Maf1 phosphorylation is regulated through the action of prefoldin-like Bud27 on PP4 phosphatase in Saccharomyces cerevisiae.

Bud27 is a prefoldin-like protein that participates in transcriptional regulation mediated by the three RNA polymerases in Saccharomyces cerevisiae. Lack of Bud27 significantly affects RNA pol III transcription, although the involved mechanisms have not been characterized. Here, we show that Bud27 regulates the phosphorylation state of the RNA pol III transcriptional repressor, Maf1, influences its nuclear localization, and likely its activity. We demonstrate that Bud27 is associated with the Maf1 main phosphatase PP4 in vivo, and that this interaction is required for proper Maf1 dephosphorylation. Lack of Bud27 decreases the interaction among PP4 and Maf1, Maf1 dephosphorylation, and its nuclear entry. Our data uncover a new nuclear function of Bud27, identify PP4 as a novel Bud27 interactor and demonstrate the effect of this prefoldin-like protein on the posttranslational regulation of Maf1. Finally, our data reveal a broader effect of Bud27 on PP4 activity by influencing, at least, the phosphorylation of Rad53.

Structural basis of Ty1 integrase tethering to RNA polymerase III for targeted retrotransposon integration.

The yeast Ty1 retrotransposon integrates upstream of genes transcribed by RNA polymerase III (Pol III). Specificity of integration is mediated by an interaction between the Ty1 integrase (IN1) and Pol III, currently uncharacterized at the atomic level. We report cryo-EM structures of Pol III in complex with IN1, revealing a 16-residue segment at the IN1 C-terminus that contacts Pol III subunits AC40 and AC19, an interaction that we validate by in vivo mutational analysis. Binding to IN1 associates with allosteric changes in Pol III that may affect its transcriptional activity. The C-terminal domain of subunit C11, involved in RNA cleavage, inserts into the Pol III funnel pore, providing evidence for a two-metal mechanism during RNA cleavage. Additionally, ordering next to C11 of an N-terminal portion from subunit C53 may explain the connection between these subunits during termination and reinitiation. Deletion of the C53 N-terminal region leads to reduced chromatin association of Pol III and IN1, and a major fall in Ty1 integration events. Our data support a model in which IN1 binding induces a Pol III configuration that may favor its retention on chromatin, thereby improving the likelihood of Ty1 integration.

A proteomic screen of Ty1 integrase partners identifies the protein kinase CK2 as a regulator of Ty1 retrotransposition.

BACKGROUND: Transposable elements are ubiquitous and play a fundamental role in shaping genomes during evolution. Since excessive transposition can be mutagenic, mechanisms exist in the cells to keep these mobile elements under control. Although many cellular factors regulating the mobility of the retrovirus-like transposon Ty1 in Saccharomyces cerevisiae have been identified in genetic screens, only very few of them interact physically with Ty1 integrase (IN). RESULTS: Here, we perform a proteomic screen to establish Ty1 IN interactome. Among the 265 potential interacting partners, we focus our study on the conserved CK2 kinase. We confirm the interaction between IN and CK2, demonstrate that IN is a substrate of CK2 in vitro and identify the modified residues. We find that Ty1 IN is phosphorylated in vivo and that these modifications are dependent in part on CK2. No significant change in Ty1 retromobility could be observed when we introduce phospho-ablative mutations that prevent IN phosphorylation by CK2 in vitro. However, the absence of CK2 holoenzyme results in a strong stimulation of Ty1 retrotransposition, characterized by an increase in Ty1 mRNA and protein levels and a high accumulation of cDNA. CONCLUSION: Our study shows that Ty1 IN is phosphorylated, as observed for retroviral INs and highlights an important role of CK2 in the regulation of Ty1 retrotransposition. In addition, the proteomic approach enabled the identification of many new Ty1 IN interacting partners, whose potential role in the control of Ty1 mobility will be interesting to study.

Ty1 integrase is composed of an active N-terminal domain and a large disordered C-terminal module dispensable for its activity in vitro.

Long-terminal repeat (LTR) retrotransposons are genetic elements that, like retroviruses, replicate by reverse transcription of an RNA intermediate into a complementary DNA (cDNA) that is next integrated into the host genome by their own integrase. The Ty1 LTR retrotransposon has proven to be a reliable working model to investigate retroelement integration site preference. However, the low yield of recombinant Ty1 integrase production reported so far has been a major obstacle for structural studies. Here we analyze the biophysical and biochemical properties of a stable and functional recombinant Ty1 integrase highly expressed in E.coli. The recombinant protein is monomeric and has an elongated shape harboring the three-domain structure common to all retroviral integrases at the N-terminal half, an extra folded region, and a large intrinsically disordered region at the C-terminal half. Recombinant Ty1 integrase efficiently catalyzes concerted integration in vitro, and the N-terminal domain displays similar activity. These studies that will facilitate structural analyses may allow elucidating the molecular mechanisms governing Ty1 specific integration into safe places in the genome.

A small targeting domain in Ty1 integrase is sufficient to direct retrotransposon integration upstream of tRNA genes.

Integration of transposable elements into the genome is mutagenic. Mechanisms targeting integrations into relatively safe locations, hence minimizing deleterious consequences for cell fitness, have emerged during evolution. In budding yeast, integration of the Ty1 LTR retrotransposon upstream of RNA polymerase III (Pol III)-transcribed genes requires interaction between Ty1 integrase (IN1) and AC40, a subunit common to Pol I and Pol III. Here, we identify the Ty1 targeting domain of IN1 that ensures (i) IN1 binding to Pol I and Pol III through AC40, (ii) IN1 genome-wide recruitment to Pol I- and Pol III-transcribed genes, and (iii) Ty1 integration only at Pol III-transcribed genes, while IN1 recruitment by AC40 is insufficient to target Ty1 integration into Pol I-transcribed genes. Swapping the targeting domains between Ty5 and Ty1 integrases causes Ty5 integration at Pol III-transcribed genes, indicating that the targeting domain of IN1 alone confers Ty1 integration site specificity.

Identification of proteins associated with RNA polymerase III using a modified tandem chromatin affinity purification.

To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

Sub1 and Maf1, two effectors of RNA polymerase III, are involved in the yeast quiescence cycle.

Sub1 and Maf1 exert an opposite effect on RNA polymerase III transcription interfering with different steps of the transcription cycle. In this study, we present evidence that Sub1 and Maf1 also exhibit an opposite role on yeast chronological life span. First, cells lacking Sub1 need more time than wild type to exit from resting and this lag in re-proliferation is correlated with a delay in transcriptional reactivation. Second, our data show that the capacity of the cells to properly establish a quiescent state is impaired in the absence of Sub1 resulting in a premature death that is dependent on the Ras/PKA and Tor1/Sch9 signalling pathways. On the other hand, we show that maf1Δ cells are long-lived mutant suggesting a connection between Pol III transcription and yeast longevity.

Maf1, repressor of tRNA transcription, is involved in the control of gluconeogenetic genes in Saccharomyces cerevisiae.

Maf1 is a negative regulator of RNA polymerase III (Pol III) in yeast. Maf1-depleted cells manifest elevated tRNA transcription and inability to grow on non-fermentable carbon source, such as glycerol. Using genomic microarray approach, we examined the effect of Maf1 deletion on expression of Pol II-transcribed genes in yeast grown in medium containing glycerol. We found that transcription of FBP1 and PCK1, two major genes controlling gluconeogenesis, was decreased in maf1Δ cells. FBP1 is located on chromosome XII in close proximity to a tRNA-Lys gene. Accordingly we hypothesized that decreased FBP1 mRNA level could be due to the effect of Maf1 on tgm silencing (tRNA gene mediated silencing). Two approaches were used to verify this hypothesis. First, we inactivated tRNA-Lys gene on chromosome XII by inserting a deletion cassette in a control wild type strain and in maf1Δ mutant. Second, we introduced a point mutation in the promoter of the tRNA-Lys gene cloned with the adjacent FBP1 in a plasmid and expressed in fbp1Δ or fbp1Δ maf1Δ cells. The levels of FBP1 mRNA were determined by RT-qPCR in each strain. Although the inactivation of the chromosomal tRNA-Lys gene increased expression of the neighboring FBP1, the mutation preventing transcription of the plasmid-born tRNA-Lys gene had no significant effect on FBP1 transcription. Taken together, those results do not support the concept of tgm silencing of FBP1. Other possible mechanisms are discussed.

Yeast RNA polymerase III transcription factors and effectors.

Recent data indicate that the well-defined transcription machinery of RNA polymerase III (Pol III) is probably more complex than commonly thought. In this review, we describe the yeast basal transcription factors of Pol III and their involvements in the transcription cycle. We also present a list of proteins detected on genes transcribed by Pol III (class III genes) that might participate in the transcription process. Surprisingly, several of these proteins are involved in RNA polymerase II transcription. Defining the role of these potential new effectors in Pol III transcription in vivo will be the challenge of the next few years. This article is part of a Special Issue entitled: Transcription by Odd Pols.

Sub1/PC4 a chromatin associated protein with multiple functions in transcription.

Yeast Sub1 and its human ortholog PC4 display multiple cellular functions in vivo. Sub1/PC4 contains a unique conserved non-specific DNA-binding domain and is involved in distinct DNA-dependent processes including replication, DNA repair and transcription. Sub1/PC4 is a non-histone chromatin-associated protein initially described as a co-activator for RNA polymerase (Pol) II transcription in vitro. Recently, biochemical and genomic studies showed that Sub1 is not restricted to Pol II transcription but is also involved in Pol III transcription revealing a more general role in transcription than anticipated. Sub1/PC4 appears to play a dual (positive and negative) complex role in gene expression and has multiple effects in distinct steps of the transcription cycle, consisting of initiation, elongation, termination and reinitiation. Here, the multiple transcriptional functions of Sub1/PC4 will be reviewed and the recent findings and their possible implications in the understanding of Sub1/PC4 function in transcription will be discussed.

Genome-wide location analysis reveals a role for Sub1 in RNA polymerase III transcription.

Human PC4 and the yeast ortholog Sub1 have multiple functions in RNA polymerase II transcription. Genome-wide mapping revealed that Sub1 is present on Pol III-transcribed genes. Sub1 was found to interact with components of the Pol III transcription system and to stimulate the initiation and reinitiation steps in a system reconstituted with all recombinant factors. Sub1 was required for optimal Pol III gene transcription in exponentially growing cells.

Maf1 is involved in coupling carbon metabolism to RNA polymerase III transcription.

RNA polymerase III (Pol III) produces essential components of the biosynthetic machinery, and therefore its activity is tightly coupled with cell growth and metabolism. In the yeast Saccharomyces cerevisiae, Maf1 is the only known global and direct Pol III transcription repressor which mediates numerous stress signals. Here we demonstrate that transcription regulation by Maf1 is not limited to stress but is important for the switch between fermentation and respiration. Under respiratory conditions, Maf1 is activated by dephosphorylation and imported into the nucleus. The transition from a nonfermentable carbon source to that of glucose induces Maf1 phosphorylation and its relocation to the cytoplasm. The absence of Maf1-mediated control of tRNA synthesis impairs cell viability in nonfermentable carbon sources. The respiratory phenotype of maf1-Delta allowed genetic suppression studies to dissect the mechanism of Maf1 action on the Pol III transcription apparatus. Moreover, in cells grown in a nonfermentable carbon source, Maf1 regulates the levels of different tRNAs to various extents. The differences in regulation may contribute to the physiological role of Maf1.

Modulation of yeast genome expression in response to defective RNA polymerase III-dependent transcription.

We used genome-wide expression analysis in Saccharomyces cerevisiae to explore whether and how the expression of protein-coding, RNA polymerase (Pol) II-transcribed genes is influenced by a decrease in RNA Pol III-dependent transcription. The Pol II transcriptome was characterized in four thermosensitive, slow-growth mutants affected in different components of the RNA Pol III transcription machinery. Unexpectedly, we found only a modest correlation between altered expression of Pol II-transcribed genes and their proximity to class III genes, a result also confirmed by the analysis of single tRNA gene deletants. Instead, the transcriptome of all of the four mutants was characterized by increased expression of genes known to be under the control of the Gcn4p transcriptional activator. Indeed, GCN4 was found to be translationally induced in the mutants, and deleting the GCN4 gene eliminated the response. The Gcn4p-dependent expression changes did not require the Gcn2 protein kinase and could be specifically counteracted by an increased gene dosage of initiator tRNA(Met). Initiator tRNA(Met) depletion thus triggers a GCN4-dependent reprogramming of genome expression in response to decreased Pol III transcription. Such an effect might represent a key element in the coordinated transcriptional response of yeast cells to environmental changes.

An Rpb4/Rpb7-like complex in yeast RNA polymerase III contains the orthologue of mammalian CGRP-RCP.

The essential C17 subunit of yeast RNA polymerase (Pol) III interacts with Brf1, a component of TFIIIB, suggesting a role for C17 in the initiation step of transcription. The protein sequence of C17 (encoded by RPC17) is conserved from yeasts to humans. However, mammalian homologues of C17 (named CGRP-RCP) are known to be involved in a signal transduction pathway related to G protein-coupled receptors, not in transcription. In the present work, we first establish that human CGRP-RCP is the genuine orthologue of C17. CGRP-RCP was found to functionally replace C17 in Deltarpc17 yeast cells; the purified mutant Pol III contained CGRP-RCP and had a decreased specific activity but initiated faithfully. Furthermore, CGRP-RCP was identified by mass spectrometry in a highly purified human Pol III preparation. These results suggest that CGRP-RCP has a dual function in mammals. Next, we demonstrate by genetic and biochemical approaches that C17 forms with C25 (encoded by RPC25) a heterodimer akin to Rpb4/Rpb7 in Pol II. C17 and C25 were found to interact genetically in suppression screens and physically in coimmunopurification and two-hybrid experiments. Sequence analysis and molecular modeling indicated that the C17/C25 heterodimer likely adopts a structure similar to that of the archaeal RpoE/RpoF counterpart of the Rpb4/Rpb7 complex. These RNA polymerase subunits appear to have evolved to meet the distinct requirements of the multiple forms of RNA polymerases.