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Los ribosomas son los orgánulos celulares que llevan a cabo el llamado proceso de traducción, en el cual las proteínas se sintetizan utilizando la información codificada en los RNA mensajeros (mRNAs). Los ribosomas están formados por dos subunidades ribosómicas, una grande, denominada 60S en eucariotas, y otra pequeña, denominada 40S en eucariotas, que a su vez están compuestas por proteínas ribosómicas y RNA ribosómicos (rRNAs). En esta Tesis, se ha utilizado el organismo modelo Saccharomyces cerevisiae para estudiar el proceso de biogénesis de los ribosomas, ya que este proceso está altamente conservado a lo largo de la evolución. En S. cerevisiae, la síntesis de ribosomas comienza en el nucléolo, continúa a través del nucleoplasma y finaliza en el citoplasma, donde los ribosomas maduros llevan a cabo la traducción. Los rRNAs se sintetizan como precursores (pre-rRNAs) que deben ser procesados y sufrir varias modificaciones para producir los diferentes rRNAs maduros (25S, 5.8S y 5S, que forman parte de la subunidad ribosómica 60S, y 18S, que forma parte de la subunidad ribosómica 40S). A lo largo del proceso de maduración de los ribosomas, se ensamblan 79 proteínas ribosómicas (46 para formar la subunidad ribosómica 60S y 33 para formar la subunidad ribosómica 40S), y se requieren más de 300 de los denominados factores de actuación en trans o factores de ensamblaje ribosómico. Estos factores garantizan la velocidad, la direccionalidad y la precisión de la síntesis de los ribosomas, se asocian a las subunidades ribosómicas nacientes y se disocian de ellas siguiendo un orden jerárquico, y no están presentes en los ribosomas maduros. A pesar del gran número de factores de ensamblaje ribosómico identificados hasta la fecha, algunos siguen siendo desconocidos y muchos están poco caracterizados. En esta Tesis, hemos estudiado la proteína de levadura Pol5, la cual fue primero identificada como la quinta DNA polimerasa esencial, y después como miembro de un complejo implic

Ribosome synthesis is a complex process that involves a large set of protein trans-acting factors, among them DEx(D/H)-box helicases. These are enzymes that carry out remodelling activities onto RNAs by hydrolysing ATP. The nucleolar DEGD-box protein Dbp7 is required for the biogenesis of large 60S ribosomal subunits. Recently, we have shown that Dbp7 is an RNA helicase that regulates the dynamic base-pairing between the snR190 small nucleolar RNA and the precursors of the ribosomal RNA within early pre-60S ribosomal particles. As the rest of DEx(D/H)-box proteins, Dbp7 has a modular organization formed by a helicase core region, which contains conserved motifs, and variable, non-conserved N- and C-terminal extensions. The role of these extensions remains unknown. Herein, we show that the N-terminal domain of Dbp7 is necessary for efficient nuclear import of the protein. Indeed, a basic bipartite nuclear localization signal (NLS) could be identified in its N-terminal domain. Removal of this putative NLS impairs, but does not abolish, Dbp7 nuclear import. Both N- and C-terminal domains are required for normal growth and 60S ribosomal subunit synthesis. Furthermore, we have studied the role of these domains in the association of Dbp7 with pre-ribosomal particles. Altogether, our results show that the N- and C-terminal domains of Dbp7 are important for the optimal function of this protein during ribosome biogenesis.

El carcinoma hepatocellular (CHC) es reconocido como el principal cáncer primario de hígado y uno de los cánceres con mayor incidencia y mortalidad a nivel mundial. Éste se desarrolla en un contexto de enfermedad hepática crónica donde la aparición, así como la progresión de la enfermedad, está influenciada por numerosos factores de riesgo. Desafortunadamente, el alto grado de heterogeneidad genética intratumoral dificulta el desarrollo de terapias verdaderamente eficaces. Sorafenib fue el primer fármaco aprobado por la FDA (Food and Drug Administration) para su uso como tratamiento en pacientes avanzados de CHC. A pesar de que otras terapias hayan sido recientemente aprobadas para su uso como primera línea de tratamiento en dichos pacientes, Sorafenib sigue siendo la opción recomendada para un porcentaje significativo de ellos. Este fármaco es un inhibidor multiquinasa con efectos anti-proliferativos, anti-angiogénicos y pro-apoptóticos. Desgraciadamente, Sorafenib cuenta con unos beneficios clínicos muy limitados ya que por un lado produce numerosos efectos secundarios y por otro, el desarrollo de resistencia al tratamiento es muy frecuente en esta enfermedad. Otras terapias basadas en diferentes inhibidores multiquinasa han sido estudiadas en ensayo clínico, pero desgraciadamente, han mostrado una eficacia inferior o igual al Sorafenib, sugiriendo que la efectividad clínica del fármaco podrías estar basada en efectos adicionales a su acción inhibidora de la actividad quinasa. El proceso de síntesis de proteínas, también conocido como traducción, consiste en la formación de nuevas cadenas polipeptídicas a partir de la información contenida en una molécula de RNA mensajero (mRNA). Dicho proceso es mediado por el ribosoma que, junto con numerosas proteínas llamadas factores de traducción, sintetizan las proteínas de novo en función de la demanda celular. El proceso de traducción constituye el principal punto de regulación de la expresión génica, donde los niveles de

Ribosomes are ribonucleoprotein complexes in charge of protein translation. Translation is an essential process in all organisms that involves decoding the information contained in mRNAs (messenger RNAs) to generate proteins. Similarly, the process of ribosome biogenesis represents a critical pathway for all cells. Ribosomes are comprised of two different subunits, one large and one small, also named 60S and 40S, respectively, in eukaryotes, among them Saccharomyces cerevisiae, which is the model organism employed in this thesis. As ribosome biogenesis is well conserved among different eukaryotes, S. cerevisiae is a perfectly appropriate organism for this study. Ribosomes of S. cerevisiae are composed of 79 ribosomal proteins and 4 ribosomal RNAs (rRNAs), three of them in the large (25S, 5.8S y 5S) and one in the small ribosomal subunit (18S). The biogenesis of ribosomes is an extraordinarily complex process, in which in addition of the four rRNAs and the 79-80 ribosomal proteins, take part about 100 small nucleolar RNAs (snoRNAs) and more than 300 assembly protein factors, which are not present in the mature subunits and therefore are also known as trans-acting factors. The synthesis of ribosomes is a sequential process highly regulated and organized that starts in the nucleolus and orderly progresses through the nucleoplasm; almost mature ribosomal particles are exported to the cytoplasm, where the last steps of maturation take place, previously to their involvement in the translation process. The role of the different components participating in the biogenesis of the ribosomal subunits has been broadly studied through last decades. However, there are numerous assembly factors and some ribosomal proteins which still await characterization. This was the case for the ribosomal proteins eL22 and eL15 during the assembly of the 60S ribosomal subunit before the study performed in this doctoral thesis. With respect to the eL22 ribosomal protein, in this thesis, we have, Los ribosomas son complejos ribonucleoproteicos encargados de la traducción de proteínas. La traducción es un proceso esencial en todos los organismos mediante el cual, la información contenida en el mRNA (RNA mensajero) se descodifica generando proteínas. El proceso de biogénesis ribosómica presenta igualmente una importancia crucial en todas las células. Los ribosomas se componen de dos subunidades diferenciadas, una llamada subunidad grande y otra llamada pequeña, también denominadas 60S y 40S en Saccharomyces cerevisiae, el organismo modelo usado en esta tesis. Como la biogénesis de los ribosomas está muy conservada entre los distintos eucariotas, S. cerevisiae es un organismo perfectamente adecuado para este estudio. Los ribosomas de S. cerevisiae están compuestos por 79 proteínas ribosómicas y 4 RNAs ribosómicos (rRNAs), tres de ellos en la subunidad grande (25S, 5.8S y 5S) y uno en la pequeña (18S). La biogénesis de los ribosomas es un proceso extraordinariamente complejo en el que intervienen los cuatro rRNAs y las 79-80 proteínas ribosómicas, del orden de 100 RNAs pequeños nucleolares (snoRNAs) y más de 300 factores proteicos de ensamblaje, que al no formar parte de las subunidades maduras también son llamados factores de actuación en trans. La síntesis de ribosomas es un proceso secuencial altamente regulado y organizado que empieza en el nucleolo y progresa ordenadamente a través del nucleoplasma; las partículas pre-ribosómicas prácticamente completas son exportadas al citoplasma donde los últimos pasos de maduración tienen lugar antes de ser reclutadas al proceso de traducción. El papel de los distintos componentes que participan en la biogénesis de las subunidades ribosómicas ha sido ampliamente estudiado en las ultimas décadas. Sin embargo, aún quedan numerosos factores de ensamblaje y algunas proteínas ribosómicas por ser caracterizadas en detalle. Este era el caso de las proteínas ribosómicas eL22 y eL15 durante el ensamblaje de la subunidad 60S ante

The small ribosomal subunit protein Rps15/uS19 is involved in early nucleolar ribosome biogenesis and subsequent nuclear export of pre-40S particles to the cytoplasm. In addition, the C-terminal tail of Rps15 was suggested to play a role in mature ribosomes, namely during translation elongation. Here, we show that Rps15 not only functions in nucleolar ribosome assembly but also in cytoplasmic pre-40S maturation, which is indicated by a strong genetic interaction between Rps15 and the 40S assembly factor Ltv1. Specifically, mutations either in the globular or C-terminal domain of Rps15 when combined with the non-essential ltv1 null allele are lethal or display a strong growth defect. However, not only rps15 ltv1 double mutants but also single rps15 C-terminal deletion mutants exhibit an accumulation of the 20S pre-rRNA in the cytoplasm, indicative of a cytoplasmic pre-40S maturation defect. Since in pre-40S particles, the C-terminal tail of Rps15 is positioned between assembly factors Rio2 and Tsr1, we further tested whether Tsr1 is genetically linked to Rps15, which indeed could be demonstrated. Thus, the integrity of the Rps15 C-terminal tail plays an important role during late pre-40S maturation, perhaps in a quality control step to ensure that only 40S ribosomal subunits with functional Rps15 C-terminal tail can efficiently enter translation. As mutations in the C-terminal tail of human RPS15 have been observed in connection with chronic lymphocytic leukaemia, it is possible that apart from defects in translation, an impaired late pre-40S maturation step in the cytoplasm could also be a reason for this disease.

Hepatocellular carcinoma (HCC) is one of the most frequent and essentially incurable cancers in its advanced stages. The tyrosine kinase inhibitor Sorafenib (Sfb) remains the globally accepted treatment for advanced HCC. However, the extent of its therapeutic benefit is limited. Sfb exerts antitumor activity through its cytotoxic, anti-proliferative and pro-apoptotic roles in HCC cells. To better understand the molecular mechanisms underlying these effects, we used RNA sequencing to generate comprehensive transcriptome profiles of HepG2 and SNU423, hepatoblastoma-(HB) and HCC-derived cell lines, respectively, following a Sfb treatment at a pharmacological dose. This resulted in similar alterations of gene expression in both cell lines. Genes functionally related to membrane trafficking, stress-responsible and unfolded protein responses, circadian clock and activation of apoptosis were predominantly upregulated, while genes involved in cell growth and cycle, DNA replication and repair, ribosome biogenesis, translation initiation and proteostasis were downregulated. Our results suggest that Sfb causes primary effects on cellular stress that lead to upregulation of selective responses to compensate for its negative effect and restore homeostasis. No significant differences were found specifically affecting each cell line, indicating the robustness of the Sfb mechanism of action despite the heterogeneity of liver cancer. We discuss our results on terms of providing rationalization for possible strategies to improve Sfb clinical outcomes.

During translation, nascent polypeptide chains travel from the peptidyl transferase center through the nascent polypeptide exit tunnel (NPET) to emerge from 60S subunits. The NPET includes portions of five of the six 25S/5.8S rRNA domains and ribosomal proteins uL4, uL22, and eL39. Internal loops of uL4 and uL22 form the constriction sites of the NPET and are important for both assembly and function of ribosomes. Here, we investigated the roles of eL39 in tunnel construction, 60S biogenesis, and protein synthesis. We show that eL39 is important for proper protein folding during translation. Consistent with a delay in processing of 27S and 7S pre-rRNAs, eL39 functions in pre-60S assembly during middle nucleolar stages. Our biochemical assays suggest the presence of eL39 in particles at these stages, although it is not visualized in them by cryo-electron microscopy. This indicates that eL39 takes part in assembly even when it is not fully accommodated into the body of pre-60S particles. eL39 is also important for later steps of assembly, rotation of the 5S ribonucleoprotein complex, likely through long range rRNA interactions. Finally, our data strongly suggest the presence of alternative pathways of ribosome assembly, previously observed in the biogenesis of bacterial ribosomal subunits.

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5′ region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.

Ubiquitin is a small protein that is highly conserved throughout eukaryotes. It operates as a reversible post-translational modifier through a process known as ubiquitination, which involves the addition of one or several ubiquitin moieties to a substrate protein. These modifications mark proteins for proteasome-dependent degradation or alter their localization or activity in a variety of cellular processes. In most eukaryotes, ubiquitin is generated by the proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a polyubiquitin precursor, or as a single N-terminal moiety to ribosomal proteins, which are practically invariably eL40 and eS31. Herein, we summarize the contribution of the ubiquitin moiety within precursors of ribosomal proteins to ribosome biogenesis and function and discuss the biological relevance of having maintained the explicit fusion to eL40 and eS31 during evolution. There are other ubiquitin-like proteins, which also work as post-translational modifiers, among them the small ubiquitin-like modifier (SUMO). Both ubiquitin and SUMO are able to modify ribosome assembly factors and ribosomal proteins to regulate ribosome biogenesis and function. Strikingly, ubiquitin-like domains are also found within two ribosome assembly factors; hence, the functional role of these proteins will also be highlighted.

The ribotoxin α-sarcin belongs to a family of ribonucleases that cleave the sarcin/ricin loop (SRL), a critical functional rRNA element within the large ribosomal subunit (60S), thereby abolishing translation. Whether α-sarcin targets the SRL only in mature 60S subunits remains unresolved. Here, we show that, in yeast, α-sarcin can cleave SRLs within late 60S pre-ribosomes containing mature 25S rRNA but not nucleolar/nuclear 60S pre-ribosomes containing 27S pre-rRNA in vivo. Conditional expression of α-sarcin is lethal, but does not impede early pre-rRNA processing, nuclear export and the cytoplasmic maturation of 60S pre-ribosomes. Thus, SRL-cleaved containing late 60S pre-ribosomes seem to escape cytoplasmic proofreading steps. Polysome analyses revealed that SRL-cleaved 60S ribosomal subunits form 80S initiation complexes, but fail to progress to the step of translation elongation. We suggest that the functional integrity of a α-sarcin cleaved SRL might be assessed only during translation.

The ribosomes are the cellular organelles responsible for translating messenger RNA (mRNA) to proteins. In eukaryotic organisms, the ribosomes are composed by two subunits: the large subunit and the small subunit that come together to make a complete ribosome. The biogenesis of eukaryotic cytoplasmic ribosomes is a very complex process. The eukaryotic organism where this process has been best studied is the budding yeast Saccharomyces cerevisiae. This microorganism is considered a model organism of study due to its low complexity, rapid growth and dispersion of its cells. In S. cerevisiae, the large subunit or 60S is composed by three rRNAs called 25S, 5.8S and 5S and about 46 ribosomal proteins; the small subunit or 40S is composed by only one rRNA called 18S and about 33 ribosomal proteins. The ribosome biogenesis process is not only complex, but it is also compartmentalized. The precursors of rRNA (pre-rRNA) are transcribed in the nucleolus by the RNA polymerase I (25S, 5.8S y 18S) and by the RNA polymerase III (5S). Along the processing of the pre-rRNAs take place accurate endo- and exonucleolytic reactions, at the same time that some molecular building blocks of pre-rRNA are modified by methylases enzymes or by small nucleolar RNA complexes (snoRNPs). The processing and modification of the pre-rRNAs do not occur in naked RNAs, conversely, these reactions take place in pre-ribosomal particles, which are complexes where the ribosomal proteins are assembling hierarchically. The restructuration of the pre-ribosomal particles and its subsequent maturation to ribosomal subunits needs a large number of protein factors, more than 300, which are known as assembly factors or trans-acting factors. These factors are not present in the mature ribosomal particles. One part of these reactions occurs in the nucleoplasm or even in the cytoplasm. The ribosomal synthesis process has not only academic, but also biomedical interest, because a set of hereditary diseases with bad pro

Background/Aims: Orthotopic liver transplantation (OLT) is the recommended treatment for patients at early stages of hepatocarcinoma (HCC) with portal hypertension and/or increased bilirubinemia, but without vascular-associated diseases. Tumor recurrence, which is the main drawback for the survival of patients submitted to OLT for HCC, has been related to tumor-related variables and the immunosuppressive therapies. We have previously shown that Tacrolimus (FK506) exerts a more potent pro-apoptotic and anti-proliferative effects than the mammalian target of rapamycin (mTOR) inhibitors (Sirolimus and Everolimus) in liver cancer cells. This study identified the role of the immunosuppressant partners such as FK506-binding proteins (FKBPs) in the induction of cell death and arrest of cell proliferation by immunosuppressants in two representative liver cancer cells. Methods: The regulation of endoplasmic reticulum (ER) stress, apoptosis/autophagy, cell proliferation, and FKBPs expression was determined in Tacrolimus-, Sirolimus- and Everolimus-treated primary human hepatocytes, and hepatoma HepG2 and Huh7 cell lines. The functional repercussion of FKBPs on cell death and proliferation was also addressed using the siRNA technology. The assessed antitumoral properties of the immunosuppressants were associated to microRNAs (miRNAs) pattern. Results: The enhanced pro-apoptotic and anti-proliferative properties of Tacrolimus versus mTOR inhibitors were associated with increased protein kinase RNA-like endoplasmic reticulum kinase (PERK)-related ER stress, Ser15 P-p53/p53 ratio and p21 protein expression that may counterbalance the risk of proliferative upregulation caused by enhanced Thr172 P-Cdk4/ Cdk4 activation in liver cancer cells. The inhibition of the mTOR pathway by Sirolimus and Everolimus was related to an induction of autophagy; and at a high dose, these drugs impaired translation likely at a very early step of the elongation phase. Tacrolimus and mTOR inhibitors in

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.

Ubiquitin is a highly conserved small eukaryotic protein. It is generated by proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a polyubiquitin precursor of head-to-tail monomers, or as a single N-terminal moiety to ribosomal proteins. Understanding the role of the ubiquitin fused to ribosomal proteins becomes relevant, as these proteins are practically invariably eS31 and eL40 in the different eukaryotes. Herein, we used the amenable yeast Saccharomyces cerevisiae to study whether ubiquitin facilitates the expression of the fused eL40 (Ubi1 and Ubi2 precursors) and eS31 (Ubi3 precursor) ribosomal proteins. We have analyzed the phenotypic effects of a genomic ubi1∆ub-HA ubi2∆ mutant, which expresses a ubiquitin-free HA-tagged eL40A protein as the sole source of cellular eL40. This mutant shows a severe slow-growth phenotype, which could be fully suppressed by increased dosage of the ubi1∆ub-HA allele, or partially by the replacement of ubiquitin by the ubiquitin-like Smt3 protein. While expression levels of eL40A-HA from ubi1∆ub-HA are low, eL40A is produced practically at normal levels from the Smt3-S-eL40A-HA precursor. Finally, we observed enhanced aggregation of eS31-HA when derived from a Ubi3∆ub-HA precursor and reduced aggregation of eL40A-HA when expressed from a Smt3-S-eL40A-HA precursor. We conclude that ubiquitin might serve as a cis-acting molecular chaperone that assists in the folding and synthesis of the fused eL40 and eS31 ribosomal proteins.

In Saccharomyces cerevisiae, more than 250 trans-acting factors are involved in the maturation of 40S and 60S ribosomal subunits. The expression of most of these factors is transcriptionally coregulated to ensure correct ribosome production under a wide variety of environmental and intracellular conditions. Here, we identified the essential nucleolar Pol5 protein as a novel trans-acting factor required for the synthesis of 60S ribosomal subunits. Pol5 weakly and/or transiently associates with early to medium pre-60S ribosomal particles. Depletion of and temperature-sensitive mutations in Pol5 result in a deficiency of 60S ribosomal subunits and accumulation of half-mer polysomes. Both processing of 27SB pre-rRNA to mature 25S rRNA and release of pre-60S ribosomal particles from the nucle(ol)us to the cytoplasm are impaired in the Pol5-depleted strain. Moreover, we identified the genes encoding ribosomal proteins uL23 and eL27A as multicopy suppressors of the slow growth of a temperature-sensitive pol5 mutant. These results suggest that Pol5 could function in ensuring the correct folding of 25S rRNA domain III; thus, favoring the correct assembly of these two ribosomal proteins at their respective binding sites into medium pre-60S ribosomal particles. Pol5 is homologous to the human tumor suppressor Myb-binding protein 1A (MYBBP1A). However, expression of MYBBP1A failed to complement the lethal phenotype of a pol5 null mutant strain though interfered with 60S ribosomal subunit biogenesis.

The contribution of most ribosomal proteins to ribo- some synthesis has been quite well analysed in Sac- charomyces cerevisiae . However, few yeast riboso- mal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre- 60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA 2 and 27SA 3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S par- ticles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S par- ticle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans -acting factors required for 27SB pre- rRNA processing but accumulate practically all fac- tors required for processing of 27SA 3 pre-rRNA. We have also analysed the functional interaction be- tween the eukaryote-specific carboxy-terminal exten- sions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alter- ations in mature 60S subunits.

Ribosome assembly requires the concerted expression of hundreds of genes, which are transcribed by all three nuclear RNA polymerases. Transcription elongation involves dynamic interactions between RNA polymerases and chromatin. We performed a synthetic lethal screening in Saccharomyces cerevisiae with a conditional allele of SPT6, which encodes one of the factors that facilitates this process. Some of these synthetic mutants corresponded to factors that facilitate pre-rRNA processing and ribosome biogenesis. We found that the in vivo depletion of one of these factors, Arb1, activated transcription elongation in the set of genes involved directly in ribosome assembly. Under these depletion conditions, Spt6 was physically targeted to the upregulated genes, where it helped maintain their chromatin integrity and the synthesis of properly stable mRNAs. The mRNA profiles of a large set of ribosome biogenesismutants confirmed the existence of a feedback regulatory network among ribosome assembly genes. The transcriptional response in this network depended on both the specific malfunction and the role of the regulated gene. In accordance with our screening, Spt6 positively contributed to the optimal operation of this global network. On the whole, this work uncovers a feedback control of ribosome biogenesis by fine-tuning transcription elongation in ribosome assembly factor-coding genes.

The synthesis of cytoplasmic eukaryotic ribosomes is an extraordinarily energy-demanding cellular activity that occurs progressively from the nucleolus to the cytoplasm. In the nucleolus, precursor rRNAs associate with a myriad of trans-acting factors and some ribosomal proteins to form pre-ribosomal particles. These factors include snoRNPs, nucleases, ATPases, GTPases, RNA helicases, and a vast list of proteins with no predicted enzymatic activity. Their coordinate activity orchestrates in a spatiotemporal manner the modification and processing of precursor rRNAs, the rearrangement reactions required for the formation of productive RNA folding intermediates, the ordered assembly of the ribosomal proteins, and the export of pre-ribosomal particles to the cytoplasm; thus, providing speed, directionality and accuracy to the overall process of formation of translation-competent ribosomes. Here, we review a particular class of trans-acting factors known as "placeholders". Placeholder factors temporarily bind selected ribosomal sites until these have achieved a structural context that is appropriate for exchanging the placeholder with another site-specific binding factor. By this strategy, placeholders sterically prevent premature recruitment of subsequently binding factors, premature formation of structures, avoid possible folding traps, and act as molecular clocks that supervise the correct progression of pre-ribosomal particles into functional ribosomal subunits. We summarize the current understanding of those factors that delay the assembly of distinct ribosomal proteins or subsequently bind key sites in pre-ribosomal particles. We also discuss recurrent examples of RNA-protein and protein-protein mimicry between rRNAs and/or factors, which have clear functional implications for the ribosome biogenesis pathway.

The archaea-/eukaryote-specific 40S-ribosomal-subunit protein S31 is expressed as an ubiquitin fusion protein in eukaryotes and consists of a conserved body and a eukaryote-specific N-terminal extension. In yeast, S31 is a practically essential protein, which is required for cytoplasmic 20S pre-rRNA maturation. Here, we have studied the role of the N-terminal extension of the yeast S31 protein. We show that deletion of this extension partially impairs cell growth and 40S subunit biogenesis and confers hypersensitivity to aminoglycoside antibiotics. Moreover, the extension harbours a nuclear localization signal that promotes active nuclear import of S31, which associates with pre-ribosomal particles in the nucleus. In the absence of the extension, truncated S31 inefficiently assembles into pre-40S particles and two subpopulations of mature small subunits, one lacking and another one containing truncated S31, can be identified. Plasmid-driven overexpression of truncated S31 partially suppresses the growth and ribosome biogenesis defects but, conversely, slightly enhances the hypersensitivity to aminoglycosides. Altogether, these results indicate that the N-terminal extension facilitates the assembly of S31 into pre-40S particles and contributes to the optimal translational activity of mature 40S subunits but has only a minor role in cytoplasmic cleavage of 20S pre-rRNA at site D

Ribosomes are the highly complex macromolecular assemblies dedicated to the synthesis of all cellular proteins from mRNA templates. The main principles underlying the making of ribosomes are conserved across eukaryotic organisms and this process has been studied in most detail in the yeast Saccharomyces cerevisiae. Yeast ribosomes are composed of four ribosomal RNAs (rRNAs) and 79 ribosomal proteins (r-proteins). Most r-proteins need to be transported from the cytoplasm to the nucleus where they get incorporated into the evolving pre-ribosomal particles. Due to the high abundance and difficult physicochemical properties of r-proteins, their correct folding and fail-safe targeting to the assembly site depends largely on general, as well as highly specialized, chaperone and transport systems. Many r-proteins contain universally conserved or eukaryote-specific internal loops and/or terminal extensions, which were shown to mediate their nuclear targeting and association with dedicated chaperones in a growing number of cases. The 60S r-protein Rpl4 is particularly interesting since it harbours a conserved long internal loop and a prominent Cterminal eukaryote-specific extension. Here we show that both the long internal loop and the C-terminal eukaryote-specific extension are strictly required for the functionality of Rpl4. While Rpl4 contains at least five distinct nuclear localization signals (NLS), the C-terminal part of the long internal loop associates with a specific binding partner, termed Acl4. Absence of Acl4 confers a severe slow-growth phenotype and a deficiency in the production of 60S subunits. Genetic and biochemical evidence indicates that Acl4 can be considered as a dedicated chaperone of Rpl4. Notably, Acl4 localizes to both the cytoplasm and nucleus and it has the capacity to capture nascent Rpl4 in a co-translational manner. Taken together, our findings indicate that the dedicated chaperone Acl4 accompanies Rpl4 from the cytoplasm to its pre-60S assembl

Ribosomal protein L3 is an evolutionarily conserved protein that participates in the assembly of early pre-60S particles. We report that the rpl3[W255C] allele, which affects the affinity and function of translation elongation factors, impairs cytoplasmic maturation of 20S pre-rRNA. This was not seen for other mutations in or depletion of L3 or other 60S ribosomal proteins. Surprisingly, pre-40S particles containing 20S pre-rRNA form translation-competent 80S ribosomes, and translation inhibition partially suppresses 20S pre-rRNA accumulation. The GTP-dependent translation initiation factor Fun12 (yeast eIF5B) shows similarin vivo binding to ribosomal particles from wild-type andrpl3[W255C] cells. However, the GTPase activity of eIF5B failed to stimulate processing of 20S pre-rRNA when assayed with ribosomal particles purified from rpl3[W255C] cells. We conclude that L3 plays an important role in the function of eIF5B in stimulating 39end processing of 18S rRNA in the context of 80S ribosomes that have not yet engaged in translation. These findings indicate that the correct conformation of the GTPase activation region is assessed in a quality control step during maturation of cytoplasmic preribosomal particles

Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATPindependent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associatesin vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity

En el Capítulo I de esta tesis se muestran los resultados de la caracterización funcional de la proteína ribosómica L35, componente esencial de la subunidad grande del ribosoma. Nuestros resultados indican que la ausencia de L35 en las células tiene como consecuencia un déficit en la producción de subunidades 60S. Tal y como se demuestra por los experimentos de pulso-caza y de hibridación Northern, este déficit se debe a una fuerte inhibición en el procesamiento del pre-rRNA 27SB, dando lugar a niveles muy reducidos de los pre-rRNAs 7S y 25.5S y, en último término, de los rRNAS maduros 5.8S y 25S respectivamente. Además, se observa un claro retraso en el procesamiento temprano en los sitios A0, A1 y A2, que tiene como última consecuencia una reducción en los niveles del rRNA 18S y la aparición del precursor aberrante 23S. Estos resultados están en consonancia con estudios sistemáticos previos (1), donde L35 aparece en el grupo de las proteínas ribosómicas que intervienen en pasos intermedios del procesamiento de los pre-rRNAs. Nuestros resultados demuestran también que la proteína ribosómica L35 se asocia de forma estable a las partículas pre-60S tempranas y que su presencia es necesaria para el transporte óptimo de las partículas pre-60S del núcleo hacia el citoplasma. En el Capítulo II se presentan los resultados de la caracterización funcional de la proteína ribosómica L26, otro componente estructural de la subunidad grande del ribosoma. Estos resultados nos han permitido demostrar la no esencialidad de L26 en el crecimiento celular, durante el proceso de biogénesis de ribosomas y en la función ribosómica. Si bien, a través de los experimentos de pulso-caza se puede observar un ligero retraso en el procesamiento del pre-rRNA 27SB en ausencia de L26. Nuestros resultados demuestran también que la proteína ribosómica L26 se ensambla establemente en partículas pre-60S tempranas y su presencia es necesaria para un transporte eficiente de las partículas pre-60S desde e

El estudio de la síntesis de los ribosomas eucariotas es un campo de investigación muy prolífico debido, sin duda, a la enorme complejidad que encierran los aspectos fundamentales de este proceso. Esta Tesis se centra en el análisis funcional, tanto de determinados factores de actuación en trans como de ciertas proteínas ribosómicas, ambos necesarios para el correcto ensamblaje de las partíc ulas prerribosómicas y en comprender cómo estas partículas adquieren su funcionalidad en el citoplasma. Como organismo de estudio se utiliza la levadura Saccharomyces cerevisiae. S. cerevisiae posee alrededor de 300 factores proteicos de actuación en trans que participan en la síntesis de ribosomas. Desconocemos la función precisa de la mayoría de estos factores para los que no existen siquiera motivos o dominios reconocibles en sus secuencias primarias de aminoácidos, más allá de motivos generales de unión a RNA presentes en algunos factores. En los últimos años se ha podido determinar la composición de algunas partículas prerribosómicas en cepas silvestres y cepas mutantes con las que se ha podido inferir al menos la posición y tiempo de acción de algunos de ellos. Otros aspectos casi completamente inexplorados son el análisis de los sustratos y las redes de interacción de los factores de actuación en trans. La mayor parte de las proteínas ribosómicas son esenciales para el procesamiento de los pre-rRNAs, y en general, para la síntesis de ribosomas. Se han llevado a cabo análisis sistemáticos y específicos del papel de numerosas proteínas ribosómicas de ambas subunidades, en la maduración y el transporte de las partículas prerribosómicas (1, 4). Un primer objetivo de este trabajo ha sido el estudio del factor de actuación en trans Nop6. En el Capítulo 1 de esta Tesis se muestran los resultados de la caracterización funcional de Nop6, un componente no esencial de partículas tempranas 90S, que es necesario para la síntesis de subunidades ribosómicas 40S (2). En el Capítulo 2 se

Ribosomal proteins play important roles in ribosome biogenesis and function. Here, we study the evolutionarily conserved L26 inSaccharomyces cerevisiae, which assembles into pre-60S ribosomal particles in the nucle(ol)us. Yeast L26 is one of the many ribosomal proteins encoded by two functional genes. We have disrupted both genes; surprisingly, the growth of the resulting rpl26null mutant is apparently identical to that of the isogenic wild-type strain. The absence of L26 minimally alters 60S ribosomal subunit biogenesis. Polysome analysis revealed the appearance of half-mers. Analysis of pre-rRNA processing indicated that L26 is mainly required to optimize 27S pre-rRNA maturation, without which the release of pre-60S particles from the nucle-(ol)us is partially impaired. Ribosomes lacking L26 exhibit differential reactivity to dimethylsulfate in domain I of 25S/5.8S rRNAs but apparently are able to support translationin vivowith wild-type accuracy. The bacterial homologue of yeast L26, L24, is a primary rRNA binding protein required for 50S ribosomal subunit assemblyin vitroandin vivo. Our results underscore potential differences between prokaryotic and eukaryotic ribosome assembly. We discuss the reasons why yeast L26 plays such an apparently nonessential role in the cell

Background: The contribution of ribosomal proteins to ribosome assembly and function is often not well understood. Results: L40 assembles within the cytoplasm into pre-60 S subunits and is required for Nmd3 and Rlp24 recycling. Conclusion: L40 contributes to formation of 60 S subunits competent for subunit joining and translation elongation. Significance: Our analysis of L40 function reveals an additional step during cytoplasmic pre-60 S maturation events

Gene expression in eukaryotes is an essential process that includes transcription, RNA processing, and export. One important player in this interface is the poly(A) + -RNA– binding protein Nab2, which regulates the mRNA poly(A) + -tail length and export. Here we show that Nab2 has additional roles during mRNA transcription, tRNA metabolism, and ribosomal subunit export. Nab2 is associated with the entire open reading frame of actively transcribed RNA polymerase (RNAP) II and III genes. As a consequence, nab2mutations confer translation defects that are detected by polysome profiling. Genome-wide analysis of expression of a conditional degron nab2mutant shows that the role of Nab2 in RNAPII transcription and RNAPIII metabolism is direct. Taken together, our results identify novel functions for Nab2 in transcription and metabolism of most types of RNAs, indicating that Nab2 function is more ubiquitous than previously anticipated, and that it is a central player in the general and coordinated control of gene expression from transcription to translation

Spb4 is a putative ATP-dependent RNA helicase that is required for proper processing of 27SB pre-rRNAs and therefore for 60S ribosomal subunit biogenesis. To define the timing of association of this protein with preribosomal particles, we have studied the composition of complexes that copurify with Spb4 tagged by tandem affinity purification (TAP-tagged Spb4). These complexes contain mainly the 27SB pre-rRNAs and about 50 ribosome biogenesis proteins, primarily components of early pre-60S ribosomal particles. To a lesser extent, some protein factors of 90S preribosomal particles and the 35S and 27SA pre-rRNAs also copurify with TAP-tagged Spb4. Moreover, we have obtained by site-directed mutagenesis an allele that results in the R360A substitution in the conserved motif VI of the Spb4 helicase domain. This allele causes a dominant-negative phenotype when overexpressed in the wild-type strain. Cells expressing Spb4(R360A) display an accumulation of 35S and 27SB pre-rRNAs and a net 40S ribosomal subunit defect. TAP-tagged Spb4(R360A) displays a greater steady-state association with 90S preribosomal particles than TAP-tagged wild-type Spb4. Together, our data indicate that Spb4 is a component of early nucle(ol)ar pre-60S ribosomal particles containing 27SB pre-rRNA. Apparently, Spb4 binds 90S preribosomal particles and dissociates from pre-60S ribosomal particles after processing of 27SB pre-rRNA

We report the characterization of the yeast Npa2p (Urb2p) protein, which is essential for 60S ribosomal subunit biogenesis. We identified this protein in a synthetic lethal screening with thersa3null allele. Rsa3p is a genetic partner of the putative RNA helicase Dbp6p. Mutation or depletion of Npa2p leads to a net deficit in 60S subunits and a decrease in the levels all 27S pre-rRNAs and mature 25S and 5.8S rRNAs. This is likely due to instability of early pre-60S particles. Consistent with a role of Npa2p in 60S subunit biogenesis, green fluorescent protein-tagged Npa2p localizes predominantly to the nucleolus and TAP-tagged Npa2p sediments with large complexes in sucrose gradients and is associated mainly with 27SA2 pre-rRNA-containing preribosomal particles. In addition, we reveal a genetic synthetic interaction between Npa2p, several factors required for early steps of 60S subunit biogenesis (Dbp6p, Dbp7p, Dbp9p, Npa1p, Nop8p, and Rsa3p), and the 60S protein Rpl3p. Furthermore, coimmunoprecipitation and gel filtration analyses demonstrated that at least Npa2p, Dbp6p, Npa1p, Nop8p, and Rsa3p are present together in a subcomplex of low molecular mass whose integrity is independent of RNA. Our results support the idea that these five factors work in concert during the early steps of 60S subunit biogenesis