Mercedes Dosil

The 90S preribosome is a multimodular structure that is assembled through a hierarchical mechanism.

ABSTRACT The 90S preribosomal particle is required for the production of the 18S rRNA from a pre-rRNA precursor. Despite the identification of the protein components of this particle, its mechanism of assembly and structural design remain unknown. In this work, we have combined biochemical studies, proteomic techniques, and bioinformatic analyses to shed light into the rules of assembly of the yeast 90S preribosome. Our results indicate that several protein subcomplexes work as discrete assembly subunits that bind in defined steps to the 35S pre-rRNA. The assembly of the t-UTP subunit is an essential step for the engagement of at least five additional subunits in two separate, and mutually independent, assembling routes. One of these routes leads to the formation of an assembly intermediate composed of the U3 snoRNP, the Pwp2p/UTP-B, subunit and the Mpp10p complex. The other assembly route involves the stepwise binding of Rrp5p and the UTP-C subunit. We also report the use of a bioinformatic approach that provides a model for the topological arrangement of protein components within the fully assembled particle. Together, our data identify the mechanism of assembly of the 90S preribosome and offer novel information about its internal architecture.

Functional characterization of Pwp2, a WD family protein essential for the assembly of the 90 S pre-ribosomal particle.

Here we report the functional characterization of Pwp2, an evolutionary conserved component of the 90 S pre-ribosome. Conditional depletion of the Pwp2 protein in yeast specifically impairs pre-rRNA proccessing at sites A0, A1, and A2, leading to a strong decrease in 18 S rRNA and 40 S ribosomal subunit levels. Pre-ribosomal particle sedimentation analysis indicated that these defects are caused by a block in the formation of 90 S pre-ribosomes. We demonstrate that in Pwp2-depleted cells the U3 small nucleolar ribonucleoprotein is not able to interact with the 35 S pre-rRNA and accumulates as a free complex. Similarly, other 90 S particle components such as Imp3 and Imp4 do not associate with the pre-rRNA precursor in the absence of Pwp2. In addition, we have found that after blocking U3 ribonucleoprotein assembly, Pwp2 predominantly accumulates as a complex in association with five proteins: Dip2, Utp6, Utp13, Utp18, and Utp21. Immunoprecipitation and gradient sedimentation analysis revealed that this Pwp2 small subcomplex is capable of interacting directly with the 35 S pre-rRNA 5′ end. Taken together, these results indicate that Pwp2 forms part of a stable particle subunit independent of the U3 small nucleolar ribonucleoprotein that is essential for the initial assembly steps of the 90 S pre-ribosome.

The C terminus of the Saccharomyces cerevisiae alpha-factor receptor contributes to the formation of preactivation complexes with its cognate G protein.

ABSTRACT Binding of the α-factor pheromone to its G-protein-coupled receptor (encoded by STE2) activates the mating pathway inMATa yeast cells. To investigate whether specific interactions between the receptor and the G protein occur prior to ligand binding, we analyzed dominant-negative mutant receptors that compete with wild-type receptors for G proteins, and we analyzed the ability of receptors to suppress the constitutive signaling activity of mutant Gα subunits in an α-factor-independent manner. Although the amino acid substitution L236H in the third intracellular loop of the receptor impairs G-protein activation, this substitution had no influence on the ability of the dominant-negative receptors to sequester G proteins or on the ability of receptors to suppress theGPA1-A345T mutant Gα subunit. In contrast, removal of the cytoplasmic C-terminal domain of the receptor eliminated both of these activities even though the C-terminal domain is unnecessary for G-protein activation. Moreover, the α-factor-independent signaling activity of ste2-P258L mutant receptors was inhibited by the coexpression of wild-type receptors but not by coexpression of truncated receptors lacking the C-terminal domain. Deletion analysis suggested that the distal half of the C-terminal domain is critical for sequestration of G proteins. The C-terminal domain was also found to influence the affinity of the receptor for α-factor in cells lacking G proteins. These results suggest that the C-terminal cytoplasmic domain of the α-factor receptor, in addition to its role in receptor downregulation, promotes the formation of receptor–G-protein preactivation complexes.

How Vav proteins discriminate the GTPases Rac1 and RhoA from Cdc42.

Vav proteins are GDP/GTP exchange factors for Rho/Rac GTPases that are activated by tyrosine phosphorylation. These proteins activate Rac1, RhoG, and RhoA but not the highly related Cdc42 protein. At present, there is no available information to explain this substrate selectivity at the structural level. Here we show that the selection of Vav proteins substrates is achieved at two different levels. On one hand, Vav proteins utilize some residues of the β2/β3 region of Rho/Rac GTPases (D49 and E54) to assure the specific binding to its substrate. In addition, these exchange factors need a second structural signal located in the β5 region of Rho/Rac proteins (residue K118) to promote proper GDP/GTP exchange. These results identify the amino acid residues that allow the discrimination of the Vav family substrates from Cdc42 and, in addition, demonstrate that the activation of specific Rho/Rac GTPases by these GEFs requires two concatenated events, binding and subsequent enzyme reaction, whose specificities are determined by two separate regions of Rho proteins.

Elucidation of the assembly events required for the recruitment of Utp20, Imp4 and Bms1 onto nascent pre-ribosomes

The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. Recently, it has been shown that the formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. However, the mode of assembly of other 90S pre-ribosome components remains obscure as yet. In this study, we have investigated the assembly of three proteins (Utp20, Imp4 and Bms1) previously regarded as potential nucleating factors of the 90S particle. Here, we demonstrate that the loading of those three proteins onto the pre-rRNA takes place independently of Rrp5/UTP-C and, instead, occurs downstream of the tUTP and U3/UTP-B subcomplexes. We also demonstrate that Bms1 and Utp20 are required for the recruitment of a subset of proteins to nascent pre-ribosomes. Finally, we show that proteins associated through secondary steps condition the stability of the two assembly branches in partially assembled pre-ribosomes. These results provide new information about the functional relationships among 90S particle components and the events that are required for their stepwise incorporation onto the primary pre-rRNA.

Tissue-specific and differential expression of prothymosin α gene during rat development

We have analyzed the RNA expression of prothymosin α (Pro Tα) gene during rat development in several tissues and compared it to that of two proteins related to cell proliferation: proliferating cell nuclear antigen (PCNA)/cyclin and histone H3 (H3). The expression of ProTα gene was found to be regulated in a developmental and tissue‐specific manner. The mRNA levels of Pro Tα followed a similar time‐course in liver, brain, kidney, and testis, being highly increased in the early periods of postnatal development. However, in thymus Pro Tα mRNA showed only moderate changes throughout development. Our findings suggest that Pro Tα participates in developmental processes like cell proliferation and/or differentiation.

Contribution of the R-Ras2 GTP-binding protein to primary breast tumorigenesis and late-stage metastatic disease

R-Ras2 is a transforming GTPase that shares downstream effectors with Ras subfamily proteins. However, little information exists about the function of this protein in tumorigenesis and its signalling overlap with classical Ras GTPases. Here we show, by combining loss- and gain-of-function studies in breast cancer cells, mammary epithelial cells and mouse models, that endogenous R-Ras2 has a role in both primary breast tumorigenesis and the late metastatic steps of cancer cells in the lung parenchyma. R-Ras2 drives tumorigenesis in a phosphatidylinostiol-3 kinase (PI3K)-dependent and signalling autonomous manner. By contrast, its prometastatic role requires other priming oncogenic signals and the engagement of several downstream elements. R-Ras2 function is required even in cancer cells exhibiting constitutive activation of classical Ras proteins, indicating that these GTPases are not functionally redundant. Our results also suggest that application of long-term R-Ras2 therapies will result in the development of compensatory mechanisms in breast tumours.

Rrp12 and the Exportin Crm1 Participate in Late Assembly Events in the Nucleolus during 40S Ribosomal Subunit Biogenesis

During the biogenesis of small ribosomal subunits in eukaryotes, the pre-40S particles formed in the nucleolus are rapidly transported to the cytoplasm. The mechanisms underlying the nuclear export of these particles and its coordination with other biogenesis steps are mostly unknown. Here we show that yeast Rrp12 is required for the exit of pre-40S particles to the cytoplasm and for proper maturation dynamics of upstream 90S pre-ribosomes. Due to this, in vivo elimination of Rrp12 leads to an accumulation of nucleoplasmic 90S to pre-40S transitional particles, abnormal 35S pre-rRNA processing, delayed elimination of processing byproducts, and no export of intermediate pre-40S complexes. The exportin Crm1 is also required for the same pre-ribosome maturation events that involve Rrp12. Thus, in addition to their implication in nuclear export, Rrp12 and Crm1 participate in earlier biosynthetic steps that take place in the nucleolus. Our results indicate that, in the 40S subunit synthesis pathway, the completion of early pre-40S particle assembly, the initiation of byproduct degradation and the priming for nuclear export occur in an integrated manner in late 90S pre-ribosomes.

Ribosome Synthesis-Unrelated Functions of the Preribosomal Factor Rrp12 in Cell Cycle Progression and the DNA Damage Response

ABSTRACT Given the high metabolic cost required to generate ribosomes, it has been assumed that proteins involved in ribosome synthesis might establish functional cross talk with other intracellular processes to efficiently couple ribosome production and cell growth. However, such interconnections have remained elusive due to the difficulty in separating the intra- and extraribosomal roles of ribosome biogenesis factors. Using a yeast functional screen, I have discovered that Rrp12, a conserved protein involved in ribosome maturation and export, plays roles in the cell cycle and the DNA damage response. These results indicate that Rrp12 participates in a karyopherin Kap121-dependent import route that is crucial for nuclear sequestration of ribonucleotide reductase subunits and, thereby, ensures the proper kinetics of deoxyribonucleotide production during the cell cycle. Within this route, Rrp12 acts as a cofactor important for the full functionality of Kap121. This activity is mechanistically different from the known roles of Rrp12 in ribosome biogenesis. I propose that the functional duality of Rrp12 may couple the control of ribosome production to the regulation of other cellular processes during cell cycle progression.

Ribosome biogenesis and cancer: basic and translational challenges

Increasing evidence suggests that alterations in ribosome biogenesis (RiBi) confer competitive advantages to cancer cells. This has led to the discovery of regulatory layers mediated by signaling proteins, oncoproteins, and tumor suppressors whose deregulation leads to increased RiBi rates in cancer cells. In addition to boosting protein synthesis, these alterations probably contribute to shape the protumorigenic proteome of cancer cells. Mutations negatively affecting RiBi are also unexpectedly found in some spontaneous and ribosomopathy-associated tumors. The advantages provided by these genetic lesions to cancer cells remain unsettled as yet. Efforts are being made nowadays to exploit RiBi-associated vulnerabilities and tumor suppressor pathways to design new therapeutic avenues. In this review, we will summarize the main developments and pending challenges in this research area.