Lucía Ramos-Alonso

Although most studies in the regulation of gene transcription in eukaryotes have focused on protein‐coding genes transcribed into mRNA by RNA polymerase II, the regulation of RNA polymerase I (RNA Pol I) transcription is a key step in the cell's adaptation to environmental or physiological changes. This polymerase transcribes rRNA, a fundamental component of ribosomes that controls the translation of mRNAs into proteins—macromolecules that are both highly abundant and energetically costly to synthesize. Using the yeast Saccharomyces cerevisiae as a model, we have previously shown that adaptation of RNA Pol I synthesis rate (SR) to large changes in cell volume exhibited by some mutants, such as cln3 , occurs over the long term through amplification of rDNA gene copies across several hundred cell divisions. However, it remains unclear what happens in the short term in de novo generated mutants that do not display rDNA amplification but exhibit all other phenotypic characteristics of the evolved cln3 mutant. Here, using an in vivo labeling procedure, we show that the RNA Pol I SR is similar in de novo and evolved cln3 mutants and that it seems not to be an alteration in the proportion of active rDNA copies or rRNA processing between both cln3 mutants. Finally, by measuring the ratio of active elongating RNA Pol I molecules to the total loaded onto rDNA genes, we conclude that short‐term adaptation occurs through an increase in their transcription efficiency that compensates the decrease in the total number of elongating RNA polymerases.

In metazoans, all cells possess an identical genetic material and a common set of general transcription factors. In theory, this suggests that all genes have the potential to be expressed in any ce...

Mitotic entry correlates with the condensation of the chromosomes, changes in histone modifications, exclusion of transcription factors from DNA, and the broad downregulation of transcription. However, whether mitotic condensation influences transcription in the subsequent interphase is unknown. Here, we show that preventing one chromosome to condense during mitosis causes it to fail resetting of transcription. Rather, in the following interphase, the affected chromosome contains unusually high levels of the transcription machinery, resulting in abnormally high expression levels of genes in cis , including various transcription factors. This subsequently causes the activation of inducible transcriptional programs in trans , such as the GAL genes, even in the absence of the relevant stimuli. Thus, mitotic chromosome condensation exerts stringent control on interphase gene expression to ensure the maintenance of basic cellular functions and cell identity across cell divisions. Together, our study identifies the maintenance of transcriptional homeostasis during interphase as an unexpected function of mitosis and mitotic chromosome condensation.

Tight control of gene expression networks required for adipose tissue formation and plasticity is essential for adaptation to energy needs and environmental cues. However, the mechanisms that orchestrate the global and dramatic transcriptional changes leading to adipocyte differentiation remain to be fully unraveled. We investigated the regulation of nascent transcription by the sumoylation pathway during adipocyte differentiation using SLAMseq and ChIPseq. We discovered that the sumoylation pathway has a dual function in differentiation; it supports the initial downregulation of pre-adipocyte-specific genes, while it promotes the establishment of the mature adipocyte transcriptional program. By characterizing endogenous sumoylome dynamics in differentiating adipocytes by mass spectrometry, we found that sumoylation of specific transcription factors like PPARγ/RXR and their co-factors are associated with the transcription of adipogenic genes. Finally, using RXR as a model, we found that sumoylation may regulate adipogenic transcription by supporting the chromatin occurrence of transcription factors. Our data demonstrate that the sumoylation pathway supports the rewiring of transcriptional networks required for formation of functional adipocytes. This study also provides the scientists in the field of cellular differentiation and development with an in-depth resource of the dynamics of the SUMO-chromatin landscape, SUMO-regulated transcription and endogenous sumoylation sites during adipocyte differentiation.

Iron is an essential element for all eukaryotic organisms because it participates as a redox active cofactor in a wide range of biological processes, including protein synthesis. Translation is probably the most energy consuming process in cells. Therefore, one of the initial responses of eukaryotic cells to stress or nutrient limitation is the arrest of mRNA translation. In first instance, the budding yeast Saccharomyces cerevisiae responds to iron deficiency by activating iron acquisition and remodeling cellular metabolism in order to prioritize essential over non-essential iron-dependent processes. We have determined that, despite a global decrease in transcription, mRNA translation is actively maintained during a short-term exposure to iron scarcity. However, a more severe iron deficiency condition induces a global repression of translation. Our results indicate that the Gcn2-eIF2α pathway limits general translation at its initiation step during iron deficiency. This bulk translational inhibition depends on the uncharged tRNA sensing Gcn1-Gcn20 complex. The involvement of the Gcn2-eIF2α pathway in the response to iron deficiency highlights its central role in the eukaryotic response to stress or nutritional deprivation, which is conserved from yeast to mammals.

Iron participates as a vital cofactor in multiple metabolic pathways. Despite its abundance, iron bioavailability is highly restricted in aerobic and alkaline environments. Therefore, living organisms have evolved multiple adaptive mechanisms to respond to iron scarcity. These strategies include a global remodeling of iron metabolism directed to optimize iron utilization. In the baker’s yeast Saccharomyces cerevisiae, this metabolic reorganization is accomplished to a large extent by an mRNA-binding protein called Cth2. Yeast Cth2 belongs to a conserved family of tandem zinc finger containing proteins that specifically bind to transcripts with AU-rich elements and promote their turnover. A recent study has revealed that Cth2 also inhibits the translation of its target mRNAs (Ramos-Alonso et al., PLoS Genet 14:e1007476, https://doi.org/10.1371/journal.pgen.1007476 , 2018 ). Interestingly, the mammalian Cth2 ortholog known as tristetraprolin (aka TTP/TIS11/ZFP36), which is also implicated in controlling iron metabolism, promotes the decay and prevents the translation of its regulated transcripts. These observations open the possibility to study the relative contribution of altering mRNA stability and translation to the physiological adaptation to iron deficiency, the function played by the different domains within the mRNA-binding protein, and the potential factors implicated in coordinating both post-transcriptional events.

All eukaryotic organisms rely on iron as an essential micronutrient for life because it participates as a redox-active cofactor in multiple biological processes. However, excess iron can generate reactive oxygen species that damage cellular macromolecules. The low solubility of ferric iron under physiological conditions increases the prevalence of iron deficiency anemia. A common strategy to treat iron deficiency consists of dietary iron supplementation. The baker's yeast Saccharomyces cerevisiae is used as a model eukaryotic organism, but also as a feed supplement. In response to iron deficiency, the yeast Aft1 transcription factor activates cellular iron acquisition. However, when constitutively active, Aft1 inhibits growth probably due to iron toxicity. In this report, we have studied the consequences of using hyperactive AFT1 alleles, including AFT1-1UP, to increase yeast iron accumulation. We first characterized the iron sensitivity of cells expressing different constitutively active AFT1 alleles. We rescued the high iron sensitivity conferred by the AFT1 alleles by deleting the sphingolipid signaling kinase YPK1. We observed that the deletion of YPK1 exerts different effects on iron accumulation depending on the AFT1 allele and the environmental iron. Moreover, we determined that the impairment of the high-affinity iron transport system partially rescues the high iron toxicity of AFT1-1UP-expressing cells. Finally, we observed that AFT1-1UP inhibits oxygen consumption through activation of the RNA-binding protein Cth2. Deletion of CTH2 partially rescues the AFT1-1UP negative respiratory effect. Collectively, these results contribute to understand how the Aft1 transcription factor functions and the multiple consequences derived from its constitutive activation.

In response to iron deficiency, the budding yeast Saccharomyces cerevisiae undergoes a metabolic remodeling in order to optimize iron utilization. The tandem zinc finger (TZF)-containing protein Cth2 plays a critical role in this adaptation by binding and promoting the degradation of multiple mRNAs that contain AU-rich elements (AREs). Here, we demonstrate that Cth2 also functions as a translational repressor of its target mRNAs. By complementary approaches, we demonstrate that Cth2 protein inhibits the translation of SDH4, which encodes a subunit of succinate dehydrogenase, and CTH2 mRNAs in response to iron depletion. Both the AREs within SDH4 and CTH2 transcripts, and the Cth2 TZF are essential for translational repression. We show that the role played by Cth2 as a negative translational regulator extends to other mRNA targets such as WTM1, CCP1 and HEM15. A structure-function analysis of Cth2 protein suggests that the Cth2 amino-terminal domain (NTD) is important for both mRNA turnover and translation inhibition, while its carboxy-terminal domain (CTD) only participates in the regulation of translation, but is dispensable for mRNA degradation. Finally, we demonstrate that the Cth2 CTD is physiologically relevant for adaptation to iron deficiency. Iron is essential for eukaryotes because it is required for many fundamental processes such as DNA replication, protein translation or respiration, but it is very insoluble and can, therefore, easily go scarce. For this reason, eukaryotic cells have developed adaptive responses to iron deficiency. Under iron limitation conditions, the yeast Saccharomyces cerevisiae induces the expression of Cth2, a protein with tandem zinc fingers that binds to adenine and uracil-rich sequences in the 3’-UTR of specific mRNAs related to iron metabolism, promoting their degradation. Here we show that Cth2 inhibits the translation of ARE-containing mRNAs, including SDH4, WTM1, HEM15 and CCP1, which encode proteins that contain iron or participate in iron-dependent pathways, and CTH2 itself, which is subjected to an autoregulatory loop that controls its expression. We also dissected different domains of Cth2 that are differentially involved in mRNA decay and translational inhibition. The involvement of Cth2 in translational control reinforces the importance of this ARE-binding protein as a post-transcriptional regulator of the iron response in yeast. By acting at different steps in the life of specific mRNA targets, Cth2 action ensures yeast cells a proper distribution of iron by optimizing its utilization in essential processes.