Structural, topological, and functional characterization of transmembrane proteins TMEM213, 207, 116, 72 and 30B provides a potential link to ccRCC etiology.

Due to their involvement in the development of various cancers Transmembrane Proteins (TMEMs) are the focus of many recent studies. Previously we reported TMEM de-regulation in clear cell Renal Cell Carcinoma (ccRCC) with TMEM213, 207, 116, 72 and 30B being among the most downregulated on mRNA level. TMEM down-regulation was also more pronounced in advanced ccRCC tumors and was potentially linked to clinical parameters such as: metastasis (TMEM72 and 116), Fuhrman grade (TMEM30B) and overall survival (TMEM30B). To further investigate these findings, first, we set off to prove experimentally that selected TMEMs are indeed membrane-bound as predicted in silico, we verified the presence of signaling peptides on their N-termini, orientation of TMEMs within the membrane and validated their predicted cellular localization. To investigate the potential role of selected TMEMs in cellular processes overexpression studies in HEK293 and HK-2 cell lines were carried out. Additionally, we tested TMEM isoform expression in ccRCC tumors, identified mutations in TMEM genes and examined chromosomal aberrations in their loci. We confirmed the membrane-bound status of all selected TMEMs, assigned TMEM213, and 207 to early endosomes, TMEM72 to early endosomes and plasma membrane, TMEM116 and 30B to the endoplasmic reticulum. The N-terminus of TMEM213 was found to be exposed to the cytoplasm, the C-terminus of TMEM207, 116 and 72 were directed toward the cytoplasm, and both termini of TMEM30B faced the cytoplasm. Interestingly, TMEM mutations and chromosomal aberrations were infrequent in ccRCC tumors, yet we identified potentially damaging mutations in TMEM213 and TMEM30B and found deletions in the TMEM30B locus in nearly 30% of the tumors. Overexpression studies suggested selected TMEMs may take part in carcinogenesis processes such as cell adhesion, regulation of epithelial cell proliferation, and regulation of adaptive immune response, which could indicate a link to the development and progression of ccRCC.

Identification of a novel alternatively spliced isoform of the ribosomal uL10 protein.

Alternative splicing is one of the key mechanisms extending the complexity of genetic information and at the same time adaptability of higher eukaryotes. As a result, the broad spectrum of isoforms produced by alternative splicing allows organisms to fine-tune their proteome; however, the functions of the majority of alternatively spliced protein isoforms are largely unknown. Ribosomal protein isoforms are one of the groups for which data are limited. Here we report characterization of an alternatively spliced isoform of the ribosomal uL10 protein, named uL10ß. The uL10 protein constitutes the core element of the ribosomal stalk structure within the GTPase associated center, which represents the landing platform for translational GTPases - trGTPases. The stalk plays an important role in the ribosome-dependent stimulation of GTP by trGTPases, which confer unidirectional trajectory for the ribosome, allosterically contributing to the speed and accuracy of translation. We have shown that the newly identified uL10ß protein is stably expressed in mammalian cells and is primarily located within the nuclear compartment with a minor signal within the cytoplasm. Importantly, uL10ß is able to bind to the ribosomal particle, but is mainly associated with 60S and 80S particles; additionally, the uL10ß undergoes re-localization into the mitochondria upon endoplasmic reticulum stress induction. Our results suggest a specific stress-related dual role of uL10ß, supporting the idea of existence of specialized ribosomes with an altered GTPase associated center.

An Improved Vector System for Homogeneous and Stable Gene Regulation.

Precise analysis of the genetic expression and functioning of proteins requires experimental approaches that, among others, enable tight control of gene expression at the transcriptional level. Doxycycline-induced Tet-On/Tet-Off expression systems provide such an opportunity, and are frequently used to regulate the activity of genes in eukaryotic cells. Since its development, the Tet-system has evolved tight gene control in mammalian cells; however, some challenges are still unaddressed. In the current set up, the establishment of the standard Tet-based system in target cells is time-consuming and laborious and has been shown to be inefficient, especially in a long-term perspective. In this work, we present an optimized inducible expression system, which enables rapid generation of doxycycline-responsive cells according to a one- or two-step protocol. The reported modifications of the Tet-On system expand the toolbox for regulated mammalian gene expression and provide high, stable, and homogenous expression of the Tet-On3G transactivator, which is of fundamental importance in the regulation of transgenes.

Phosphorylation of the N-terminal domain of ribosomal P-stalk protein uL10 governs its association with the ribosome.

The uL10 protein is the main constituent of the ribosomal P-stalk, anchoring the whole stalk to the ribosome through interactions with rRNA. The P-stalk is the core of the GTPase-associated center (GAC), a critical element for ribosome biogenesis and ribosome translational activity. All P-stalk proteins (uL10, P1, and P2) undergo phosphorylation within their C termini. Here, we show that uL10 has multiple phosphorylation sites, mapped also within the N-terminal rRNA-binding domain. Our results reveal that the introduction of a negative charge within the N terminus of uL10 impairs its association with the ribosome. These findings demonstrate that uL10 N-terminal phosphorylation has regulatory potential governing the uL10 interaction with the ribosome and may control the activity of GAC.

The influence of ricin-mediated rRNA depurination on the translational machinery in vivo - New insight into ricin toxicity.

The generally accepted model of ricin intoxication assumes that direct inactivation of ribosomes by depurination of a specific adenine residue within the sarcin-ricin-loop (SRL) on the 60S ribosomal subunit is a major source of its toxicity. The model proposes that SRL depurination leads to protein synthesis inhibition, evoking ribotoxic stress with concomitant induction of numerous metabolic pathways, which lead to cell death. However, the direct relationship between the depurination and its impact on the translational machinery in vivo has never been satisfactorily explained. In this work, we approached a long-standing question about the influence of SRL depurination on the functioning of the translational machinery in vivo. We have shown that an already low level of depurinated ribosomes exert an effect on cell metabolism, indicating that minute modification within the ribosomal pool is sufficient to elicit a toxic effect. Importantly, depurination does not affect notably any particular step of translation, and translational slowdown caused by ricin is not a direct consequence of depurination and cannot be considered as the sole source of cell death. Instead, SRL depurination in a small fraction of ribosomes blocks cell cycle progression with no effect on cell viability. In this work, we have provided a comprehensive picture of the impact of SRL depurination on the translational apparatus in vivo. We propose that ribosomes with depurinated SRL represent a small imprinted ribosomal pool, which generates a specific signal for the cell to halt the cell cycle.

The uL10 protein, a component of the ribosomal P-stalk, is released from the ribosome in nucleolar stress.

The ribosomal uL10 protein, formerly known as P0, is an essential element of the ribosomal GTPase-associated center responsible for the interplay with translational factors during various stages of protein synthesis. In eukaryotic cells, uL10 binds two P1/P2 protein heterodimers to form a pentameric P-stalk, described as uL10-(P1-P2)2, which represents the functional form of these proteins on translating ribosomes. Unlike most ribosomal proteins, which are incorporated into pre-ribosomal particles during early steps of ribosome biogenesis in the nucleus, P-stalk proteins are attached to the 60S subunit in the cytoplasm. Although the primary role of the P-stalk is related to the process of translation, other extraribosomal functions of its constituents have been proposed, especially for the uL10 protein; however, the list of its activities beyond the ribosome is still an open question. Here, by the combination of biochemical and advanced fluorescence microscopy techniques, we demonstrate that upon nucleolar stress induction the uL10 protein accumulates in the cytoplasm of mammalian cells as a free, ribosome-unbound protein. Importantly, using a novel approach, FRAP-AC (FRAP after photoConversion), we have shown that the ribosome-free pool of uL10 represents a population of proteins released from pre-existing ribosomes. Taken together, our data indicate that the presence of uL10 on the ribosomes is affected in stressed cells, thus it might be considered as a regulatory element responding to environmental fluctuations.

Multiplication of Ribosomal P-Stalk Proteins Contributes to the Fidelity of Translation.

The P-stalk represents a vital element within the ribosomal GTPase-associated center, which represents a landing platform for translational GTPases. The eukaryotic P-stalk exists as a uL10-(P1-P2)2 pentameric complex, which contains five identical C-terminal domains, one within each protein, and the presence of only one such element is sufficient to stimulate factor-dependent GTP hydrolysis in vitro and to sustain cell viability. The functional contribution of the P-stalk to the performance of the translational machinery in vivo, especially the role of P-protein multiplication, has never been explored. Here, we show that ribosomes depleted of P1/P2 proteins exhibit reduced translation fidelity at elongation and termination steps. The elevated rate of the decoding error is inversely correlated with the number of the P-proteins present on the ribosome. Unexpectedly, the lack of P1/P2 has little effect in vivo on the efficiency of other translational GTPase (trGTPase)-dependent steps of protein synthesis, including translocation. We have shown that loss of accuracy of decoding caused by P1/P2 depletion is the major cause of translation slowdown, which in turn affects the metabolic fitness of the yeast cell. We postulate that the multiplication of P-proteins is functionally coupled with the qualitative aspect of ribosome action, i.e., the recoding phenomenon shaping the cellular proteome.

Functional analysis of the uL11 protein impact on translational machinery.

The ribosomal GTPase associated center constitutes the ribosomal area, which is the landing platform for translational GTPases and stimulates their hydrolytic activity. The ribosomal stalk represents a landmark structure in this center, and in eukaryotes is composed of uL11, uL10 and P1/P2 proteins. The modus operandi of the uL11 protein has not been exhaustively studied in vivo neither in prokaryotic nor in eukaryotic cells. Using a yeast model, we have brought functional insight into the translational apparatus deprived of uL11, filling the gap between structural and biochemical studies. We show that the uL11 is an important element in various aspects of 'ribosomal life'. uL11 is involved in 'birth' (biogenesis and initiation), by taking part in Tif6 release and contributing to ribosomal subunit-joining at the initiation step of translation. uL11 is particularly engaged in the 'active life' of the ribosome, in elongation, being responsible for the interplay with eEF1A and fidelity of translation and contributing to a lesser extent to eEF2-dependent translocation. Our results define the uL11 protein as a critical GAC element universally involved in trGTPase 'productive state' stabilization, being primarily a part of the ribosomal element allosterically contributing to the fidelity of the decoding event.

Molecular behavior of human Mrt4 protein, MRTO4, in stress conditions is regulated by its C-terminal region.

Protein Mrt4 is one of trans-acting factors involved in ribosome biogenesis, which in higher eukaryotic cells contains a C-terminal extension similar to the C-terminal part of ribosomal P proteins. We show that human Mrt4 (hMrt4/MRTO4) undergoes phosphorylation in vivo and that serines S229, S233, and S235, placed within its acidic C-termini, have been phosphorylated by CK2 kinase in vitro. Such modification does not alter the subcellular distribution of hMrt4 in standard conditions but affects its molecular behavior during ActD induced nucleolar stress. Thus, we propose a new regulatory element important for the stress response pathway connecting ribosome biogenesis with cellular metabolism.