Fig 1.
Position of r-proteins studied in this work in ribosomes and overview of early yeast LSU rRNA processing events.
The yeast LSU is shown in A) and B) viewed from the solvent exposed side with LSU rRNA domain II in yellow, LSU rRNA expansion segment ES7 in orange, rpL4/uL4 in dark blue, rpL7/uL30 in light blue, rpL16/uL13 in light green, rpL18/eL18 in red, rpL20/eL20 in dark green, rpL32/eL32 in purple and rpL33/eL33 in brown. Other parts of the LSU are shown in grey in A) and are hidden in B). In C) is shown an overview of yeast LSU rRNA processing highlighting early events leading to formation of the 5.8S rRNA 5’ end at sites B1S and B1L. The scheme was adapted and modified from [9]. Scissors and pacman symbols illustrate that a pre-rRNA processing event is endonucleolytic or exonucleolytic, respectively. Enzymes catalyzing the respective processing events are indicated, if known, according to [9]. Major pre-rRNAs co-purifying with Noc2 are highlighted on the right.
Fig 2.
Association of Noc2 with early LSU precursors after in vivo depletion of LSU r-proteins required for early LSU pre-rRNA processing.
The indicated yeast strains, expressing a chromosomally-encoded TAP-tagged version of the LSU biogenesis factor Noc2, together with either the indicated or no LSU r-protein gene under control of the galactose-inducible GAL1/10 promoter, were cultivated for four hours in glucose-containing medium to shut down expression of the respective LSU r-protein gene. Noc2-TAP and associated pre-ribosomal particles were then affinity purified from the corresponding cellular extracts as described in Materials and Methods. The (pre-) rRNA content of total cellular extracts (“Input” lanes 1–12) or of parts of the affinity purified fractions (“IP”lanes 13–24) was analyzed by northern blotting. (pre-) rRNAs detected by DNA oligonucleotide probes shown on the right are denoted on the left. Equal signal intensities in the Input and IP fractions indicate that 4% of the respective (pre-)rRNA population co-purified with Noc2-TAP.
Fig 3.
Changes in the association of LSU biogenesis factors with early LSU precursors after in vivo depletion of LSU r-proteins required for early LSU pre-rRNA processing.
Conditional r-protein expression mutants or wild type cells were cultivated as described in Fig 2. Noc2-TAP and associated pre-ribosomal particles were then affinity purified from corresponding cellular extracts (see Materials and Methods). Proteins in Noc2-TAP fractions were identified and quantified by mass spectrometry. Isobaric labeling of peptides (iTRAQ, see Materials and Methods) was used to directly compare levels of individual proteins in Noc2-TAP fractions from wild type cells with the respective levels in Noc2-TAP fractions from conditional r-protein expression mutants. In A) is shown the average proteome composition of nine Noc2-TAP fractions analyzed in this study by mass spectrometry. In B) the changes in levels of individual LSU biogenesis factors in Noc2-TAP fractions from mutant versus wild type cells as determined by iTRAQ analyzes are summarized. Each pair wise comparison (“mutant versus wild type”) was performed at least twice starting from independent cell cultures. Data from 17 pairwise comparisons were used to detect co-behaving groups of LSU biogenesis factors by a clustering algorithm (see Materials and Methods). Similar behaving proteins are grouped in the same branch of the dendrogram depicted on the left. The iTRAQ ratios of LSU biogenesis factors for each individual pairwise comparison are shown as heat map using the color code depicted on the upper right. Only LSU biogenesis factors were included in these analyses, which were identified in at least 70% of the 17 pairwise comparisons. Groups of LSU biogenesis factors mentioned in the text are highlighted by bars on the right.
Fig 4.
Changes in r-protein assembly states of early LSU precursors after in vivo depletion of LSU r-proteins required for early LSU pre-rRNA processing.
The dataset generated as described in Fig 3 was analyzed in respect to changes in r-protein levels in Noc2-TAP fractions isolated from wild type cells or from r-protein expression mutants. Observed changes in levels of r-proteins and the results of clustering analyses are visualized as described in Fig 3B. Groups of r-proteins mentioned in the text are highlighted by bars on the right. Only r-proteins which were identified in at least 70% of the 17 pairwise comparisons were included in these analyses. The r-proteins whose expression was shut down in the respective experiment (“mutant versus wild type”) are highlighted by a red box.
Fig 5.
Interactions of dII/dVI cluster r-proteins with rRNA in mature ribosomes.
The yeast LSU is shown as in Fig 1 viewed from the solvent exposed side in A), C) and E). It is rotated by approximately 90 degree around a horizontal axis in B), D) and F). In A)–F) LSU rRNA domain II is colored in yellow, LSU rRNA expansion segment 7 in orange, LSU rRNA domain VI in blue, 5.8S rRNA in red and other parts of the LSU rRNA in grey. In A) and B) dII/dVI cluster r-proteins are the only r-proteins shown and colored in shades of green. In C) and D) only rRNA is visualized and in E) and F) rpL16/uL13 is the only r-protein shown, with its globular domain in light green and its C-terminal clamp-like domain in dark green.
Fig 6.
Impact of the C-terminal clamp-like domain of rpL16 on yeast growth and LSU rRNA processing.
A) The wild type (WT) RPL16A, RPL16B yeast strain BY4741 and transformants of yeast strain TY931 expressing the full length allele of RPL16B under control of the GAL1/10 promoter (pGAL-RPL16B), and containing the indicated RPL16Balleles on low copy (lc) plasmids or the FLAG-tagged versions of RPL16B alleles on high copy (hc) plasmids, were grown in selective complete medium with galactose as carbon source (SCGal-U) to an OD600 of 0.6 at 30°C. The same amount of cells of each culture was spotted on solid YPD medium in a 1:10 serial dilution series and incubated for 3 days at 30°C. B) and C) strains from A) harbouring the lc plasmids coding for the indicated truncated protein variants of rpL16B were grown at 28°C in SCGal-U and then cultivated in glucose containing medium (YPD). Total RNA was extracted from cells at the indicated time points, and steady-state levels of pre-rRNAs were assayed by northern blotting B), or primer extension and denaturing gel electrophoresis C).
Fig 7.
Impact of the C-terminal clamp-like domain of rpL16/uL13 on recruitment into early yeast LSU precursors.
Yeast strains Hm653, Hm654 and Hm655 ectopically expressing only the indicated FLAG tagged variants of rpL16/uL13 were grown in selective complete medium with galactose as carbon source and were then cultivated for the indicated times in glucose containing medium. Cells were harvested and subjected to affinity purification using an anti-FLAG matrix as described in Materials and Methods. Wild type yeast strain BY4741 expressing only untagged rpL16/uL13 was included as control in these analyses. The (pre-) rRNA content of the total cellular extracts (“Input” lanes) or of parts of the affinity purified fractions (“IP” lanes) were analyzed by northern blotting using the indicated probes.
Fig 8.
Contacts between LSU rRNA domains II and VI and position of the LSU rRNA 5’ end in bacterial, archaeal and eukaryotic ribosomes.
Structure models are shown of the LSU of the bacterium Escherichia coli in A) and B), the archaeon Pyrococcus furiosus in C) and D), and the two eukaryotes Saccharomyces cerevisiae in E) and F) and Sus scrofa in G) and H). LSU rRNA expansion segments ES7 and ES39, and dII/dVI cluster r-proteins are colored in yellow, except the universal conserved globular domain of rpL16/uL13 which is shown in green. 5.8S rRNA is highlighted in red and other parts of LSU rRNA, except ES7 and ES39, are grey. In A), C), E) and G) only rRNA is shown and in B), D), F) and H) dII/dVI cluster r-proteins are the only r-proteins visualized.