Mutations in a Peptidylprolyl-cis/trans-isomerase Gene Lead to a Defect in 3′-End Formation of a Pre-mRNA inSaccharomyces cerevisiae

Abstract

In a genetic screen aimed at the identification of trans-acting factors involved in mRNA 3-end processing of budding yeast, we have previously isolated two temperature-sensitive mutants with an apparent defect in the 3-end formation of a plasmid-derived pre-mRNA. Surprisingly, both mutants were rescued by the essential gene ESS1/PTF1 that encoded a putative peptidylprolyl-cis/trans-isomerase (PPIase) (Hani, J., Stumpf, G., and Domdey, H. (1995) FEBS Lett. 365, 198 -202). Such enzymes, which catalyze the cis/trans-interconversion of peptide bonds N-terminal of prolines, are suggested to play a role in protein folding or trafficking. Here we report that Ptf1p shows PPIase activity in vitro, displaying an unusual substrate specificity for peptides with phosphorylated serine and threonine residues preceding proline. Both mutations were found to result in amino acid substitutions of highly conserved residues within the PPIase domain, causing a marked decrease in PPIase activity of the mutant enzymes. Our results are suggestive of a so far unknown involvement of a PPIase in mRNA 3-end formation in Saccharomyces cerevisiae.Despite intensive efforts to unravel the complex process of mRNA 3Ј-end formation in Saccharomyces cerevisiae, the list of participating factors still awaits its completion.We have recently isolated a gene complementing the phenotype of two temperature-sensitive yeast mutants that were impaired in mRNA 3Ј-end formation. This gene, designated PTF1 (processing/termination factor 1; identical with the previously described ESS1 (1)), encodes a protein that, by virtue of sequence similarity, was identified as a peptidylprolyl-cis/ trans-isomerase (PPIase) 1 (2). PPIases are ubiquitous enzymes that catalyze the interconversion from cis to trans of peptide bonds preceding a proline and are thought to accelerate this often rate-limiting step in the folding of a number of proteins in vivo (3-6).PPIases are divided in three families, based on their sensitivities toward two clinically relevant immunosuppressants: the cyclosporin A-binding proteins (cyclophilins), the FK506-binding proteins, and a third family, named after the Escherichia coli protein parvulin, which is not inhibited by either of the two drugs (for review see Refs. 3-6). In addition, the members of each family are characterized by conserved but distinct amino acid domains. By this criterion, PTF1 was predicted to belong to the parvulin family of PPIases (2).Although disruption of PPIase genes did not generally impair cell growth (7-8), PTF1 was the first PPIase gene shown to be essential for cell viability (1). In fact, PTF1 is the only essential PPIase gene in S. cerevisiae as demonstrated more recently by the viability of a yeast mutant lacking the remaining 12 PPIases, members of the other two immunosuppressant binding families. (8). So far, the only other example of an essential PPIase is the recently discovered PIN1, a human protein, that is structurally and functionally related to Ptf1p (9 -10).In this paper we describe the genetic screen ...

Functional Conservation of Phosphorylation-specific Prolyl Isomerases in Plants

“…Furthermore, in neurons of Alzheimer's disease patients, depletion of Pin1 is associated with hyperphosphorylated tau and neuronal loss (29). In addition, ESS1/PTF1 seems to play an important role in mRNA maturation, because its mutation leads to a defect in 3Ј-end formation of pre-mRNAs (13). Recently, a link between ESS1/PTF1 and the general transcription machinery via its interaction with the phosphorylated COOH-terminal domain of RNA polymerase II has been described (30).…”

“…Furthermore, Pin1 regulates the functions of its binding proteins, including inhibiting the mitosis-promoting activity of Cdc25C (28) and restoring the biological activity of Alzheimerassociated tau protein (29). Depletion or mutations of Pin1 induce premature mitotic entry and mitotic arrest in yeast, HeLa cells, and Xenopus egg extracts (11,13,16,17,28). Pin1 is also required for the replication checkpoint in Xenopus extracts (16).…”

“…Among this family are the highly conserved Pin1-type PPIases (10,11), which are the only PPIases that seem to be essential for cell survival, at least in budding yeast and HeLa cells. The Pin1-type PPIases, such as the human Pin1 (11), Saccharomyces cerevisiae ESS1/Ptf1 (10,12,13), Drosophila Dodo (14), Neurospora crassa Ssp1 (15), Xenopus Pin1 (16), Aspergillus nidulans Pin1 (17), and others, consist of an amino-terminal WW domain and a carboxyl-terminal PPIase domain. WW domains, which are characterized by two invariant tryptophans, are present in a variety of signaling and regulatory proteins and were originally identified as protein interaction modules that bind to proline-rich regions in protein targets (18,19).…”

Phospho-Carboxyl-Terminal Domain Binding and the Role of a Prolyl Isomerase in Pre-mRNA 3′-End Formation

“…All three proteins have a small 31-residue WW domain amino-terminal to the isomerase domain. Studies of the peptidyl prolyl isomerase activity of recombinant Ess1 (21) and Pin1 (26) indicate these enzymes display more than a 1000-fold higher specificity toward substrates in which the other amino acid at the isomerizable bond is negatively charged (Glu, phospho-Ser, and phospho-Thr). In higher eukaryotes a variety of techniques have been used to show that Pin1 stably associates with phosphorylated but not unphosphorylated mitotic regulators that are targets of Cdc2/ cyclinB (26 -29).…”

“…Recently, mutations in the peptidyl prolyl isomerase, ESS1/ PTF1, were found to be responsible for the temperature-sensitive phenotypes of two yeast mutants that displayed a pre-mRNA 3Ј-end formation defect (21). Yeast ESS1 was originally identified as essential gene (22), and is in fact the only essential peptidyl prolyl isomerase (PPIase) in Saccharomyces cerevisiae (23).…”