ROLE OF RIBOSOMAL PROTEIN L4 IN RIBOSOME BIOGENEIS AND CELL CYCLE PROGRESSION IN S.CEREVISIAE

Ribosomal protein L4 (RPL4) is a large ribosomal subunit protein that is structurally conserved in all kingdoms of life. This protein is a component of the 90S pre-ribosomal particle that initiates ribosomal assembly on the primary (35S) transcript. Here I show that in vivo repression of Rpl4p synthesis in S. cerevisiae results in severe loss of 60S ribosomal subunits and affects progression of the cell cycle. Analysis of rRNA processing suggests that these effects are associated with a block in the processing of the 27SA3 precursor RNA into 5.8S and 25S rRNA as well as a delay in processing of 35S precursor. More surprisingly, depletion of Rpl4p results in a unique bi-budded phenotype, with multiple cell cycle defects mainly affecting mitotic exit. To further characterize the role of RPL4 in cell cycle progression, I isolated temperature-sensitive L4 mutants. To date I have analyzed one of these mutants. Six hours after a temperature shift of this mutant, cells are uniformly arrested in SG2 progression with single large bud, unseparated genetic material, and emerging spindle apparatus. Interestingly, the mutation lies in the extended tentacles of RPL4, which is disposable in prokaryotes, but appears to play a key role in the functioning of this protein in eukaryotes. All the above data indicate an important extra-ribosomal function of RPL4 in orchestrating ribosome biogenesis with cell cycle progression in S.cerevisiae. Currently, we are attempting to define more precisely how Rpl4p is involved in these essential cellular phenomena

ROLE OF RIBOSOMAL PROTEIN L4 IN RIBOSOME BIOGENEIS AND CELL CYCLE PROGRESSION IN S.CEREVISIAE

Ribosomal protein L4 (RPL4) is a large ribosomal subunit protein that is structurally conserved in all kingdoms of life. This protein is a component of the 90S pre-ribosomal particle that initiates ribosomal assembly on the primary (35S) transcript. Here I show that in vivo repression of Rpl4p synthesis in S. cerevisiae results in severe loss of 60S ribosomal subunits and affects progression of the cell cycle. Analysis of rRNA processing suggests that these effects are associated with a block in the processing of the 27SA3 precursor RNA into 5.8S and 25S rRNA as well as a delay in processing of 35S precursor. More surprisingly, depletion of Rpl4p results in a unique bi-budded phenotype, with multiple cell cycle defects mainly affecting mitotic exit. To further characterize the role of RPL4 in cell cycle progression, I isolated temperature-sensitive L4 mutants. To date I have analyzed one of these mutants. Six hours after a temperature shift of this mutant, cells are uniformly arrested in SG2 progression with single large bud, unseparated genetic material, and emerging spindle apparatus. Interestingly, the mutation lies in the extended tentacles of RPL4, which is disposable in prokaryotes, but appears to play a key role in the functioning of this protein in eukaryotes. All the above data indicate an important extra-ribosomal function of RPL4 in orchestrating ribosome biogenesis with cell cycle progression in S.cerevisiae. Currently, we are attempting to define more precisely how Rpl4p is involved in these essential cellular phenomena

Analysis of cell cycle parameters during the transition from unhindered growth to ribosomal and translational stress conditions

<div><p>Abrogation of ribosome synthesis (ribosomal stress) leads to cell cycle arrest. However, the immediate cell response to cessation of ribosome formation and the transition from normal cell proliferation to cell cycle arrest have not been characterized. Furthermore, there are conflicting conclusions about whether cells are arrested in G2/M or G1, and whether the cause is dismantling ribosomal assembly per se, or the ensuing decreased number of translating ribosomes. To address these questions, we have compared the time kinetics of key cell cycle parameters after inhibiting ribosome formation or function in <i>Saccharomyces cerevisiae</i>. Within one-to-two hours of repressing genes for individual ribosomal proteins or Translation Elongation factor 3, configurations of spindles, spindle pole bodies began changing. Actin began depolarizing within 4 hours. Thus the loss of ribosome formation and function is sensed immediately. After several hours no spindles or mitotic actin rings were visible, but membrane ingression was completed in most cells and Ace2 was localized to daughter cell nuclei demonstrating that the G1 stage was reached. Thus cell division was completed without the help of a contractile actin ring. Moreover, cell wall material held mother and daughter cells together resulting in delayed cell separation, suggesting that expression or function of daughter gluconases and chitinases is inhibited. Moreover, cell development changes in very similar ways in response to inhibition of ribosome formation and function, compatible with the notion that decreased translation capacity contributes to arresting the cell cycle after abrogation of ribosome biogenesis. Potential implications for the mechanisms of diseases caused by mutations in ribosomal genes (ribosomopathies) are discussed.</p></div

Structure of mother-daughter complexes.

<p>(A) Ace2 in Pgal-eL43 was tagged with GFP and Spc42 with RFP. (i) Mother cell with daughter cell during growth in galactose medium. (ii) Mother cells with daughter cell 6 hours after switch to glucose medium. Note the cell wall tether in the bright field image (arrow). (iii) Mother cell with two daughter cells after 16 hours of growth in glucose medium. Note that Ace2 is only in one of the buds. (B) Effect of zymolyase digestion on distribution of single cells, mother cells with bud or daughter, and mother with two daughters or buds in Pgal-eL43 and Pgal-uL30 cultures shifted from galactose to glucose medium for 20 hours. At least 100 cells were counted for each histogram. Raw values for the counts unbudded, single budded and dibudded cells is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186494#pone.0186494.s009" target="_blank">S2 Table</a>.</p

Dynamics of cell membranes during ribosomal and translational stress.

<p>The plasma membrane protein Ras2 was tagged by GFP and the SPB protein Spc42 was tagged by RFP. (A) Pgal-eL43 growing unhindered in galactose medium. (B-C) Mother-daughter complexes of Pgal-eL43 after 16 hours in glucose medium. (D) Mother-daughter complexes of Pgal-eEF3 synthesis after 31 hours in glucose medium. Arrows point to SPBs.</p

Quantification of cell cycle stages classified by spindle and membrane structure, and position of SPB(s).

<p>Data were obtained from classifying and counting cells on field images collected at different times beginning one hour after the repression of the genes for uS4, eL43, or eEF3. A cell is counted as an individual cell, if it is surrounded by a completed plasma membrane, whether it is in mother-daughter complexes or a single cell. Cells in each classification are normalized to the total number of cells. (A) Single cells (i.e. cells not in mother daughter complexes) with one bud and one SPB. (B) Single cells with two SPBs with no spindle or spindles shorter than an anaphase spindle. (C) Anaphase cells with two SPBs, one at each end of the mother-daughter axis and connected by a long spindle. (D) Mother cells with attached daughters surrounded by a complete plasma membrane, i.e. “mother-daughter complexes”. (E) Total number of cells surrounded by a plasma membrane in mother-daughter complexes. F. Total number of unbudded cells (single cells or in mother daughter complexes). The number of cells counted for each time point was 101–295. (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186494#pone.0186494.s008" target="_blank">S1 Table</a> for raw cell counts.) Blue triangles: repressed uS4 synthesis; red and green circles: biological replicates of repressed eL43 synthesis; light and dark blue diamonds: biological replicates of repressed eEF3 synthesis.</p

Quantification of polarized and dispersed actin patches.

<p>(A-C) The number of cells in each state was normalized to the total number of cells. (A) Polarized to budsite. (B) Dispersed. (C) Polarized to budneck. Between 58 and 446 cells were counted for each strain and time point. Raw data and calculation of the aggregate categories plotted in the figure are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186494#pone.0186494.s010" target="_blank">S3 Table</a> and an overview of the characteristics of the classifications used is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186494#pone.0186494.s011" target="_blank">S4 Table</a>.</p

Comparison of repression of r-proteins and translation elongation factor 3 (eEF3).

<p>Strains in which uL4, uL18, eL43, uS4, or eEF3 synthesis is under control of the <i>GAL1/10</i> promoter were grown in galactose medium and shifted to glucose medium for the indicated length of time. (A) Western analysis of eEF3 before and after switching strains expressing eEF3 from the Gal1/10 promoter to glucose medium. The chromosomal gene of TEF3, or both TEF3 and HEF3, were deleted. R-protein uL18 was used as a loading standard. (B) Western analysis of eEF3 before and after glucose repression of the genes for uL4, eEF3, or uS4. (C) Sucrose gradient analysis of extracts prepared before and after repression of eL43 for 16 hours and eEF3 for 26 hours. (D) Flow cytometry (cell number vs. DNA content) of Pgal-uL4B, -uL18, -eL43, and–uS4 growing in galactose or shifted to glucose for the indicated times. Brackets R2, R3, and R4 correspond to 1N, 2N and 3N amounts of DNA, respectively.</p

Dynamics of spindle and spindle pole body (SPB) during ribosomal stress.

<p>The Pgal-eL43 strain tagged with (A-B) Tub1-GFP or (C-D) Tub1-GFP and Spc42-RFP were grown in galactose (A and C) and shifted to glucose for 16 hours (B and D). (A) Spindle structures in cells growing in galactose. The white arrows in point to the long astral microtubules and red arrows point to anaphase spindles. (B) Merge of bright field and Tub1-GFP after 16 hours in glucose medium. Note the long astral microtubules in most cells. (C-D) Merges of Tub1-GFP and Spc42-RFP in cells growing in (C) galactose or (D) shifted to glucose for 16 hours. The top left shows Tub1-GFP, top right shows Spc42-RFP, bottom left shows the brightfield image, and right bottom shows the merged images.</p

Dynamics of actin patches during ribosomal and translational stress.

<p>Actin was stained with rhodamine-phalloidin. (A) Pgal-eL43 tagged with Tub1-GFP was grown in galactose medium and shifted to glucose medium. (i) Cells grown in galactose. (ii) Cells switched to glucose medium for 6 hours. Note the large astral microtubules and the lack of actin rings. (B) Pgal-eL43 strain tagged with GFP-Ras2. (i) Cells growing in galactose. Note the actin rings in cells after membrane ingression. (ii) Cell switched to glucose for 6 hours. Note the lack of actin rings in mother and the two post-mitotic daughter cells. (C) Pgal-eEF3 strain stained with phalloidin. (i) Cells grown in galactose. (ii) Cells after growth in glucose for 31 hours. The image shows a rare example of polarization to budsite. (D) Distribution of actin patches after cycloheximide inhibition of translation. (i) Pgal-uS4 cells grown in galactose without CHX (left), (ii) grown in galactose with 100 ug/ml CHX for 1hr. White arrows point to actin polarized to bud sites. Red arrows show actin rings in mother and daughter cells. The yellow arrow shows a spindle that does not stretch to the ends of the mother daughter axis (pre-anaphase); note that no actin rings are visible at this stage.</p