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Nature Structural & Molecular Biology May 2023During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large...
During transcription of eukaryotic ribosomal DNA in the nucleolus, assembly checkpoints exist that guarantee the formation of stable precursors of small and large ribosomal subunits. While the formation of an early large subunit assembly checkpoint precedes the separation of small and large subunit maturation, its mechanism of action and function remain unknown. Here, we report the cryo-electron microscopy structure of the yeast co-transcriptional large ribosomal subunit assembly intermediate that serves as a checkpoint. The structure provides the mechanistic basis for how quality-control pathways are established through co-transcriptional ribosome assembly factors, that structurally interrogate, remodel and, together with ribosomal proteins, cooperatively stabilize correctly folded pre-ribosomal RNA. Our findings thus provide a molecular explanation for quality control during eukaryotic ribosome assembly in the nucleolus.
Topics: Cryoelectron Microscopy; RNA, Ribosomal; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ribosomal Proteins; Ribosome Subunits, Large; Ribosome Subunits, Large, Eukaryotic; Ribosome Subunits, Small, Eukaryotic
PubMed: 37037974
DOI: 10.1038/s41594-023-00947-3 -
Nucleic Acids Research Sep 2023Ribosome biogenesis is one of the biggest consumers of cellular energy. More than 20 genetic diseases (ribosomopathies) and multiple cancers arise from defects in the...
Ribosome biogenesis is one of the biggest consumers of cellular energy. More than 20 genetic diseases (ribosomopathies) and multiple cancers arise from defects in the production of the 40S (SSU) and 60S (LSU) ribosomal subunits. Defects in the production of either the SSU or LSU result in p53 induction through the accumulation of the 5S RNP, an LSU assembly intermediate. While the mechanism is understood for the LSU, it is still unclear how SSU production defects induce p53 through the 5S RNP since the production of the two subunits is believed to be uncoupled. Here, we examined the response to SSU production defects to understand how this leads to the activation of p53 via the 5S RNP. We found that p53 activation occurs rapidly after SSU production is blocked, prior to changes in mature ribosomal RNA (rRNA) levels but correlated with early, middle and late SSU pre-rRNA processing defects. Furthermore, both nucleolar/nuclear LSU maturation, in particular late stages in 5.8S rRNA processing, and pre-LSU export were affected by SSU production defects. We have therefore uncovered a novel connection between the SSU and LSU production pathways in human cells, which explains how p53 is induced in response to SSU production defects.
Topics: Humans; Ribosomal Proteins; Ribosome Subunits, Large; Ribosome Subunits, Small; Ribosomes; RNA, Ribosomal; Tumor Suppressor Protein p53
PubMed: 37526268
DOI: 10.1093/nar/gkad637 -
Science Advances Jun 2017Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating...
Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNA to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap.
Topics: Models, Molecular; Peptides; Protein Biosynthesis; Protein Conformation; RNA, Messenger; Ribosome Subunits, Small, Bacterial; Ribosomes; Structure-Activity Relationship
PubMed: 28630923
DOI: 10.1126/sciadv.1700147 -
Plant Physiology May 2018Ribosome biogenesis is crucial for plant growth and environmental acclimation. Processing of ribosomal RNAs (rRNAs) is an essential step in ribosome biogenesis and...
Ribosome biogenesis is crucial for plant growth and environmental acclimation. Processing of ribosomal RNAs (rRNAs) is an essential step in ribosome biogenesis and begins with transcription of the rDNA. The resulting precursor-rRNA (pre-rRNA) transcript undergoes systematic processing, where multiple endonucleolytic and exonucleolytic cleavages remove the external and internal transcribed spacers (ETS and ITS). The processing sites and pathways for pre-rRNA processing have been deciphered in and, to some extent, in , mammalian cells, and Arabidopsis (). However, the processing sites and pathways remain largely unknown in crops, particularly in monocots such as rice (), one of the most important food resources in the world. Here, we identified the rRNA precursors produced during rRNA biogenesis and the critical endonucleolytic cleavage sites in the transcribed spacer regions of pre-rRNAs in rice. We further found that two pre-rRNA processing pathways, distinguished by the order of 5' ETS removal and ITS1 cleavage, coexist in vivo. Moreover, exposing rice to chilling stress resulted in the inhibition of rRNA biogenesis mainly at the pre-rRNA processing level, suggesting that these energy-intensive processes may be reduced to increase acclimation and survival at lower temperatures. Overall, our study identified the pre-rRNA processing pathway in rice and showed that ribosome biogenesis is quickly inhibited by low temperatures, which may shed light on the link between ribosome biogenesis and environmental acclimation in crop plants.
Topics: Cold Temperature; Models, Biological; Oryza; RNA Precursors; RNA Processing, Post-Transcriptional; RNA, Messenger; RNA, Ribosomal; RNA, Ribosomal, 18S; Ribosome Subunits, Large; Ribosome Subunits, Small; Stress, Physiological
PubMed: 29555785
DOI: 10.1104/pp.17.01714 -
Molecular Microbiology Oct 2018The organization of the chromosomal DNA and ribosomes in living Escherichia coli is compared under two growth conditions: 'fast' (50 min doubling time) and 'slow'... (Comparative Study)
Comparative Study
The organization of the chromosomal DNA and ribosomes in living Escherichia coli is compared under two growth conditions: 'fast' (50 min doubling time) and 'slow' (147 min doubling time). Superresolution fluorescence microscopy reveals strong DNA-ribosome segregation in both cases. In both fast and slow growth, free ribosomal subunits evidently must circulate between the nucleoid (where they initiate co-transcriptional translation) and ribosome-rich regions (where most translation occurs). Single-molecule diffusive behavior dissects the ribosome copies into translating 70S polysomes and free 30S subunits, providing separate spatial distributions for each. In slow growth, ~21,000 total 30S copies/cell comprise ~65% translating 70S ribosomes and ~35% free 30S subunits. The ratio of 70S ribosomes to free 30S subunits is ~2.5 outside the nucleoid and ~0.50 inside the nucleoid. This new level of quantitative detail may motivate development of comprehensive, three-dimensional reaction-diffusion models of ribosome, DNA, mRNA and RNAP spatial distributions and dynamics within the E. coli cytoplasm.
Topics: Bacterial Proteins; Cytoplasm; DNA, Bacterial; DNA-Binding Proteins; DNA-Directed RNA Polymerases; Escherichia coli; Escherichia coli Proteins; Fluorescent Dyes; Polyribosomes; Protein Biosynthesis; RNA, Messenger; Ribosome Subunits; Single Molecule Imaging
PubMed: 30107639
DOI: 10.1111/mmi.14103 -
Nucleic Acids Research Jun 2021Mitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components...
Mitochondria contain their own translation apparatus which enables them to produce the polypeptides encoded in their genome. The mitochondrially-encoded RNA components of the mitochondrial ribosome require various post-transcriptional processing steps. Additional protein factors are required to facilitate the biogenesis of the functional mitoribosome. We have characterized a mitochondrially-localized protein, YbeY, which interacts with the assembling mitoribosome through the small subunit. Loss of YbeY leads to a severe reduction in mitochondrial translation and a loss of cell viability, associated with less accurate mitochondrial tRNASer(AGY) processing from the primary transcript and a defect in the maturation of the mitoribosomal small subunit. Our results suggest that YbeY performs a dual, likely independent, function in mitochondria being involved in precursor RNA processing and mitoribosome biogenesis. Issue Section: Nucleic Acid Enzymes.
Topics: Amino Acid Sequence; Cell Survival; Gene Knockout Techniques; HEK293 Cells; Humans; Immunohistochemistry; Mass Spectrometry; Mitochondria; Mitochondrial Proteins; Mitochondrial Ribosomes; Protein Biosynthesis; RNA Processing, Post-Transcriptional; RNA, Transfer; Ribonucleases; Ribosome Subunits, Small; Sequence Alignment
PubMed: 34037799
DOI: 10.1093/nar/gkab404 -
Nucleic Acids Research Aug 2021In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal...
In the cell, stalled ribosomes are rescued through ribosome-associated protein quality-control (RQC) pathways. After splitting of the stalled ribosome, a C-terminal polyalanine 'tail' is added to the unfinished polypeptide attached to the tRNA on the 50S ribosomal subunit. In Bacillus subtilis, polyalanine tailing is catalyzed by the NEMF family protein RqcH, in cooperation with RqcP. However, the mechanistic details of this process remain unclear. Here we demonstrate that RqcH is responsible for tRNAAla selection during RQC elongation, whereas RqcP lacks any tRNA specificity. The ribosomal protein uL11 is crucial for RqcH, but not RqcP, recruitment to the 50S subunit, and B. subtilis lacking uL11 are RQC-deficient. Through mutational mapping, we identify critical residues within RqcH and RqcP that are important for interaction with the P-site tRNA and/or the 50S subunit. Additionally, we have reconstituted polyalanine-tailing in vitro and can demonstrate that RqcH and RqcP are necessary and sufficient for processivity in a minimal system. Moreover, the in vitro reconstituted system recapitulates our in vivo findings by reproducing the importance of conserved residues of RqcH and RqcP for functionality. Collectively, our findings provide mechanistic insight into the role of RqcH and RqcP in the bacterial RQC pathway.
Topics: Bacillus subtilis; DNA Helicases; Peptides; RNA, Transfer; Ribosomal Proteins; Ribosome Subunits, Large, Bacterial; Ribosomes
PubMed: 34255840
DOI: 10.1093/nar/gkab589 -
Nucleic Acids Research Feb 2021Ribosome stalling at tandem CGA codons or poly(A) sequences activates quality controls for nascent polypeptides including ribosome-associated quality control (RQC) and...
Ribosome stalling at tandem CGA codons or poly(A) sequences activates quality controls for nascent polypeptides including ribosome-associated quality control (RQC) and no-go mRNA decay (NGD). In RQC pathway, Hel2-dependent uS10 ubiquitination and the RQC-trigger (RQT) complex are essential for subunit dissociation, and Ltn1-dependent ubiquitination of peptidyl-tRNA in the 60S subunit requires Rqc2. Here, we report that polytryptophan sequences induce Rqc2-independent RQC. More than 11 consecutive tryptophan residues induced RQC in a manner dependent on Hel2-mediated ribosome ubiquitination and the RQT complex. Polytryptophan sequence-mediated RQC was not coupled with CAT-tailing, and Rqc2 was not required for Ltn1-dependent degradation of the arrest products. Eight consecutive tryptophan residues located at the region proximal to the peptidyl transferase center in the ribosome tunnel inhibited CAT-tailing by tandem CGA codons. Polytryptophan sequences also induced Hel2-mediated canonical RQC-coupled NGD and RQC-uncoupled NGD outside the stalled ribosomes. We propose that poly-tryptophan sequences induce Rqc2-independent RQC, suggesting that CAT-tailing in the 60S subunit could be modulated by the polypeptide in the ribosome exit tunnel.
Topics: Codon; Peptides; Protein Biosynthesis; RNA Stability; RNA, Messenger; RNA, Transfer, Amino Acyl; RNA-Binding Proteins; Ribosome Subunits, Large, Eukaryotic; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tryptophan; Ubiquitination
PubMed: 33511411
DOI: 10.1093/nar/gkab005 -
Methods (San Diego, Calif.) Mar 2017Polysomes are macromolecular complexes made up of multiple ribosomes simultaneously translating a single mRNA into polypeptide chains. Together, the cellular mRNAs... (Review)
Review
Polysomes are macromolecular complexes made up of multiple ribosomes simultaneously translating a single mRNA into polypeptide chains. Together, the cellular mRNAs translated in this way are referred to 'translatome.' Translation determines a cell's overall gene expression profile. Studying translatome leads to a better understanding of the translational machinery and of its complex regulatory pathways. Given its fundamental role in cell homeostasis and division, bacterial translation is an important target for antibiotics. However, there are no detailed protocols for polysome purification from Staphylococcus aureus, the human pathogen responsible for the majority of multi-drug resistance issues. We therefore developed methods for the isolation of active polysomes, ribosomes, and ribosomal subunits, examining the purity and quality of each fraction and monitoring polysomal activity during protein synthesis. These steps are mandatory for the use of purified S. aureus polysomes and ribosomes for structural studies or for genome-scale analysis of most translated mRNAs.
Topics: Cell Fractionation; Electrophoresis, Agar Gel; Microscopy, Electron; Polyribosomes; Protein Biosynthesis; RNA, Messenger; Ribosome Subunits, Large, Bacterial; Ribosome Subunits, Small, Bacterial; Staphylococcus aureus
PubMed: 27729294
DOI: 10.1016/j.ymeth.2016.10.003 -
Journal of Molecular Biology May 2019rRNA is the single most abundant polymer in most cells. Mammalian rRNAs are nearly twice as large as those of prokaryotes. Differences in rRNA size are due to expansion...
rRNA is the single most abundant polymer in most cells. Mammalian rRNAs are nearly twice as large as those of prokaryotes. Differences in rRNA size are due to expansion segments, which contain extended tentacles in metazoans. Here we show that the terminus of an rRNA tentacle of Homo sapiens contains 10 tandem G-tracts that form highly stable G-quadruplexes in vitro. We characterized rRNA of the H. sapiens large ribosomal subunit by computation, circular dichroism, UV melting, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts, and blotting. We investigated Expansion Segment 7 (ES7), oligomers derived from ES7, intact 28S rRNA, 80S ribosomes, and polysomes. We used mass spectrometry to identify proteins that bind to rRNA G-quadruplexes in cell lysates. These proteins include helicases (DDX3, CNBP, DDX21, DDX17) and heterogeneous nuclear ribonucleoproteins. Finally, by multiple sequence alignments, we observe that G-quadruplex-forming sequences are a general feature of LSU rRNA of Chordata but not, as far as we can tell, of other species. Chordata ribosomes present polymorphic tentacles with the potential to switch between inter- and intramolecular G-quadruplexes. To our knowledge, G-quadruplexes have not been reported previously in ribosomes.
Topics: Animals; Base Sequence; Circular Dichroism; Electrophoretic Mobility Shift Assay; G-Quadruplexes; Humans; Nucleic Acid Conformation; RNA, Ribosomal; Ribosome Subunits, Large; Sequence Alignment
PubMed: 30885721
DOI: 10.1016/j.jmb.2019.03.010