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.

Transcription by the Three RNA Polymerases under the Control of the TOR Signaling Pathway in Saccharomyces cerevisiae.

Ribosomes are the basis for protein production, whose biogenesis is essential for cells to drive growth and proliferation. Ribosome biogenesis is highly regulated in accordance with cellular energy status and stress signals. In eukaryotic cells, response to stress signals and the production of newly-synthesized ribosomes require elements to be transcribed by the three RNA polymerases (RNA pols). Thus, cells need the tight coordination of RNA pols to adjust adequate components production for ribosome biogenesis which depends on environmental cues. This complex coordination probably occurs through a signaling pathway that links nutrient availability with transcription. Several pieces of evidence strongly support that the Target of Rapamycin (TOR) pathway, conserved among eukaryotes, influences the transcription of RNA pols through different mechanisms to ensure proper ribosome components production. This review summarizes the connection between TOR and regulatory elements for the transcription of each RNA pol in the budding yeast Saccharomyces cerevisiae. It also focuses on how TOR regulates transcription depending on external cues. Finally, it discusses the simultaneous coordination of the three RNA pols through common factors regulated by TOR and summarizes the most important similarities and differences between S. cerevisiae and mammals.

Emerging Role of Plant-Based Dietary Components in Post-Translational Modifications Associated with Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide. Its main modifiable risk factors are diet, alcohol consumption, and smoking. Thus, the right approach through lifestyle changes may lead to its prevention. In fact, some natural dietary components have exhibited chemopreventive activity through modulation of cellular processes involved in CRC development. Although cancer is a multi-factorial process, the study of post-translational modifications (PTMs) of proteins associated with CRC has recently gained interest, as inappropriate modification is closely related to the activation of cell signalling pathways involved in carcinogenesis. Therefore, this review aimed to collect the main PTMs associated with CRC, analyse the relationship between different proteins that are susceptible to inappropriate PTMs, and review the available scientific literature on the role of plant-based dietary compounds in modulating CRC-associated PTMs. In summary, this review suggested that some plant-based dietary components such as phenols, flavonoids, lignans, terpenoids, and alkaloids may be able to correct the inappropriate PTMs associated with CRC and promote apoptosis in tumour cells.

A High-Copy Suppressor Screen Reveals a Broad Role of Prefoldin-like Bud27 in the TOR Signaling Pathway in Saccharomyces cerevisiae.

Bud27 is a prefoldin-like, a member of the family of ATP-independent molecular chaperones that associates with RNA polymerases I, II, and III in Saccharomyces cerevisiae. Bud27 and its human ortholog URI perform several functions in the cytoplasm and the nucleus. Both proteins participate in the TOR signaling cascade by coordinating nutrient availability with gene expression, and lack of Bud27 partially mimics TOR pathway inactivation. Bud27 regulates the transcription of the three RNA polymerases to mediate the synthesis of ribosomal components for ribosome biogenesis through the TOR cascade. This work presents a high-copy suppression screening of the temperature sensitivity of the bud27Δ mutant. It shows that Bud27 influences different TOR-dependent processes. Our data also suggest that Bud27 can impact some of these TOR-dependent processes: cell wall integrity and autophagy induction.

The Association of Rpb4 with RNA Polymerase II Depends on CTD Ser5P Phosphatase Rtr1 and Influences mRNA Decay in Saccharomyces cerevisiae.

Rtr1 is an RNA polymerase II (RNA pol II) CTD-phosphatase that influences gene expression during the transition from transcription initiation to elongation and during transcription termination. Rtr1 interacts with the RNA pol II and this interaction depends on the phosphorylation state of the CTD of Rpb1, which may influence dissociation of the heterodimer Rpb4/7 during transcription. In addition, Rtr1 was proposed as an RNA pol II import factor in RNA pol II biogenesis and participates in mRNA decay by autoregulating the turnover of its own mRNA. Our work shows that Rtr1 acts in RNA pol II assembly by mediating the Rpb4/7 association with the rest of the enzyme. RTR1 deletion alters RNA pol II assembly and increases the amount of RNA pol II associated with the chromatin that lacks Rpb4, decreasing Rpb4-mRNA imprinting and, consequently, increasing mRNA stability. Thus, Rtr1 interplays RNA pol II biogenesis and mRNA decay regulation. Our data also indicate that Rtr1 mediates mRNA decay regulation more broadly than previously proposed by cooperating with Rpb4. Interestingly, our data include new layers in the mechanisms of gene regulation and in the crosstalk between mRNA synthesis and decay by demonstrating how the association of Rpb4/7 to the RNA pol II influences mRNA decay.

Synergistic Effect of Squalene and Hydroxytyrosol on Highly Invasive MDA-MB-231 Breast Cancer Cells.

Several studies relate Mediterranean diet and virgin olive oil (VOO) intake with lower risk of several chronic diseases, including breast cancer. Many of them described antitumor properties of isolated minor compounds present in VOO, but beneficial properties of VOO arise from the effects of all its compounds acting together. The aim of the present study was to test the antitumor effects of two minor compounds from VOO (hydroxytyrosol (HT) and squalene (SQ)) on highly metastatic human breast tumor cells (MDA-MB-231) when acting in combination. Both isolated compounds were previously analyzed without showing any antitumoral effect on highly invasive MDA-MB-231 breast cancer cells, but the present results show that HT at 100 µM, combined with different concentrations of SQ, could exert antitumor effects. When they are combined, HT and SQ are able to inhibit cell proliferation, promoting apoptosis and DNA damage in metastatic breast cancer cells. Therefore, our results suggest that the health-promoting properties of VOO may be due, at least in part, to the combined action of these two minor compounds.

Biogenesis of RNA Polymerases in Yeast.

Eukaryotic RNA polymerases (RNA pols) transcriptional processes have been extensively investigated, and the structural analysis of eukaryotic RNA pols has been explored. However, the global assembly and biogenesis of these heteromultimeric complexes have been narrowly studied. Despite nuclear transcription being carried out by three RNA polymerases in eukaryotes (five in plants) with specificity in the synthesis of different RNA types, the biogenesis process has been proposed to be similar, at least for RNA pol II, to that of bacteria, which contains only one RNA pol. The formation of three different interacting subassembly complexes to conform the complete enzyme in the cytoplasm, prior to its nuclear import, has been assumed. In Saccharomyces cerevisiae, recent studies have examined in depth the biogenesis of RNA polymerases by characterizing some elements involved in the assembly of these multisubunit complexes, some of which are conserved in humans. This study reviews the latest studies governing the mechanisms and proteins described as being involved in the biogenesis of RNA polymerases in yeast.

Prefoldin-like Bud27 influences the transcription of ribosomal components and ribosome biogenesis in Saccharomyces cerevisiae.

Understanding the functional connection that occurs for the three nuclear RNA polymerases to synthesize ribosome components during the ribosome biogenesis process has been the focal point of extensive research. To preserve correct homeostasis on the production of ribosomal components, cells might require the existence of proteins that target a common subunit of these RNA polymerases to impact their respective activities. This work describes how the yeast prefoldin-like Bud27 protein, which physically interacts with the Rpb5 common subunit of the three RNA polymerases, is able to modulate the transcription mediated by the RNA polymerase I, likely by influencing transcription elongation, the transcription of the RNA polymerase III, and the processing of ribosomal RNA. Bud27 also regulates both RNA polymerase II-dependent transcription of ribosomal proteins and ribosome biogenesis regulon genes, likely by occupying their DNA ORFs, and the processing of the corresponding mRNAs. With RNA polymerase II, this association occurs in a transcription rate-dependent manner. Our data also indicate that Bud27 inactivation alters the phosphorylation kinetics of ribosomal protein S6, a readout of TORC1 activity. We conclude that Bud27 impacts the homeostasis of the ribosome biogenesis process by regulating the activity of the three RNA polymerases and, in this way, the synthesis of ribosomal components. This quite likely occurs through a functional connection of Bud27 with the TOR signaling pathway.

A Yeast Chromatin-enriched Fractions Purification Approach, yChEFs, from Saccharomyces cerevisiae.

We have adapted a previous procedure and improved an approach that we named yChEFs (yeast Chromatin Enriched Fractions) for purifying chromatin fractions. This methodology allows the easy, reproducible and scalable recovery of proteins associated with chromatin. By using yChEFs, we bypass subcellular fractionation requirements involved when using zymolyase to obtain the spheroplast, which is employed in many other procedures. Employing small amount of culture cells and small volumes of solutions during the yChEFs procedure is very useful to allow many samples to be handled at the same time, and also reduces costs and efforts. The purified proteins associated with chromatin fractions obtained by yChEFs can be analyzed by Western blot (Figure 1) or combined with mass spectrometry for proteomic analyses.