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

Abstract

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), 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.

Cell surface expression of HLA I molecules as a marker of young platelets

“…Flow cytometric analyses showed that RNaseA treatment significantly decreased the MFI of TO on platelets from control and patient P1 but only moderately affected this value on platelets from patient P2. Therefore, the high TO staining in P2 platelets as compared to that in control platelets was not due to a higher RNA content, but probably to a greater number of dense granules that contributed to TO fluorescence . In contrast, in P1 platelets the TO staining was partly due to a higher proportion of young platelets, confirming the observed elevated expression of HLA I (Figure D, right panel).…”

“…Ribosomal P‐antigen is present on the C‐terminal part of three ribosomal subunits (RLP 0, 2, and 3) known to be necessary for messenger RNA (mRNA) translation . Since mRNA translation occurs mainly in platelets that are a few hours old, one would expect to detect ribosomal P‐antigen in TO bright cells.…”

uL3 Mediated Nucleolar Stress Pathway as a New Mechanism of Action of Antiproliferative G-quadruplex TBA Derivatives in Colon Cancer Cells

“…In this context, the results of MTT assays that suggest a role of uL3 in the cytotoxic activity of LQ1 led us to hypothesize that the activity of this ODN could be mediated by a nucleolar stress pathway involving uL3. To test this hypothesis, we analyzed the expression profile of uL3 and a subset of ribosomal proteins known to be involved in the nucleolar stress response, including uL5, uL11, uL18, uS12 [21,35,36], together with the nucleolar marker B23/NPM after treatment of the cells with LQ1. To this aim, HCT 116 p53−/− and uL3∆HCT 116 p53−/− cells were treated with 10 µM of LQ1, and, 48 h later, the relative abundance of uL3, uL5, uL11, uL18, uS12, and B23/NPM mRNAs was determined by RT-qPCR with specific primers ( Table 2).…”

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

“…Genetic manipulations were performed on a pEGFP-C1-uL10 plasmid described previously [45][46][47]. To obtain phospho-and dephosphomimetic variants of GFP-tagged uL10 protein, the pEGFP-C1-uL10 plasmid was mutated by replacing the TAT triplet coding Y24, the ACC triplet coding T59, or TCG coding S304 and S307 in the DNA sequence for the uL10 protein with GAA to obtain Y24E, T59E, and S304E/S307E (phosphomimetic) and with TTT, GCT, and GCG to obtain Y24F, T59A, and S304A/S307A (dephosphomimetic), respectively.…”