scholarly journals Structural Probing with MNase Tethered to Ribosome Assembly Factors Resolves Flexible RNA Regions within the Nascent Pre-Ribosomal RNA

2022 ◽  
Vol 8 (1) ◽  
pp. 1
Author(s):  
Tom Dielforder ◽  
Christina Maria Braun ◽  
Fabian Hölzgen ◽  
Shuang Li ◽  
Mona Thiele ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Dynamic Information ◽  
Nascent Transcript ◽  
Ribonucleoprotein Complexes ◽  
U3 Snorna ◽  
Assembly Factors ◽  
Compositional Changes ◽  
Ribosomal Precursor

The synthesis of ribosomes involves the correct folding of the pre-ribosomal RNA within pre-ribosomal particles. The first ribosomal precursor or small subunit processome assembles stepwise on the nascent transcript of the 35S gene. At the earlier stages, the pre-ribosomal particles undergo structural and compositional changes, resulting in heterogeneous populations of particles with highly flexible regions. Structural probing methods are suitable for resolving these structures and providing evidence about the architecture of ribonucleoprotein complexes. Our approach used MNase tethered to the assembly factors Nan1/Utp17, Utp10, Utp12, and Utp13, which among other factors, initiate the formation of the small subunit processome. Our results provide dynamic information about the folding of the pre-ribosomes by elucidating the relative organization of the 5′ETS and ITS1 regions within the 35S and U3 snoRNA around the C-terminal domains of Nan1/Utp17, Utp10, Utp12, and Utp13.

scholarly journals Conformational switches control early maturation of the eukaryotic small ribosomal subunit

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mirjam Hunziker ◽  
Jonas Barandun ◽  
Olga Buzovetsky ◽  
Caitlin Steckler ◽  
Henrik Molina ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Conformational Changes ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Early Maturation ◽  
External Transcribed Spacer ◽  
Assembly Factors

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5’ external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5’ external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5’ external transcribed spacer ribonucleoprotein – provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

scholarly journals UtpA and UtpB chaperone nascent pre-ribosomal RNA and U3 snoRNA to initiate eukaryotic ribosome assembly

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Mirjam Hunziker ◽  
Jonas Barandun ◽  
Elisabeth Petfalski ◽  
Dongyan Tan ◽  
Clémentine Delan-Forino ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosome Assembly ◽  
Eukaryotic Ribosome ◽  
U3 Snorna

scholarly journals A network of assembly factors is involved in remodeling rRNA elements during preribosome maturation

2014 ◽  
Vol 207 (4) ◽  
pp. 481-498 ◽  
Author(s):  
Jochen Baßler ◽  
Helge Paternoga ◽  
Iris Holdermann ◽  
Matthias Thoms ◽  
Sander Granneman ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Ribosome Assembly ◽  
Functional Importance ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Eukaryotic Ribosome ◽  
Assembly Factors

Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation.

Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery

Science ◽  
2019 ◽  
Vol 365 (6458) ◽  
pp. 1144-1149 ◽  
Author(s):  
Martin Saurer ◽  
David J. F. Ramrath ◽  
Moritz Niemann ◽  
Salvatore Calderaro ◽  
Céline Prange ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Conformational Changes ◽  
Large Scale ◽  
Small Subunit ◽  
Ribosomal Assembly ◽  
Mitochondrial Ribosomes ◽  
Ribonucleoprotein Complexes ◽  
Cryo Electron Microscopy ◽  
Number Of Factors ◽  
Cellular Machinery

Mitochondrial ribosomes (mitoribosomes) are large ribonucleoprotein complexes that synthesize proteins encoded by the mitochondrial genome. An extensive cellular machinery responsible for ribosome assembly has been described only for eukaryotic cytosolic ribosomes. Here we report that the assembly of the small mitoribosomal subunit in Trypanosoma brucei involves a large number of factors and proceeds through the formation of assembly intermediates, which we analyzed by using cryo–electron microscopy. One of them is a 4-megadalton complex, referred to as the small subunit assemblosome, in which we identified 34 factors that interact with immature ribosomal RNA (rRNA) and recognize its functionally important regions. The assembly proceeds through large-scale conformational changes in rRNA coupled with successive incorporation of mitoribosomal proteins, providing an example for the complexity of the ribosomal assembly process in mitochondria.

scholarly journals Co-Assembly of 40S and 60S Ribosomal Proteins in Early Steps of Eukaryotic Ribosome Assembly

2019 ◽  
Vol 20 (11) ◽  
pp. 2806 ◽  
Author(s):  
Jesse M. Fox ◽  
Rebekah L. Rashford ◽  
Lasse Lindahl
Keyword(s):  
Ribosomal Rna ◽  
Ribosomal Proteins ◽  
Ribosome Assembly ◽  
Relative Timing ◽  
Ribosomal Subunits ◽  
Eukaryotic Ribosome ◽  
Rrna Molecule ◽  
Ribosomal Precursor ◽  
Its1 And Its2

In eukaryotes three of the four ribosomal RNA (rRNA) molecules are transcribed as a long precursor that is processed into mature rRNAs concurrently with the assembly of ribosomal subunits. However, the relative timing of association of ribosomal proteins with the ribosomal precursor particles and the cleavage of the precursor rRNA into the subunit-specific moieties is not known. To address this question, we searched for ribosomal precursors containing components from both subunits. Particles containing specific ribosomal proteins were targeted by inducing synthesis of epitope-tagged ribosomal proteins followed by pull-down with antibodies targeting the tagged protein. By identifying other ribosomal proteins and internal rRNA transcribed spacers (ITS1 and ITS2) in the immuno-purified ribosomal particles, we showed that eS7/S7 and uL4/L4 bind to nascent ribosomes prior to the separation of 40S and 60S specific segments, while uS4/S9, uL22, and eL13/L13 are bound after, or simultaneously with, the separation. Thus, the incorporation of ribosomal proteins from the two subunits begins as a co-assembly with a single rRNA molecule, but is finished as an assembly onto separate precursors for the two subunits.

scholarly journals Molecular architecture of the 90S small subunit pre-ribosome

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Qi Sun ◽  
Xing Zhu ◽  
Jia Qi ◽  
Weidong An ◽  
Pengfei Lan ◽  
...  
Keyword(s):  
Small Subunit ◽  
Molecular Architecture ◽  
Ribosomal Subunits ◽  
Cryo Electron Microscopy ◽  
U3 Snorna ◽  
Density Maps ◽  
External Transcribed Spacer ◽  
Assembly Factors ◽  
Protein Components ◽  
Insight Into

Eukaryotic small ribosomal subunits are first assembled into 90S pre-ribosomes. The complete 90S is a gigantic complex with a molecular mass of approximately five megadaltons. Here, we report the nearly complete architecture of Saccharomyces cerevisiae 90S determined from three cryo-electron microscopy single particle reconstructions at 4.5 to 8.7 angstrom resolution. The majority of the density maps were modeled and assigned to specific RNA and protein components. The nascent ribosome is assembled into isolated native-like substructures that are stabilized by abundant assembly factors. The 5' external transcribed spacer and U3 snoRNA nucleate a large subcomplex that scaffolds the nascent ribosome. U3 binds four sites of pre-rRNA, including a novel site on helix 27 but not the 3' side of the central pseudoknot, and crucially organizes the 90S structure. The 90S model provides significant insight into the principle of small subunit assembly and the function of assembly factors.

scholarly journals Nucleolar proteins Bfr2 and Enp2 interact with DEAD-box RNA helicase Dbp4 in two different complexes

2013 ◽  
Vol 42 (5) ◽  
pp. 3194-3206 ◽  
Author(s):  
Sahar Soltanieh ◽  
Martin Lapensée ◽  
François Dragon
Keyword(s):  
Ribosomal Rna ◽  
Ribosome Biogenesis ◽  
Rna Helicase ◽  
Small Subunit ◽  
Specific Protein ◽  
18S Ribosomal Rna ◽  
Dead Box ◽  
Nucleolar Proteins ◽  
U3 Snorna ◽  
Ssu Processome

AbstractDifferent pre-ribosomal complexes are formed during ribosome biogenesis, and the composition of these complexes is highly dynamic. Dbp4, a conserved DEAD-box RNA helicase implicated in ribosome biogenesis, interacts with nucleolar proteins Bfr2 and Enp2. We show that, like Dbp4, Bfr2 and Enp2 are required for the early processing steps leading to the production of 18S ribosomal RNA. We also found that Bfr2 and Enp2 associate with the U3 small nucleolar RNA (snoRNA), the U3-specific protein Mpp10 and various pre-18S ribosomal RNA species. Thus, we propose that Bfr2, Dbp4 and Enp2 are components of the small subunit (SSU) processome, a large complex of ∼80S. Sucrose gradient sedimentation analyses indicated that Dbp4, Bfr2 and Enp2 sediment in a peak of ∼50S and in a peak of ∼80S. Bfr2, Dbp4 and Enp2 associate together in the 50S complex, which does not include the U3 snoRNA; however, they associate with U3 snoRNA in the 80S complex (SSU processome). Immunoprecipitation experiments revealed that U14 snoRNA associates with Dbp4 in the 50S complex, but not with Bfr2 or Enp2. The assembly factor Tsr1 is not part of the ‘50S’ complex, indicating this complex is not a pre-40S ribosome. A combination of experiments leads us to propose that Bfr2, Enp2 and Dbp4 are recruited at late steps during assembly of the SSU processome.

scholarly journals Bud23 promotes the final disassembly of the small subunit Processome in Saccharomyces cerevisiae

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1009215
Author(s):  
Joshua J. Black ◽  
Richa Sardana ◽  
Ezzeddine W. Elmir ◽  
Arlen W. Johnson
Keyword(s):  
Binding Site ◽  
Ribosomal Rna ◽  
Rna Processing ◽  
Interaction Network ◽  
Small Subunit ◽  
Physical Interaction ◽  
U3 Snorna ◽  
Genes Encoding ◽  
Ribosomal Rna Processing ◽  
Ssu Processome

The first metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU) is the SSU Processome, a large complex of RNA and protein factors that is thought to represent an early checkpoint in the assembly pathway. Transition of the SSU Processome towards continued maturation requires the removal of the U3 snoRNA and biogenesis factors as well as ribosomal RNA processing. While the factors that drive these events are largely known, how they do so is not. The methyltransferase Bud23 has a role during this transition, but its function, beyond the nonessential methylation of ribosomal RNA, is not characterized. Here, we have carried out a comprehensive genetic screen to understand Bud23 function. We identified 67 unique extragenic bud23Δ-suppressing mutations that mapped to genes encoding the SSU Processome factors DHR1, IMP4, UTP2 (NOP14), BMS1 and the SSU protein RPS28A. These factors form a physical interaction network that links the binding site of Bud23 to the U3 snoRNA and many of the amino acid substitutions weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the essential GTPase Bms1, which acts late in the disassembly pathway, and the RNA helicase Dhr1, which catalyzes U3 snoRNA removal. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and ribosomal RNA processing defects. We propose a model in which Bud23 dissociates factors surrounding its binding site to promote SSU Processome progression.

scholarly journals High Diversity of Cryptosporidium Species and Subtypes Identified in Cryptosporidiosis Acquired in Sweden and Abroad

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 523
Author(s):  
Marianne Lebbad ◽  
Jadwiga Winiecka-Krusnell ◽  
Christen Rune Stensvold ◽  
Jessica Beser
Keyword(s):  
Cryptosporidium Parvum ◽  
Ribosomal Rna ◽  
Diarrheal Disease ◽  
Protozoan Parasite ◽  
Small Subunit ◽  
Cryptosporidium Species ◽  
Intestinal Protozoan ◽  
Genotype I ◽  
Glycoprotein Gene ◽  
High Diversity

The intestinal protozoan parasite Cryptosporidium is an important cause of diarrheal disease worldwide. The aim of this study was to expand the knowledge on the molecular epidemiology of human cryptosporidiosis in Sweden to better understand transmission patterns and potential zoonotic sources. Cryptosporidium-positive fecal samples were collected between January 2013 and December 2014 from 12 regional clinical microbiology laboratories in Sweden. Species and subtype determination was achieved using small subunit ribosomal RNA and 60 kDa glycoprotein gene analysis. Samples were available for 398 patients, of whom 250 (63%) and 138 (35%) had acquired the infection in Sweden and abroad, respectively. Species identification was successful for 95% (379/398) of the samples, revealing 12 species/genotypes: Cryptosporidium parvum (n = 299), C. hominis (n = 49), C. meleagridis (n = 8), C. cuniculus (n = 5), Cryptosporidium chipmunk genotype I (n = 5), C. felis (n = 4), C. erinacei (n = 2), C. ubiquitum (n = 2), and one each of C. suis, C. viatorum, C. ditrichi, and Cryptosporidium horse genotype. One patient was co-infected with C. parvum and C. hominis. Subtyping was successful for all species/genotypes, except for C. ditrichi, and revealed large diversity, with 29 subtype families (including 4 novel ones: C. parvum IIr, IIs, IIt, and Cryptosporidium horse genotype VIc) and 81 different subtypes. The most common subtype families were IIa (n = 164) and IId (n = 118) for C. parvum and Ib (n = 26) and Ia (n = 12) for C. hominis. Infections caused by the zoonotic C. parvum subtype families IIa and IId dominated both in patients infected in Sweden and abroad, while most C. hominis cases were travel-related. Infections caused by non-hominis and non-parvum species were quite common (8%) and equally represented in cases infected in Sweden and abroad.

scholarly journals A discontinuous small subunit ribosomal RNA in Tetrahymena pyriformis mitochondria.

1986 ◽  
Vol 261 (11) ◽  
pp. 5187-5193
Author(s):  
M N Schnare ◽  
T Y Heinonen ◽  
P G Young ◽  
M W Gray
Keyword(s):  
Ribosomal Rna ◽  
Tetrahymena Pyriformis ◽  
Small Subunit ◽  
Small Subunit Ribosomal Rna
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