The role of phosphatases in TOR signaling in yeast.

The TOR pathway controls cellular functions necessary for cell growth and proliferation of yeast and larger eukaryotes. The search for members of the TOR signaling cascade in yeast led to the discovery of type 2A protein phosphatases (PP2A) as important players within the pathway. We describe the roles in yeast of PP2A and the closely related phosphatase, Sit4, and then focus on complexes formed between the catalytic subunit of these phosphatases and Tap42, a direct target of the Tor protein kinases in yeast. Recent results suggest that Tap42 mediates many of the Tor functions in yeast, especially those involved in transcriptional modulation. However, whether Tap42 executes its function by inhibiting phosphatase activity or by activating phosphatases is still uncertain. In addition, Tor affects some transcriptional and physiological processes through Tap42 independent pathways. Thus, Tor proteins use multiple mechanisms to regulate transcriptional and physiological processes in yeast.

Regulation of the Aspergillus nidulans hisB gene by histidine starvation.

The hisB gene of the filamentous fungus Aspergillus nidulans encodes imidazole glycerol-phosphate dehydratase (E.C. 4.2.1.19), which catalyses the seventh enzymatic step in histidine biosynthesis. The gene was isolated and its deduced peptide sequence of 247 amino acids showed up to 54% identity with the IGPD enzymes of organisms comprising all three kingdoms. Expression of hisB cDNA in a Saccharomyces cerevisiae his3delta mutant strain functionally complemented the growth phenotype under histidine limitation. Addition of histidine did not affect hisB mRNA levels in A. nidulans wild-type cells. Histidine starvation conditions increased the hisB transcript level four-fold, suggesting regulation by a cross-pathway regulatory network. Deletion of the complete hisB open reading frame in A. nidulans strain A234 resulted in histidine auxotrophy. Additionally, hisB deletion strains were blocked from sexual fruiting body formation on medium containing low concentrations of histidine. This developmental phenotype of the hisB deletion mutant strain correlated with the induction of the cross-pathway control system.

Different positioning elements select poly(A) sites at the 3'-end of GCN4 mRNA in the yeast Saccharomyces cerevisiae.

Cleavage and polyadenylation of eukaryotic mRNA requires efficiency and positioning elements in the 3'-untranslated region (3'-UTR) of the mRNA. Specific point mutations were introduced into the yeast GCN4 3'-UTR to detect sequence motifs which are involved in the positioning of the poly(A) site. 3'-End proces-sing activities of different GCN4 3'-UTR alleles were measured in an in vivo test system. Point mutations in an AAGAA motif defocussed selection of the poly(A) sites of the GCN4 3'-UTR to various additional poly(A) sites instead of the single site of the wild-type GCN4 3'-UTR. A strain with an intact wild-type GCN4 3'-UTR but impaired in RNA15 encoding an RNA-binding processing factor showed a similar defocussed pattern of poly(A) site selection. Remarkably, two additional sequence motifs upstream of the AAGAA motif which resemble yeast efficiency motifs independently affected poly(A) site positioning but not efficiency of 3'-end processing. Mutations in one motif resulted in an additional upstream poly(A) site. Alterations of the other motif shifted the poly(A) sites exclusively to two downstream poly(A) sites. These data suggest several contact points between the precursor mRNA and the polyadenylation machinery in yeast.

A single point mutation in the yeast TRP4 gene affects efficiency of mRNA 3' end processing and alters selection of the poly(A) site.

The yeast TRP4 mRNA 3' end formation element is a bidirectional element which functions in both orien-tations in an artificial in vivo test system. In this study, the role of 3' end formation was analysed in the context of the entire TRP4 gene. The 3' untranslated region (3'UTR) of TRP4 was altered and changes were analysed for their influence on TRP4 gene expression. The 3'UTR in reverse orientation was fully functional and did not affect TRP4 gene expression. Exchanging the TRP4 3'UTR by the bidirectional ARO4 or the unidirectional GCN4 3' end formation element allowed efficient gene expression. Deletion of the entire TRP4 3'UTR resulted in 70% reduction of TRP4 mRNA and 50% reduced specific Trp4 enzyme activity in comparison to wild-type. A single point mutation within the TRP4 3'UTR caused the same effect on gene expression. This point mutation did not only affect the efficiency of 3' end formation, but also produced new poly(A) sites which were situated upstream of the wild-type poly(A) sites. Therefore this sequence motif in the TRP4 3'UTR acts simultaneously as both an efficiency and positioning element.

Sequence requirements of the bidirectional yeast TRP4 mRNA 3'-end formation signal.

The yeast TRP4 3'-end formation signal functions in both orientations in an in vivo test system. We show here that the TRP4 3'-end formation element consists of two functionally different sequence regions. One region of approximately 70 nucleotides is located in the untranslated region between the translational stop codon and the major poly(A) site. The major poly(A) site is not part of this region and can be deleted without a decrease in TRP4 3'-end formation. 5'and 3'deletions and point mutations within this region affected 3'-end formation similarly in both orientations. In the center of this region the motif TAGT is located on the antisense strand. Point mutations within this motif resulted in a drastic reduce of 3'-end formation activity in both orientations. A second region consists of the 3'-end of the TRP4 open reading frame and is required for 3'-end formation in forward orientation. A single point mutation in a TAGT motif of the TRP4 open reading frame abolished TRP4 mRNA 3'-end formation in forward orientation and had no effect on the reverse orientation.