Crucial role of the Rcl1p–Bms1p interaction for yeast pre-ribosomal RNA processing

Abstract The essential Rcl1p and Bms1p proteins form a complex required for 40S ribosomal subunit maturation. Bms1p is a GTPase and Rcl1p has been proposed to catalyse the endonucleolytic cleavage at site A2 separating the pre-40S and pre-60S maturation pathways. We determined the 2.0 Å crystal structure of Bms1p associated with Rcl1p. We demonstrate that Rcl1p nuclear import depends on Bms1p and that the two proteins are loaded into pre-ribosomes at a similar stage of the maturation pathway and remain present within pre-ribosomes after cleavage at A2. Importantly, GTP binding to Bms1p is not required for the import in the nucleus nor for the incorporation of Rcl1p into pre-ribosomes, but is essential for early pre-rRNA processing. We propose that GTP binding to Bms1p and/or GTP hydrolysis may induce conformational rearrangements within the Bms1p-Rcl1p complex allowing the interaction of Rcl1p with its RNA substrate.

Download Full-text

Structure of the eukaryotic cytoplasmic pre-40S ribosomal subunit

10.1101/216713 ◽

2017 ◽

Author(s):

Alain Scaiola ◽

Cohue Peña ◽

Melanie Weisser ◽

Daniel Böhringer ◽

Marc Leibundgut ◽

...

Keyword(s):

Electron Microscopy ◽

Ribosomal Rna ◽

Ribosomal Subunit ◽

Mature Form ◽

Structural Framework ◽

Cryo Electron Microscopy ◽

40S Ribosomal Subunit ◽

Final Maturation ◽

Assembly Factors ◽

Conformational Rearrangements

AbstractFinal maturation of eukaryotic ribosomes occurs in the cytoplasm and requires the sequential removal of associated assembly factors and processing of the immature 20S pre-RNA. Using cryo-electron microscopy (cryo-EM), we have determined the structure of a cytoplasmic pre-40S particle poised to initiate final maturation at a resolution of 3.4 Å. The structure reveals the extent of conformational rearrangements of the 3’ major and 3’ minor domains of the ribosomal RNA that take place during maturation, as well as the roles of the assembly factors Enp1, Ltv1, Rio2, Tsr1, and Pno1 in the process. Altogether, we provide a structural framework for the coordination of the final maturation events that drive a pre-40S particle towards the mature form capable of engaging in translation.

Download Full-text

Cryo-EM reconstruction of the human 40S ribosomal subunit at 2.15 Å resolution

10.1101/2022.01.16.475527 ◽

2022 ◽

Author(s):

Simone Pellegrino ◽

Kyle C Dent ◽

Tobias Spikes ◽

Alan J Warren

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Ribosomal Subunit ◽

Magnesium Ions ◽

Rna Modifications ◽

Post Translational Modifications ◽

The Core ◽

40S Ribosomal Subunit ◽

Structural Details

The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialization in development and disease. However, the inability to accurately visualize these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 Å resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualize post-transcriptional modifications within the 18S rRNA and post-translational modifications at the N-termini of two ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilization and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unraveling the functional role of ribosomal RNA modifications.

Download Full-text

The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

10.1101/2021.09.28.462141 ◽

2021 ◽

Author(s):

Haina Huang ◽

Melissa Parker ◽

Katrin Karbstein

Keyword(s):

Quality Control ◽

Binding Site ◽

Ribosomal Rna ◽

Ribosomal Proteins ◽

Kinase Activity ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Yeast Strains

AbstractRibosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

Download Full-text

Ribosomal RNA processing and the role of SmMAK16 in ribosome biogenesis in Schistosoma mansoni

Molecular and Biochemical Parasitology ◽

10.1016/j.molbiopara.2003.08.006 ◽

2003 ◽

Vol 132 (2) ◽

pp. 67-74 ◽

Cited By ~ 9

Author(s):

Elizabeth E. Capowski ◽

James W. Tracy

Keyword(s):

Schistosoma Mansoni ◽

Ribosomal Rna ◽

Rna Processing ◽

Ribosome Biogenesis ◽

Ribosomal Rna Processing

Download Full-text

DER containing two consecutive GTP-binding domains plays an essential role in chloroplast ribosomal RNA processing and ribosome biogenesis in higher plants

Journal of Experimental Botany ◽

10.1093/jxb/ert360 ◽

2013 ◽

Vol 65 (1) ◽

pp. 117-130 ◽

Cited By ~ 15

Author(s):

Young Jeon ◽

Chang Sook Ahn ◽

Hyun Ju Jung ◽

Hunseung Kang ◽

Guen Tae Park ◽

...

Keyword(s):

Ribosomal Rna ◽

Rna Processing ◽

Ribosome Biogenesis ◽

Essential Role ◽

Higher Plants ◽

Binding Domains ◽

Gtp Binding ◽

Ribosomal Rna Processing

Download Full-text

Essential role of NbNOG1 in ribosomal RNA processing

Journal of Integrative Plant Biology ◽

10.1111/jipb.12691 ◽

2018 ◽

Vol 60 (11) ◽

pp. 1018-1022 ◽

Cited By ~ 1

Author(s):

Jiangbo Guo ◽

Shaojie Han ◽

Jinping Zhao ◽

Cuihua Xin ◽

Xiyin Zheng ◽

...

Keyword(s):

Ribosomal Rna ◽

Rna Processing ◽

Essential Role ◽

Ribosomal Rna Processing

Download Full-text

The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

RNA ◽

10.1261/rna.078994.121 ◽

2022 ◽

pp. rna.078994.121

Author(s):

Haina Huang ◽

Melissa D Parker ◽

Katrin Karbstein

Keyword(s):

Quality Control ◽

Binding Site ◽

Ribosomal Rna ◽

Ribosomal Proteins ◽

Kinase Activity ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Yeast Strains

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

Download Full-text

Heat-sensitive mutant strain of Neurospora crassa, 4M(t), conditionally defective in 25S ribosomal ribonucleic acid production.

Molecular and Cellular Biology ◽

10.1128/mcb.1.3.199 ◽

1981 ◽

Vol 1 (3) ◽

pp. 199-207 ◽

Cited By ~ 5

Author(s):

M W Loo ◽

N S Schricker ◽

P J Russell

Keyword(s):

Neurospora Crassa ◽

Mutant Strain ◽

Ribosomal Rna ◽

Ribonucleic Acid ◽

Ribosomal Subunit ◽

Macromolecular Synthesis ◽

Sensitive Mutant ◽

Ribosomal Subunits ◽

Sequence Of Events ◽

Ribosomal Rna Processing

A heat-sensitive mutant strain of Neurospora crassa, 4M(t), was studied in an attempt to define its molecular lesion. The mutant strain is inhibited in conidial germination and mycelial extension at the nonpermissive temperature (37 degrees C). Macromolecular synthesis studies showed that both ribonucleic acid (RNA) and protein syntheses are inhibited when 4-h cultures are shifted from 20 to 37 degrees C. Density gradient analysis of ribosomal subunits made at 37 degrees C indicated that strain 4M(t) is deficient in the accumulation of 60S ribosomal subunits in that the ratio of 60S/37S subunits was 0.29:1 compared with 1.6:1 for the parental strain. This phenotype was shown to be the result of a slow rate of processing of, and a deficiency in the amount of, the immediate precursor to 25S ribosomal RNA (the large RNA of the 60S subunit) in the sequence of events constituting the production of mature ribosomal RNAs from the primary transcript of the ribosomal deoxyribonucleic acid, the precursor ribosomal RNA molecule. Analysis of polysomes suggested that the heat-sensitive gene product might function in both the assembly and the function of the 60S ribosomal subunit, since there was a smaller proportion of newly made 60S subunits synthesized at 37 degrees C in the polysome region of the gradients than in the monosome-plus-subunit region. The ribosomal RNA processing defect is apparently responsible for the observed defects in germination and macromolecular synthesis at 37 degrees C, but the precise molecular lesion is not known. On the basis of these results, the heat-sensitive mutant allele in the 4M(t) strain is considered to define the rip1 (ribosome production) gene locus.

Download Full-text

Recent Advances on the Structure and Function of RNA Acetyltransferase Kre33/NAT10

Cells ◽

10.3390/cells8091035 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1035 ◽

Cited By ~ 8

Author(s):

Sophie Sleiman ◽

Francois Dragon

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

40S Ribosomal Subunit ◽

Functional Features ◽

And Function ◽

Progeria Syndrome ◽

Hutchinson Gilford Progeria Syndrome ◽

Nucleolar Rna

Ribosome biogenesis is one of the most energy demanding processes in the cell. In eukaryotes, the main steps of this process occur in the nucleolus and include pre-ribosomal RNA (pre-rRNA) processing, post-transcriptional modifications, and assembly of many non-ribosomal factors and ribosomal proteins in order to form mature and functional ribosomes. In yeast and humans, the nucleolar RNA acetyltransferase Kre33/NAT10 participates in different maturation events, such as acetylation and processing of 18S rRNA, and assembly of the 40S ribosomal subunit. Here, we review the structural and functional features of Kre33/NAT10 RNA acetyltransferase, and we underscore the importance of this enzyme in ribosome biogenesis, as well as in acetylation of non-ribosomal targets. We also report on the role of human NAT10 in Hutchinson–Gilford progeria syndrome.

Download Full-text