The Arabidopsis 2′-O-Ribose-Methylation and Pseudouridylation Landscape of rRNA in Comparison to Human and Yeast

Eukaryotic ribosome assembly starts in the nucleolus, where the ribosomal DNA (rDNA) is transcribed into the 35S pre-ribosomal RNA (pre-rRNA). More than two-hundred ribosome biogenesis factors (RBFs) and more than two-hundred small nucleolar RNAs (snoRNA) catalyze the processing, folding and modification of the rRNA in Arabidopsis thaliana. The initial pre-ribosomal 90S complex is formed already during transcription by association of ribosomal proteins (RPs) and RBFs. In addition, small nucleolar ribonucleoprotein particles (snoRNPs) composed of snoRNAs and RBFs catalyze the two major rRNA modification types, 2′-O-ribose-methylation and pseudouridylation. Besides these two modifications, rRNAs can also undergo base methylations and acetylation. However, the latter two modifications have not yet been systematically explored in plants. The snoRNAs of these snoRNPs serve as targeting factors to direct modifications to specific rRNA regions by antisense elements. Today, hundreds of different sites of modifications in the rRNA have been described for eukaryotic ribosomes in general. While our understanding of the general process of ribosome biogenesis has advanced rapidly, the diversities appearing during plant ribosome biogenesis is beginning to emerge. Today, more than two-hundred RBFs were identified by bioinformatics or biochemical approaches, including several plant specific factors. Similarly, more than two hundred snoRNA were predicted based on RNA sequencing experiments. Here, we discuss the predicted and verified rRNA modification sites and the corresponding identified snoRNAs on the example of the model plant Arabidopsis thaliana. Our summary uncovers the plant modification sites in comparison to the human and yeast modification sites.

Download Full-text

Principles of 60S ribosomal subunit assembly emerging from recent studies in yeast

Biochemical Journal ◽

10.1042/bcj20160516 ◽

2017 ◽

Vol 474 (2) ◽

pp. 195-214 ◽

Cited By ~ 44

Author(s):

Salini Konikkat ◽

John L. Woolford,

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Small Nucleolar Rnas ◽

Large Ribosomal Subunit ◽

Yeast Saccharomyces Cerevisiae ◽

Subunit Assembly ◽

Nucleolar Rnas

Ribosome biogenesis requires the intertwined processes of folding, modification, and processing of ribosomal RNA, together with binding of ribosomal proteins. In eukaryotic cells, ribosome assembly begins in the nucleolus, continues in the nucleoplasm, and is not completed until after nascent particles are exported to the cytoplasm. The efficiency and fidelity of ribosome biogenesis are facilitated by >200 assembly factors and ∼76 different small nucleolar RNAs. The pathway is driven forward by numerous remodeling events to rearrange the ribonucleoprotein architecture of pre-ribosomes. Here, we describe principles of ribosome assembly that have emerged from recent studies of biogenesis of the large ribosomal subunit in the yeast Saccharomyces cerevisiae. We describe tools that have empowered investigations of ribosome biogenesis, and then summarize recent discoveries about each of the consecutive steps of subunit assembly.

Download Full-text

Eukaryotic Ribosome Assembly

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-013118-110817 ◽

2019 ◽

Vol 88 (1) ◽

pp. 281-306 ◽

Cited By ~ 60

Author(s):

Jochen Baßler ◽

Ed Hurt

Keyword(s):

Ribosomal Rna ◽

Nuclear Export ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Cryo Electron Microscopy ◽

A Cell ◽

Cellular Pathways ◽

Nucleolar Rnas ◽

Shed Light

Ribosomes, which synthesize the proteins of a cell, comprise ribosomal RNA and ribosomal proteins, which coassemble hierarchically during a process termed ribosome biogenesis. Historically, biochemical and molecular biology approaches have revealed how preribosomal particles form and mature in consecutive steps, starting in the nucleolus and terminating after nuclear export into the cytoplasm. However, only recently, due to the revolution in cryo–electron microscopy, could pseudoatomic structures of different preribosomal particles be obtained. Together with in vitro maturation assays, these findings shed light on how nascent ribosomes progress stepwise along a dynamic biogenesis pathway. Preribosomes assemble gradually, chaperoned by a myriad of assembly factors and small nucleolar RNAs, before they reach maturity and enter translation. This information will lead to a better understanding of how ribosome synthesis is linked to other cellular pathways in humans and how it can cause diseases, including cancer, if disturbed.

Download Full-text

The Host Gene for Intronic U17 Small Nucleolar RNAs in Mammals Has No Protein-Coding Potential and Is a Member of the 5′-Terminal Oligopyrimidine Gene Family

Molecular and Cellular Biology ◽

10.1128/mcb.18.8.4509 ◽

1998 ◽

Vol 18 (8) ◽

pp. 4509-4518 ◽

Cited By ~ 75

Author(s):

Pawel Pelczar ◽

Witold Filipowicz

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Transcription Unit ◽

Host Gene ◽

Small Nucleolar Rnas ◽

Ribosomal Protein Genes ◽

Protein Coding ◽

Genes Encoding ◽

Terminal Oligopyrimidine ◽

Nucleolar Rnas

ABSTRACT Intron-encoded U17a and U17b RNAs are members of the H/ACA-box class of small nucleolar RNAs (snoRNAs) participating in rRNA processing and modification. We have investigated the organization and expression of the U17 locus in human cells and found that intronic U17a and U17b sequences are transcribed as part of the three-exon transcription unit, named U17HG, positioned approximately 9 kb upstream of the RCC1 locus. Comparison of the human and mouse U17HG genes has revealed that snoRNA-encoding intron sequences but not exon sequences are conserved between the two species and that neither human nor mouse spliced U17HGpoly(A)+ RNAs have the potential to code for proteins. Analyses of polysome profiles and effects of translation inhibitors on the abundance of U17HG RNA in HeLa cells indicated that despite its cytoplasmic localization, little if any U17HGRNA is associated with polysomes. This distinguishes U17HGRNA from another non-protein-coding snoRNA host gene product,UHG RNA, described previously (K. T. Tycowski, M. D. Shu, and J. A. Steitz, Nature 379:464–466, 1996). Determination of the 5′ terminus of the U17HG RNA revealed that transcription of the U17HG gene starts with a C residue followed by a polypyrimidine tract, making this gene a member of the 5′-terminal oligopyrimidine (5′TOP) family, which includes genes encoding ribosomal proteins and some translation factors. Interestingly, other known snoRNA host genes, including theUHG gene (Tycowski et al., op. cit.), have features of the 5′TOP genes. Similar characteristics of the transcription start site regions in snoRNA host and ribosomal protein genes raise the possibility that expression of components of ribosome biogenesis and translational machineries is coregulated.

Download Full-text

Emerging Data on the Diversity of Molecular Mechanisms Involving C/D snoRNAs

Non-Coding RNA ◽

10.3390/ncrna7020030 ◽

2021 ◽

Vol 7 (2) ◽

pp. 30

Author(s):

Laeya Baldini ◽

Bruno Charpentier ◽

Stéphane Labialle

Keyword(s):

Ribosomal Rna ◽

Molecular Mechanisms ◽

Molecular Level ◽

Accurate Description ◽

Small Nucleolar Rnas ◽

Age Of Maturity ◽

Non Coding Rnas ◽

And Function ◽

Nucleolar Rnas

Box C/D small nucleolar RNAs (C/D snoRNAs) represent an ancient family of small non-coding RNAs that are classically viewed as housekeeping guides for the 2′-O-methylation of ribosomal RNA in Archaea and Eukaryotes. However, an extensive set of studies now argues that they are involved in mechanisms that go well beyond this function. Here, we present these pieces of evidence in light of the current comprehension of the molecular mechanisms that control C/D snoRNA expression and function. From this inventory emerges that an accurate description of these activities at a molecular level is required to let the snoRNA field enter in a second age of maturity.

Download Full-text

Ubiquitin and Ubiquitin-Like Proteins and Domains in Ribosome Production and Function: Chance or Necessity?

International Journal of Molecular Sciences ◽

10.3390/ijms22094359 ◽

2021 ◽

Vol 22 (9) ◽

pp. 4359

Author(s):

Sara Martín-Villanueva ◽

Gabriel Gutiérrez ◽

Dieter Kressler ◽

Jesús de la Cruz

Keyword(s):

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosome Assembly ◽

Cellular Processes ◽

Substrate Protein ◽

Precursor Proteins ◽

Assembly Factors ◽

Ubiquitin Moiety ◽

And Function

Ubiquitin is a small protein that is highly conserved throughout eukaryotes. It operates as a reversible post-translational modifier through a process known as ubiquitination, which involves the addition of one or several ubiquitin moieties to a substrate protein. These modifications mark proteins for proteasome-dependent degradation or alter their localization or activity in a variety of cellular processes. In most eukaryotes, ubiquitin is generated by the proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a polyubiquitin precursor, or as a single N-terminal moiety to ribosomal proteins, which are practically invariably eL40 and eS31. Herein, we summarize the contribution of the ubiquitin moiety within precursors of ribosomal proteins to ribosome biogenesis and function and discuss the biological relevance of having maintained the explicit fusion to eL40 and eS31 during evolution. There are other ubiquitin-like proteins, which also work as post-translational modifiers, among them the small ubiquitin-like modifier (SUMO). Both ubiquitin and SUMO are able to modify ribosome assembly factors and ribosomal proteins to regulate ribosome biogenesis and function. Strikingly, ubiquitin-like domains are also found within two ribosome assembly factors; hence, the functional role of these proteins will also be highlighted.

Download Full-text

Proteotoxicity from aberrant ribosome biogenesis compromises cell fitness

eLife ◽

10.7554/elife.43002 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 25

Author(s):

Blake W Tye ◽

Nicoletta Commins ◽

Lillia V Ryazanova ◽

Martin Wühr ◽

Michael Springer ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Ribosome Assembly ◽

Maximal Growth ◽

Proliferating Cells ◽

Cellular Processes ◽

Direct Contribution ◽

The Face ◽

Cellular Phenotypes

To achieve maximal growth, cells must manage a massive economy of ribosomal proteins (r-proteins) and RNAs (rRNAs) to produce thousands of ribosomes every minute. Although ribosomes are essential in all cells, natural disruptions to ribosome biogenesis lead to heterogeneous phenotypes. Here, we model these perturbations in Saccharomyces cerevisiae and show that challenges to ribosome biogenesis result in acute loss of proteostasis. Imbalances in the synthesis of r-proteins and rRNAs lead to the rapid aggregation of newly synthesized orphan r-proteins and compromise essential cellular processes, which cells alleviate by activating proteostasis genes. Exogenously bolstering the proteostasis network increases cellular fitness in the face of challenges to ribosome assembly, demonstrating the direct contribution of orphan r-proteins to cellular phenotypes. We propose that ribosome assembly is a key vulnerability of proteostasis maintenance in proliferating cells that may be compromised by diverse genetic, environmental, and xenobiotic perturbations that generate orphan r-proteins.

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

Genome-Wide Analysis of mRNA Stability Using Transcription Inhibitors and Microarrays Reveals Posttranscriptional Control of Ribosome Biogenesis Factors

Molecular and Cellular Biology ◽

10.1128/mcb.24.12.5534-5547.2004 ◽

2004 ◽

Vol 24 (12) ◽

pp. 5534-5547 ◽

Cited By ~ 224

Author(s):

Jörg Grigull ◽

Sanie Mnaimneh ◽

Jeffrey Pootoolal ◽

Mark D. Robinson ◽

Timothy R. Hughes

Keyword(s):

Heat Stress ◽

Microarray Data ◽

Mrna Stability ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Transcriptional Response ◽

Rrna Synthesis ◽

Ribosome Assembly ◽

Genome Wide ◽

Genes Encoding

ABSTRACT Using DNA microarrays, we compared global transcript stability profiles following chemical inhibition of transcription to rpb1-1 (a temperature-sensitive allele of yeast RNA polymerase II). Among the five inhibitors tested, the effects of thiolutin and 1,10-phenanthroline were most similar to rpb1-1. A comparison to various microarray data already in the literature revealed similarity between mRNA stability profiles and the transcriptional response to stresses such as heat shock, consistent with the fact that the general stress response includes a transient shutoff of general mRNA transcription. Genes encoding factors involved in rRNA synthesis and ribosome assembly, which are often observed to be coordinately down-regulated in yeast microarray data, were among the least stable transcripts. We examined the effects of deletions of genes encoding deadenylase components Ccr4p and Pan2p and putative RNA-binding proteins Pub1p and Puf4p on the genome-wide pattern of mRNA stability after inhibition of transcription by chemicals and/or heat stress. This examination showed that Ccr4p, the major yeast mRNA deadenylase, contributes to the degradation of transcripts encoding both ribosomal proteins and rRNA synthesis and ribosome assembly factors and mediates a large part of the transcriptional response to heat stress. Pan2p and Puf4p also contributed to the degradation rate of these mRNAs following transcriptional shutoff, while Pub1p preferentially stabilized transcripts encoding ribosomal proteins. Our results indicate that the abundance of ribosome biogenesis factors is controlled at the level of mRNA stability.

Download Full-text

SnoRNA microarray analysis reveals changes in H/ACA and C/D RNA levels caused by dyskerin ablation in mouse liver

Biochemical Journal ◽

10.1042/bj20091898 ◽

2010 ◽

Vol 429 (1) ◽

pp. 33-41 ◽

Cited By ~ 7

Author(s):

Jingping Ge ◽

Seth D. Crosby ◽

Michael E. Heinz ◽

Monica Bessler ◽

Philip J. Mason

Keyword(s):

Microarray Analysis ◽

Mouse Liver ◽

Cellular Response ◽

Ribosome Biogenesis ◽

Microarray Platform ◽

Protein Component ◽

Small Nucleolar Rnas ◽

Ribosome Synthesis ◽

Nucleolar Rnas ◽

Host Genes

snoRNAs (small nucleolar RNAs) are key components of snoRNP (small nucleolar ribonucleoprotein) particles involved in modifying specific residues of ribosomal and other RNAs by pseudouridylation (H/ACA snoRNAs) or methylation (C/D snoRNAs). They are encoded within the introns of host genes, which tend to be genes whose products are involved in ribosome biogenesis or function. Although snoRNPs are abundant, ubiquitous and their components highly conserved, information concerning their expression during development or how their expression is altered in diseased states is sparse. To facilitate these studies we have developed a snoRNA microarray platform for the analysis of the abundance of snoRNAs in different RNA samples. In the present study we show that the microarray is sensitive and specific for the detection of snoRNAs. A mouse snoRNA microarray was used to monitor changes in abundance of snoRNAs after ablation of dyskerin, an H/ACA snoRNA protein component, from mouse liver, which causes a decrease in ribosome production. H/ACA snoRNAs were decreased in abundance in these livers while, unexpectedly, C/D snoRNAs were increased. The increase in C/D snoRNAs corresponded with an increase in the abundance of the mRNAs transcribed from snoRNA host genes, suggesting the increase may be part of a cellular response to defective ribosome synthesis.

Download Full-text

Identification of Genes That Function in the Biogenesis and Localization of Small Nucleolar RNAs in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.01115-07 ◽

2008 ◽

Vol 28 (11) ◽

pp. 3686-3699 ◽

Cited By ~ 13

Author(s):

Hui Qiu ◽

Julia Eifert ◽

Ludivine Wacheul ◽

Marc Thiry ◽

Adam C. Berger ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosome Biogenesis ◽

Large Scale ◽

Cajal Body ◽

Temperature Sensitive ◽

Small Nucleolar Rnas ◽

Animal Cells ◽

U3 Snorna ◽

Genes Encoding ◽

Nucleolar Rnas

ABSTRACT Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.

Download Full-text