Saturation mutagenesis of the human 5S rRNA gene defines expression- and ribosome-incorporation-defective variants
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE339543 | GEO | 2026/09/02
REPOSITORIES: GEO
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Project description:In eukaryotes, three of the four ribosomal RNAs (rRNAs), the 5.8S, 18S and 25S/28S rRNAs, are processed from a single pre-rRNA transcript and assembled into ribosomes. The fourth rRNA, the 5S rRNA, is transcribed by RNA polymerase III and is assembled into the 5S ribonucleoprotein particle (5S RNP), containing ribosomal proteins Rpl5/uL18 and Rpl11/uL5, prior to its incorporation into pre-ribosomes. In mammals the 5S RNP is also central regulator of the homeostasis of the tumour suppressor p53. The nucleolar localisation of the 5S RNP and its assembly into pre-ribosomes is performed by a specialised complex composed of Rpf2 and Rrs1 in yeast or Bxdc1 and hRrs1 in humans. Here we report the structural and functional characterisation of the Rpf2-Rrs1 complex alone, in complex with the 5S RNA and within pre-60S ribosomes. We show that the Rpf2-Rrs1 complex contains a specialised 5S RNA E loop binding module, contacts the Rpl5 protein and also contacts the ribosome assembly factor Rsa4 and the 25S RNA. We propose that the Rpf2-Rrs1 complex establishes a network of interactions that guide the incorporation of the 5S RNP in pre-ribosomes in the initial conformation prior to its rotation to form the central protuberance found in the mature large ribosomal subunit.
2015-07-20 | GSE68431 | GEO
Project description:The 5S RNP is assembled from its three components (5S rRNA, Rpl5/uL18, and Rpl11/uL5) before being incorporated into the pre-60S subunit. However, when ribosome synthesis is disturbed, a free 5S RNP can enter the MDM2–p53 pathway to regulate cell cycle and apoptotic signaling. Here we reconstitute and determine the cryo-EM structure of the conserved hexameric 5S RNP with fungal or human factors. This reveals how the nascent 5S rRNA associates with the initial nuclear import complex Syo1–uL18–uL5, and upon further recruitment of two nucleolar factors Rpf2–Rrs1 develops into the 5S RNP precursor that can assemble into the pre-ribosome. In addition, we elucidate the structure of another 5S RNP intermediate, carrying the human ubiquitin ligase Mdm2, which unraveled how this enzyme can be sequestered from its target substrate p53. Our data provide molecular insight into how the 5S RNP can mediate between ribosome biogenesis and cell proliferation.
2023-06-13 | PXD040087 | Pride
Project description:While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.Ribosome biogenesis is a dynamic process that involves the ordered assembly of ribosomal proteins and numerous RNA structural rearrangements. Here the authors apply ChemModSeq, a high-throughput RNA structure probing method, to quantitatively measure changes in RNA flexibility during the nucleolar stages of 60S assembly in yeast.
2017-10-04 | GSE83821 | GEO
Project description:Assembly of eukaryotic ribosomes begins in the nucleolus, a compartmentalized membraneless organelle. Although the two ribosomal subunits, 40S and 60S, assemble independently, it remains unknown if these particles are physically sorted as they assemble and how they partition from the central chromatin compartment into the outer nucleolar regions, where maturation occurs. In this study, we show that nucleophosmin specifically mediates the assembly of nascent 60S subunits and that this specificity is determined by its chromatin localization at the rDNA sites encoding for 60S subunit rRNA. Nucleophosmin dissociates from chromatin to bind nascent 60S subunits, causing their partitioning away from chromatin and from nascent 40S subunits through liquid-liquid phase separation. This directs translocation of nascent 60S subunits towards the nucleophosmin-rich granular component, where biogenesis continues. Notably, this compartmentalization increases the efficiency of 60S subunit assembly, specifically the incorporation of the 60S domain III. Our data reveal that the chromatin localization of nucleophosmin determines its specificity in sorting and coordinates the movement of ribosomal subunits into specialized assembly compartments.
2023-03-01 | GSE156203 | GEO
Project description:The 120-nt long 5S rRNA, is an indispensable component of cytoplasmic ribosomes in all living organisms. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether maintaining 5S rRNA as an independent molecule is critical for ribosome function and cell survival. By fusing circularly permutated 5S rRNA (cp5S) with 23S rRNA and deleting all wild type 5S rRNA genes, we generated an Escherichia coli strain completely devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is not required for cell growth at 37°C and is unlikely to have essential functions outside the ribosome. The fully-assembled ribosomes carrying 23S-cp5S hybrid rRNA and lacking free 5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits that are unable to form stable 70S ribosomes. Cryo-EM analysis revealed a dramatically malformed peptidyl transferase center in the misassembled 50S subunits. The results of our experiments argue that the key evolutionary force preserving the autonomous nature of the smallest rRNA is its role in ribosome biogenesis.
2020-06-18 | PXD019058 | Pride
Project description:Ribosome assembly, which requires the temporal and spatial coordination of multiple interconnected processes, is essential for life in every known organism. The large ribosomal subunit (LSU) of Escherichia coli (E. coli) contains two RNA molecules, 23S and 5S, which serve as a platform for assembly. Ribosomal RNA (rRNA) modifications and processing are key steps in this process. We simultaneously detect and quantify eight classes of 23S rRNA modifications in the mature 50S LSU from E. coli cells expressing wild-type DbpA, the 50S LSU from cells expressing the helicase-inactive R331A DbpA construct, and two LSU intermediates, 35S and 45S, which accumulate in distinct ribosome maturation pathways in R331A DbpA-expressing cells. Additionally, we analyze the 3′-end processing of 23S and 5S rRNA in all these particles. Most 23S rRNA modifications are incorporated into the 35S and 45S intermediates at levels comparable to those in the 50S LSU from both wild-type and R331A DbpA-expressing cells, indicating that modification enzymes complete their functions before 35S and 45S accumulation. However, three enzymes exhibit differential modification patterns. RlmN incorporates N²-methyladenosine (m²A) at position 2507 more extensively in the 50S LSU from wild-type DbpA-expressing cells than in the 35S, 45S, and 50S LSU from R331A DbpA-expressing cells. Conversely, RluC incorporates pseudouridine (Ψ) at position 2508 at lower levels in the 50S LSU from wild-type DbpA-expressing cells than in the 35S, 45S, and 50S LSU from R331A DbpA-expressing cells. The RluF enzyme incorporates Ψ at position 2508 at a lower level in the 50S LSU from R331A DbpA-expressing cells than in the 35S and 45S intermediates and the 50S LSU from wild-type DbpA-expressing cells. Since the pathways leading to 35S and 45S account for only ~50% of total ribosome assembly in R331A DbpA-expressing cells, these findings reveal that RluF activity is reduced in a subset of maturation pathways. The 3′ end of 23S rRNA is less mature in the early-stage LSU intermediate, 35S, and more mature in the late-stage LSU intermediate, 45S. Interestingly, the 3′ end of 5S rRNA is less processed in the 45S than in the 35S, suggesting that 5S rRNA in the 45S intermediate matures during the final steps of large subunit ribosome assembly. A complete understanding of ribosome assembly demands a precise characterization of ribosome assembly pathways and processes across diverse cellular conditions. This study reveals critical insights into rRNA modifications and processing dynamics in bacterial ribosomes under stress.
2026-08-07 | GSE289243 | GEO
Project description:We purified pre60S ribosomes from yeast using Nsa1-FtpA as bait in Tandem Affinity Purification experiments. We tested the depletion of RPF2, using a Galactose-depletion system, to obtain ribosomal particles depleted of 5S RNP complexes. Sample A1: Control group (wild type strain) grown in YPGal; Sample A2: Control group (wild type strain) grown in YPGlu; Sample A3: Control group (Gal::HA-RPF2 strain) grown in YPGal; and Sample A4: Test group (Gal::HA-RPF2) shifted from YPGal to YPGlu for 6 hours to achieve 5S RNP depletion. Sample N1: Purified pre-60S Nsa1-associated with 5S RNP depleted after Sucrose gradient separation; Sample N2: Purified pre-60S Nsa1-associated with 5S RNP reconstituted after Sucrose gradient separation.
2023-10-24 | PXD040306 | Pride
Project description:The eukaryotic ribosome biogenesis is a highly orchestrated multistep process that starts at the nucleolus with the transcription of pre-rRNAs 5S and 35S. The latter comprises the mature 18S, 5.8S and 25S rRNAs separated by internal transcribed spacers (ITS1 and ITS2) and externally flanked by the 5’ETS and 3’ETS. The 35S pre-rRNA undergoes several co- and post-transcriptional processing events, which will enable the pre-60S and pre-40S particles to take independent maturation routes. Hundreds of assembly factors (AF) are required, being recruited and released hierarchically, for the proper folding of the rRNAs and correct positioning of the ribosomal proteins. One of the most intricate events that have recently been described is the removal of the ITS2-containing structure called pre-60S foot, which happens in a step-wise manner. Nop53 is an essential 60S AF that binds close to the ITS2 and plays a fundamental role in recruiting the RNA exosome for the 7S pre-rRNA processing, thereby dismantling the foot structure. Here we characterize the impact of Nop53 binding to the pre-60S on the compositional changes that happen during 60S assembly. For this purpose, preribosomes were affinity-purified with TAP-tagged 60S AFs (Nop7, Erb1, Rsa4, Arx1, Nmd3, Yvh1, and Lsg1) representative of different maturation stages both in the presence and absence of Nop53. Nop7 particles were also coimmunoprecipitated in the presence of Nop53 mutants incapable of recruiting the exosome (Nop53∆1-71, Nop53∆48-98) to compare with Nop53 depletion. The isolated preribosomes were analyzed by label-free MS/MS-based quantitative proteomics, revealing early and late-stage specific effects of Nop53 depletion.
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Project description:Total (rRNA-depleted) RNAseq of mouse ESCs carrying a homozygous KO mutation in setd2. Homozygous cells were also treated with the DNa methylation inhibitor 5-azacytidine. Total RNA was isolated from cell pellets using NEB Monarch® Total RNA Miniprep Kit and rRNA removed with NEBNext® rRNA Depletion Kit v2 (Human/Mouse/Rat). Libraries were prepared using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB E7760L, input: 500 ng of total RNA). Libraries were multiplexed and sequenced on a NextSeq500 (Paired-End; read length 40). Each sample is present in 3 biological replicates.
2023-11-15 | E-MTAB-13468 | biostudies-arrayexpress
Project description:The 17-subunit RNA polymerase III (RNAP III) synthesizes essential untranslated RNAs such as tRNAs and 5S rRNA. In yeast and vertebrates, subunit C82 forms a stable subcomplex with C34 and C31 that is necessary for promoter-specific transcription initiation. Little is known about RNAP III transcription in trypanosomatid parasites. To narrow this knowledge gap, we characterized the C82 subunit in Trypanosoma brucei and Leishmania major. Bioinformatic analyses showed that the four distinctive winged-helix domains and the coiled-coil motif are present in C82 in these microorganisms, despite the observation of certain unique traits. Also, their projected three-dimensional structure is comparable to that of the human orthologue. C82 localizes to the nucleus and binds to RNAP III-dependent genes in the insect stages of both parasites. In T. brucei, knock-down of C82 by RNA interference significantly reduced the levels of tRNAs and 5S rRNA and led to the death of procyclic forms of the pathogen. Tandem affinity purifications with both parasites allowed the identification of several C82-interacting partners, including C34 and some genus-specific putative regulators of transcription. However, the orthologue of C31 was not found in trypanosomatids. Interestingly, our data suggest a strong association of C82 with TFIIIC subunits in T. brucei, but not in L. major.
2024-11-13 | PXD051017 | Pride