under aerobic and hypoxic conditions, in wild-type and mutant backgrounds obtained by the disruption of the genes encoding the regulatory proteins Hap1, Rox1 and the Srb10 and Rox3 subunits of RNA polymerase II holoenzyme. None of the mutations studied was able to fully overcome the wild-type hypoxic response. Deletion of the hap1 gene changed the expression profiles of individual open reading frames (ORFs) under both aerobic and hypoxic conditions. Major changes associated with rox3 deletion were related to the hypoxic activation. Rox3 also caused a repressor effect (oxygen-independent) on a subset of genes related to subtelomeric proteins. With regard to the effect brought about by the deletion of rox1 and srb10, correspondence cluster analysis revealed that the transcriptome profile in aerobic conditions is very similar in the wild-type and both deletion strains. In contrast, however, differences were found during hypoxia between the subgroup formed by wild-type and the Deltarox1 deletant compared with the Deltasrb10 deletant. An analysis of selected ORFs responding to hypoxia, in association with a dependence on the regulatory factors studied, made it possible to identify the clusters that are related to different regulatory circuits.