Sec61 channel subunit Sbh1/Sec61ß promotes ER translocation of proteins with suboptimal targeting sequences and is fine-tuned by phosphorylation.

The highly conserved endoplasmic reticulum (ER) protein translocation channel contains one nonessential subunit, Sec61ß/Sbh1, whose function is poorly understood so far. Its intrinsically unstructured cytosolic domain makes transient contact with ER-targeting sequences in the cytosolic channel vestibule and contains multiple phosphorylation sites suggesting a potential for regulating ER protein import. In a microscopic screen, we show that 12% of a GFP-tagged secretory protein library depends on Sbh1 for translocation into the ER. Sbh1-dependent proteins had targeting sequences with less pronounced hydrophobicity and often no charge bias or an inverse charge bias which reduces their insertion efficiency into the Sec61 channel. We determined that mutating two N-terminal, proline-flanked phosphorylation sites in the Sbh1 cytosolic domain to alanine phenocopied the temperature-sensitivity of a yeast strain lacking SBH1 and its ortholog SBH2. The phosphorylation site mutations reduced translocation into the ER of a subset of Sbh1-dependent proteins, including enzymes whose concentration in the ER lumen is critical for ER proteostasis. In addition, we found that ER import of these proteins depended on the activity of the phospho-S/T-specific proline isomerase Ess1 (PIN1 in mammals). We conclude that Sbh1 promotes ER translocation of substrates with suboptimal targeting sequences and that its activity can be regulated by a conformational change induced by N-terminal phosphorylation.

A universal RNA polymerase II CTD cycle is orchestrated by complex interplays between kinase, phosphatase, and isomerase enzymes along genes.

Transcription by RNA polymerase II (RNAPII) is coupled to mRNA processing and chromatin modifications via the C-terminal domain (CTD) of its largest subunit, consisting of multiple repeats of the heptapeptide YSPTSPS. Pioneering studies showed that CTD serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Genome-wide analyses challenged this idea, suggesting that this cycle is not uniform among different genes. Moreover, the respective role of enzymes responsible for CTD modifications remains controversial. Here, we systematically profiled the location of the RNAPII phosphoisoforms in wild-type cells and mutants for most CTD modifying enzymes. Together with results of in vitro assays, these data reveal a complex interplay between the modifying enzymes, and provide evidence that the CTD cycle is uniform across genes. We also identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes.

The Ess1 prolyl isomerase is required for transcription termination of small noncoding RNAs via the Nrd1 pathway.

Genome-wide studies have identified abundant small, noncoding RNAs, including small nuclear RNAs, small nucleolar RNAs (snoRNAs), cryptic unstable transcripts (CUTs), and upstream regulatory RNAs (uRNAs), that are transcribed by RNA polymerase II (pol II) and terminated by an Nrd1-dependent pathway. Here, we show that the prolyl isomerase Ess1 is required for Nrd1-dependent termination of noncoding RNAs. Ess1 binds the carboxy-terminal domain (CTD) of pol II and is thought to regulate transcription by conformational isomerization of Ser-Pro bonds within the CTD. In ess1 mutants, expression of approximately 10% of the genome was altered, due primarily to defects in termination of snoRNAs, CUTs, stable unannotated transcripts, and uRNAs. Ess1 promoted dephosphorylation of Ser5 (but not Ser2) within the CTD, most likely by the Ssu72 phosphatase. We also provide evidence for a competition between Nrd1 and Pcf11 for CTD binding that is regulated by Ess1. These data indicate that a prolyl isomerase is required for specifying the "CTD code."

Prolyl isomerases in gene transcription.

BACKGROUND: Peptidyl-prolyl isomerases (PPIases) are enzymes that assist in the folding of newly-synthesized proteins and regulate the stability, localization, and activity of mature proteins. They do so by catalyzing reversible (cis-trans) rotation about the peptide bond that precedes proline, inducing conformational changes in target proteins. SCOPE OF REVIEW: This review will discuss how PPIases regulate gene transcription by controlling the activity of (1) DNA-binding transcription regulatory proteins, (2) RNA polymerase II, and (3) chromatin and histone modifying enzymes. MAJOR CONCLUSIONS: Members of each family of PPIase (cyclophilins, FKBPs, and parvulins) regulate gene transcription at multiple levels. In all but a few cases, the exact mechanisms remain elusive. Structure studies, development of specific inhibitors, and new methodologies for studying cis/trans isomerization in vivo represent some of the challenges in this new frontier that merges two important fields. GENERAL SIGNIFICANCE: Prolyl isomerases have been found to play key regulatory roles in all phases of the transcription process. Moreover, PPIases control upstream signaling pathways that regulate gene-specific transcription during development, hormone response and environmental stress. Although transcription is often rate-limiting in the production of enzymes and structural proteins, post-transcriptional modifications are also critical, and PPIases play key roles here as well (see other reviews in this issue). This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.

Rpb4/7 facilitates RNA polymerase II CTD dephosphorylation.

The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAPII) participate in a variety of processes from transcription, DNA repair, mRNA export and decay, to translation regulation and stress response. However, their mechanism(s) of action remains unclear. Here, we show that the Rpb4/7 heterodimer in Saccharomyces cerevisiae plays a key role in controlling phosphorylation of the carboxy terminal domain (CTD) of the Rpb1 subunit of RNAPII. Proper phosphorylation of the CTD is critical for the synthesis and processing of RNAPII transcripts. Deletion of RPB4, and mutations that disrupt the integrity of Rpb4/7 or its recruitment to the RNAPII complex, increased phosphorylation of Ser2, Ser5, Ser7 and Thr4 within the CTD. RPB4 interacted genetically with genes encoding CTD phosphatases (SSU72, FCP1), CTD kinases (KIN28, CTK1, SRB10) and a prolyl isomerase that targets the CTD (ESS1). We show that Rpb4 is important for Ssu72 and Fcp1 phosphatases association, recruitment and/or accessibility to the CTD, and that this correlates strongly with Ser5P and Ser2P levels, respectively. Our data also suggest that Fcp1 is the Thr4P phosphatase in yeast. Based on these and other results, we suggest a model in which Rpb4/7 helps recruit and potentially stimulate the activity of CTD-modifying enzymes, a role that is central to RNAPII function.

The Ess1 prolyl isomerase: traffic cop of the RNA polymerase II transcription cycle.

Ess1 is a prolyl isomerase that regulates the structure and function of eukaryotic RNA polymerase II. Ess1 works by catalyzing the cis/trans conversion of pSer5-Pro6 bonds, and to a lesser extent pSer2-Pro3 bonds, within the carboxy-terminal domain (CTD) of Rpb1, the largest subunit of RNA pol II. Ess1 is conserved in organisms ranging from yeast to humans. In budding yeast, Ess1 is essential for growth and is required for efficient transcription initiation and termination, RNA processing, and suppression of cryptic transcription. In mammals, Ess1 (called Pin1) functions in a variety of pathways, including transcription, but it is not essential. Recent work has shown that Ess1 coordinates the binding and release of CTD-binding proteins that function as co-factors in the RNA pol II complex. In this way, Ess1 plays an integral role in writing (and reading) the so-called CTD code to promote production of mature RNA pol II transcripts including non-coding RNAs and mRNAs.

Catalytic activity of the Bin3/MePCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster.

Methylphosphate Capping Enzyme (MePCE) monomethylates the gamma phosphate at the 5' end of the 7SK noncoding RNA, a modification thought to protect 7SK from degradation. 7SK serves as a scaffold for assembly of a snRNP complex that inhibits transcription by sequestering the positive elongation factor P-TEFb. While much is known about the biochemical activity of MePCE in vitro, little is known about its functions in vivo, or what roles-if any-there are for regions outside the conserved methyltransferase domain. Here, we investigated the role of Bin3, the Drosophila ortholog of MePCE, and its conserved functional domains in Drosophila development. We found that bin3 mutant females had strongly reduced rates of egg-laying, which was rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 promotes fecundity by repressing P-TEFb. bin3 mutants also exhibited neuromuscular defects, analogous to a patient with MePCE haploinsufficiency. These defects were also rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 and MePCE have conserved roles in promoting neuromuscular function by repressing P-TEFb. Unexpectedly, we found that a Bin3 catalytic mutant (Bin3Y795A) could still bind and stabilize 7SK and rescue all bin3 mutant phenotypes, indicating that Bin3 catalytic activity is dispensable for 7SK stability and snRNP function in vivo. Finally, we identified a metazoan-specific motif (MSM) outside of the methyltransferase domain and generated mutant flies lacking this motif (Bin3ΔMSM). Bin3ΔMSM mutant flies exhibited some-but not all-bin3 mutant phenotypes, suggesting that the MSM is required for a 7SK-independent, tissue-specific function of Bin3.

Catalytic activity of the Bin3/MEPCE methyltransferase domain is dispensable for 7SK snRNP function in Drosophila melanogaster.

Methylphosphate Capping Enzyme (MEPCE) monomethylates the gamma phosphate at the 5' end of the 7SK noncoding RNA, a modification thought to protect 7SK from degradation. 7SK serves as a scaffold for assembly of a snRNP complex that inhibits transcription by sequestering the positive elongation factor P-TEFb. While much is known about the biochemical activity of MEPCE in vitro, little is known about its functions in vivo, or what roles- if any-there are for regions outside the conserved methyltransferase domain. Here, we investigated the role of Bin3, the Drosophila ortholog of MEPCE, and its conserved functional domains in Drosophila development. We found that bin3 mutant females had strongly reduced rates of egg-laying, which was rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 promotes fecundity by repressing P-TEFb. bin3 mutants also exhibited neuromuscular defects, analogous to a patient with MEPCE haploinsufficiency. These defects were also rescued by genetic reduction of P-TEFb activity, suggesting that Bin3 and MEPCE have conserved roles in promoting neuromuscular function by repressing P-TEFb. Unexpectedly, we found that a Bin3 catalytic mutant (Bin3Y795A) could still bind and stabilize 7SK and rescue all bin3 mutant phenotypes, indicating that Bin3 catalytic activity is dispensable for 7SK stability and snRNP function in vivo. Finally, we identified a metazoan-specific motif (MSM) outside of the methyltransferase domain and generated mutant flies lacking this motif (Bin3ΔMSM). Bin3ΔMSM mutant flies exhibited some-but not all-bin3 mutant phenotypes, suggesting that the MSM is required for a 7SK-independent, tissue-specific function of Bin3.

The Bin3 RNA methyltransferase is required for repression of caudal translation in the Drosophila embryo.

Bin3 was first identified as a Bicoid-interacting protein in a yeast two-hybrid screen. In human cells, a Bin3 ortholog (BCDIN3) methylates the 5' end of 7SK RNA, but its role in vivo is unknown. Here, we show that in Drosophila, Bin3 is important for dorso-ventral patterning in oogenesis and for anterior-posterior pattern formation during embryogenesis. Embryos that lack Bin3 fail to repress the translation of caudal mRNA and exhibit head involution defects. bin3 mutants also show (1) a severe reduction in the level of 7SK RNA, (2) reduced binding of Bicoid to the caudal 3' UTR, and (3) genetic interactions with bicoid, and with genes encoding eIF4E, Larp1, polyA binding protein (PABP), and Ago2. 7SK RNA coimmunoprecipitated with Bin3 and is present in Bicoid complexes. These data suggest a model in which Bicoid recruits Bin3 to the caudal 3' UTR. Bin3's role is to bind and stabilize 7SK RNA, thereby promoting formation of a repressive RNA-protein complex that includes the RNA-binding proteins Larp1, PABP, and Ago2. This complex would prevent translation by blocking eIF4E interactions required for initiation. Our results, together with prior network analysis in human cells, suggest that Bin3 interacts with multiple partner proteins, methylates small non-coding RNAs, and plays diverse roles in development.

Coevolution of the Ess1-CTD axis in polar fungi suggests a role for phase separation in cold tolerance.

Most of the world's biodiversity lives in cold (-2° to 4°C) and hypersaline environments. To understand how cells adapt to such conditions, we isolated two key components of the transcription machinery from fungal species that live in extreme polar environments: the Ess1 prolyl isomerase and its target, the carboxy-terminal domain (CTD) of RNA polymerase II. Polar Ess1 enzymes are conserved and functional in the model yeast, Saccharomyces cerevisiae. By contrast, polar CTDs diverge from the consensus (YSPTSPS)26 and are not fully functional in S. cerevisiae. These CTDs retain the critical Ess1 Ser-Pro target motifs, but substitutions at Y1, T4, and S7 profoundly affected their ability to undergo phase separation in vitro and localize in vivo. We propose that environmentally tuned phase separation by the CTD and other intrinsically disordered regions plays an adaptive role in cold tolerance by concentrating enzymes and substrates to overcome energetic barriers to metabolic activity.

Restricted domain mobility in the Candida albicans Ess1 prolyl isomerase.

Ess1 is a peptidyl prolyl cis/trans isomerase that is required for virulence of the pathogenic fungi Candida albicans and Cryptococcus neoformans. The enzyme isomerizes the phospho-Ser-Pro linkages in the C-terminal domain of RNA polymerase II. Its human homolog, Pin1, has been implicated in a wide range of human diseases, including cancer and Alzheimer's disease. Crystallographic and NMR studies have demonstrated that the sequence linking the catalytic isomerase domain and the substrate binding WW domain of Pin1 is unstructured and that the two domains are only loosely associated in the absence of the substrate. In contrast, the crystal structure of C. albicans Ess1 revealed a highly ordered linker that contains a three turn alpha-helix and extensive association between the two tightly juxtaposed domains. In part to address the concern that the marked differences in the domain interactions for the human and fungal structures might reflect crystal lattice effects, NMR chemical shift analysis and 15N relaxation measurements have been employed to confirm that the linker of the fungal protein is highly ordered in solution. With the exception of two loops within the active site of the isomerase domain, the local backbone geometry observed in the crystal structure appears to be well preserved throughout the protein chain. The marked differences in interdomain interactions and linker flexibility between the human and fungal enzymes provide a structural basis for therapeutic targeting of the fungal enzymes.

Milestones in Candida albicans gene manipulation.

In the United States, candidemia is one of the most common hospital-acquired infections and is estimated to cause 10,000 deaths per year. The species Candida albicans is responsible for the majority of these cases. As C. albicans is capable of developing resistance against the currently available drugs, understanding the molecular basis of drug resistance, finding new cellular targets, and further understanding the overall mechanism of C. albicans pathogenesis are important goals. To study this pathogen it is advantageous to manipulate its genome. Numerous strategies of C. albicans gene manipulation have been introduced. This review evaluates a majority of these strategies and should be a helpful guide for researchers to identify gene targeting strategies to suit their requirements.

Structure analysis suggests Ess1 isomerizes the carboxy-terminal domain of RNA polymerase II via a bivalent anchoring mechanism.

Accurate gene transcription in eukaryotes depends on isomerization of serine-proline bonds within the carboxy-terminal domain (CTD) of RNA polymerase II. Isomerization is part of the "CTD code" that regulates recruitment of proteins required for transcription and co-transcriptional RNA processing. Saccharomyces cerevisiae Ess1 and its human ortholog, Pin1, are prolyl isomerases that engage the long heptad repeat (YSPTSPS)26 of the CTD by an unknown mechanism. Here, we used an integrative structural approach to decipher Ess1 interactions with the CTD. Ess1 has a rigid linker between its WW and catalytic domains that enforces a distance constraint for bivalent interaction with the ends of long CTD substrates (≥4-5 heptad repeats). Our binding results suggest that the Ess1 WW domain anchors the proximal end of the CTD substrate during isomerization, and that linker divergence may underlie evolution of substrate specificity.

The Drosophila calcipressin sarah is required for several aspects of egg activation.

Activation of mature oocytes initiates development by releasing the prior arrest of female meiosis, degrading certain maternal mRNAs while initiating the translation of others, and modifying egg coverings. In vertebrates and marine invertebrates, the fertilizing sperm triggers activation events through a rise in free calcium within the egg. In insects, egg activation occurs independently of sperm and is instead triggered by passage of the egg through the female reproductive tract ; it is unknown whether calcium signaling is involved. We report here that mutations in sarah, which encodes an inhibitor of the calcium-dependent phosphatase calcineurin, disrupt several aspects of egg activation in Drosophila. Eggs laid by sarah mutant females arrest in anaphase of meiosis I and fail to fully polyadenylate and translate bicoid mRNA. Furthermore, sarah mutant eggs show elevated cyclin B levels, indicating a failure to inactivate M-phase promoting factor (MPF). Taken together, these results demonstrate that calcium signaling is involved in Drosophila egg activation and suggest a molecular mechanism for the sarah phenotype. We also find the conversion of the sperm nucleus into a functional male pronucleus is compromised in sarah mutant eggs, indicating that the Drosophila egg's competence to support male pronuclear maturation is acquired during activation.

dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element.

It was described earlier that the Drosophila GAGA factor [Trithorax-like (Trl)] interacts with dSAP18, which, in mammals, was reported to be a component of the Sin3-HDAC co-repressor complex. GAGA-dSAP18 interaction was proposed to contribute to the functional regulation of the bithorax complex (BX-C). Here, we show that mutant alleles of Trl, dsap18 and drpd3/hdac1 enhance A6-to-A5 transformation indicating a contribution to the regulation of Abd-B expression at A6. In A6, expression of Abd-B is driven by the iab-6 enhancer, which is insulated from iab-7 by the Fab-7 element. Here, we report that GAGA, dSAP18 and dRPD3/HDAC1 co-localize to ectopic Fab-7 sites in polytene chromosomes and that mutant Trl, dsap18 and drpd3/hdac1 alleles affect Fab-7-dependent silencing. Consistent with these findings, chromatin immunoprecipitation analysis shows that, in Drosophila embryos, the endogenous Fab-7 element is hypoacetylated at histones H3 and H4. These results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the regulation of Fab-7 function.

Genetic interactions with C-terminal domain (CTD) kinases and the CTD of RNA Pol II suggest a role for ESS1 in transcription initiation and elongation in Saccharomyces cerevisiae.

Ess1 is an essential prolyl isomerase that binds the C-terminal domain (CTD) of Rpb1, the large subunit of RNA polymerase II. Ess1 is proposed to control transcription by isomerizing phospho-Ser-Pro peptide bonds within the CTD repeat. To determine which step(s) in the transcription cycle might require Ess1, we examined genetic interactions between ESS1 and genes encoding the known CTD kinases (KIN28, CTK1, BUR1, and SRB10). Although genetic interactions were identified between ESS1 and all four kinases, the clearest interactions were with CTK1 and SRB10. Reduced dosage of CTK1 rescued the growth defect of ess1(ts) mutants, while overexpression of CTK1 enhanced the growth defects of ess1(ts) mutants. Deletion of SRB10 suppressed ess1(ts) and ess1Delta mutants. The interactions suggest that Ess1 opposes the functions of these kinases, which are thought to function in preinitiation and elongation. Using a series of CTD substitution alleles, we also identified Ser5-Pro6 as a potential target for Ess1 isomerization within the first "half" of the CTD repeats. On the basis of the results, we suggest a model in which Ess1-directed conformational changes promote dephosphorylation of Ser5 to stimulate preinitiation complex formation and, later, to inhibit elongation.

The Ess1 prolyl isomerase is required for growth and morphogenetic switching in Candida albicans.

Prolyl-isomerases (PPIases) are found in all organisms and are important for the folding and activity of many proteins. Of the 13 PPIases in Saccharomyces cerevisiae only Ess1, a parvulin-class PPIase, is essential for growth. Ess1 is required to complete mitosis, and Ess1 and its mammalian homolog, Pin1, interact directly with RNA polymerase II. Here, we isolate the ESS1 gene from the pathogenic fungus Candida albicans and show that it is functionally homologous to the S. cerevisiae ESS1. We generate conditional-lethal (ts) alleles of C. albicans ESS1 and use these mutations to demonstrate that ESS1 is essential for growth in C. albicans. We also show that reducing the dosage or activity of ESS1 blocks morphogenetic switching from the yeast to the hyphal and pseudohyphal forms under certain conditions. Analysis of double mutants of ESS1 and TUP1 or CPH1, two genes known to be involved in morphogenetic switching, suggests that ESS1 functions in the same pathway as CPH1 and upstream of or in parallel to TUP1. Given that switching is important for virulence of C. albicans, inhibitors of Ess1 might be useful as antifungal agents.

The ESS1 prolyl isomerase and its suppressor BYE1 interact with RNA pol II to inhibit transcription elongation in Saccharomyces cerevisiae.

Transcription by RNA polymerase II (pol II) requires the ordered binding of distinct protein complexes to catalyze initiation, elongation, termination, and coupled mRNA processing events. One or more proteins from each complex are known to bind pol II via the carboxy-terminal domain (CTD) of the largest subunit, Rpb1. How binding is coordinated is not known, but it might involve conformational changes in the CTD induced by the Ess1 peptidyl-prolyl cis/trans isomerase. Here, we examined the role of ESS1 in transcription by studying one of its multicopy suppressors, BYE1. We found that Bye1 is a negative regulator of transcription elongation. This led to the finding that Ess1 also inhibits elongation; Ess1 opposes elongation factors Dst1 and Spt4/5, and overexpression of ESS1 makes cells more sensitive to the elongation inhibitor 6-AU. In reporter gene assays, ess1 mutations reduce the ability of elongation-arrest sites to stall polymerase. We also show that Ess1 acts positively in transcription termination, independent of its role in elongation. We propose that Ess1-induced conformational changes attenuate pol II elongation and help coordinate the ordered assembly of protein complexes on the CTD. In this way, Ess1 might regulate the transition between multiple steps of transcription.

Prolyl isomerases in yeast.

Prolyl isomerases are enzymes that catalyze cis-trans isomerization of peptidyl-prolyl bonds and span three structurally unrelated protein families: the cyclophilins, FKBPs, and parvulins. The genome of the budding yeast Saccharomyces cerevisiae encodes eight different cyclophilins (Cpr1 to Cpr8), four FKBPs (Fpr1 to Fpr4), and a single parvulin (Ess1). Remarkably, two of these proteins, cyclophilin A and FKBP12, are conserved from yeast to humans and mediate virtually all of the intracellular actions of the immunosuppressive antifungal drugs cyclosporin A, FK506, and rapamycin. The study of prolyl isomerases in S. cerevisiae has proven invaluable to understand the elusive functions of these proteins, and continues to provide new insights into their diverse cellular roles. Here we review the current state of knowledge about prolyl-isomerases in this model organism.

The yeast Ess1 prolyl isomerase controls Swi6 and Whi5 nuclear localization.

The Ess1 prolyl isomerase from Saccharomyces cerevisiae and its human ortholog, Pin1, play critical roles in transcription by regulating RNA polymerase II. In human cells, Pin1 also regulates a variety of signaling proteins, and Pin1 misexpression is linked to several human diseases. To gain insight into Ess1/Pin1 function, we carried out a synthetic genetic array screen to identify novel targets of Ess1 in yeast. We identified potential targets of Ess1 in transcription, stress, and cell-cycle pathways. We focused on the cell-cycle regulators Swi6 and Whi5, both of which show highly regulated nucleocytoplasmic shuttling during the cell cycle. Surprisingly, Ess1 did not control their transcription but instead was necessary for their nuclear localization. Ess1 associated with Swi6 and Whi5 in vivo and bound directly to peptides corresponding to their nuclear localization sequences in vitro. Binding by Ess1 was significant only if the Swi6 and Whi5 peptides were phosphorylated at Ser-Pro motifs, the target sites of cyclin-dependent kinases. On the basis of these results, we propose a model in which Ess1 induces a conformational switch (cis-trans isomerization) at phospho-Ser-Pro sites within the nuclear targeting sequences of Swi6 and Whi5. This switch would promote nuclear entry and/or retention during late M and G1 phases and might work by stimulating dephosphorylation at these sites by the Cdc14 phosphatase. This is the first study to identify targets of Ess1 in yeast other than RNA polymerase II.