The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei

The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

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The Nucleolar DExD/H Protein Hel66 is Involved in Ribosome Biogenesis in Trypanosoma Brucei

10.21203/rs.3.rs-612898/v1 ◽

2021 ◽

Author(s):

Majeed Bakari-Soale ◽

Nonso Josephat Ikenga ◽

Marion Scheibe ◽

Falk Butter ◽

Nicola Gail Jones ◽

...

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Growth Defect ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Comprehensive Picture ◽

Severe Growth Defect ◽

H Protein ◽

Severe Growth

Abstract The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process.

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NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3865-3871.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3865-3871

Author(s):

W C Lee ◽

D Zabetakis ◽

T Mélèse

Keyword(s):

Nuclear Localization ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Protein Translation ◽

Striking Similarity ◽

Growth Defect ◽

Nuclear Localization Sequence ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Localization Sequence

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.

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NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3865 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3865-3871 ◽

Cited By ~ 76

Author(s):

W C Lee ◽

D Zabetakis ◽

T Mélèse

Keyword(s):

Nuclear Localization ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Protein Translation ◽

Striking Similarity ◽

Growth Defect ◽

Nuclear Localization Sequence ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Localization Sequence

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.

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Intranucleolar sites of ribosome biogenesis defined by the localization of early binding ribosomal proteins

The Journal of Cell Biology ◽

10.1083/jcb.200612048 ◽

2007 ◽

Vol 177 (4) ◽

pp. 573-578 ◽

Cited By ~ 38

Author(s):

Tim Krüger ◽

Hanswalter Zentgraf ◽

Ulrich Scheer

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Live Cell Imaging ◽

Ribosome Biogenesis ◽

Cell Imaging ◽

Rrna Processing ◽

Dense Fibrillar Component ◽

Assembly Pathway ◽

Fibrillar Component ◽

Processing Steps

Considerable efforts are being undertaken to elucidate the processes of ribosome biogenesis. Although various preribosomal RNP complexes have been isolated and molecularly characterized, the order of ribosomal protein (r-protein) addition to the emerging ribosome subunits is largely unknown. Furthermore, the correlation between the ribosome assembly pathway and the structural organization of the dedicated ribosome factory, the nucleolus, is not well established. We have analyzed the nucleolar localization of several early binding r-proteins in human cells, applying various methods, including live-cell imaging and electron microscopy. We have located all examined r-proteins (S4, S6, S7, S9, S14, and L4) in the granular component (GC), which is the nucleolar region where later pre-ribosomal RNA (rRNA) processing steps take place. These results imply that early binding r-proteins do not assemble with nascent pre-rRNA transcripts in the dense fibrillar component (DFC), as is generally believed, and provide a link between r-protein assembly and the emergence of distinct granules at the DFC–GC interface.

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Recent studies implicate the nucleolus as the major site of nuclear translation

Biochemical Society Transactions ◽

10.1042/bst20140062 ◽

2014 ◽

Vol 42 (4) ◽

pp. 1224-1228 ◽

Cited By ~ 16

Author(s):

Tina McLeod ◽

Akilu Abdullahi ◽

Min Li ◽

Saverio Brogna

Keyword(s):

Ribosomal Rna ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Nuclear Protein ◽

Substantial Evidence ◽

Major Site ◽

Ribosomal Subunits ◽

Late Maturation ◽

Mrna Binding ◽

Nuclear Translation

The nucleolus is the most prominent morphological feature within the nucleus of eukaryotic cells and is best known for its role in ribosome biogenesis. It forms around highly transcribed ribosomal RNA gene repeats which yield precursor rRNAs that are co-transcriptionally processed, folded and, while still within the nucleolus, associate with most of the ribosomal proteins. The nucleolus is therefore often thought of as a factory for making ribosomal subunits, which are exported as inactive precursors to the cytoplasm where late maturation makes them capable of mRNA binding and translation initiation. However, recent studies have shown substantial evidence for the presence of functional, translation competent ribosomal subunits within the nucleus, particularly in the nucleolus. These observations raise the intriguing possibility that the nucleolus, as well as being a ribosome factory, is also an important nuclear protein-synthesis plant.

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Species Specificity in Ribosome Biogenesis: a Nonconserved Phosphoprotein Is Required for Formation of the Large Ribosomal Subunit in Trypanosoma brucei

Eukaryotic Cell ◽

10.1128/ec.4.1.30-35.2005 ◽

2005 ◽

Vol 4 (1) ◽

pp. 30-35 ◽

Cited By ~ 27

Author(s):

Bryan C. Jensen ◽

Deirdre L. Brekken ◽

Amber C. Randall ◽

Charles T. Kifer ◽

Marilyn Parsons

Keyword(s):

Trypanosoma Brucei ◽

Small Rnas ◽

Ribosome Biogenesis ◽

Large Subunit ◽

Ribosomal Subunit ◽

Protozoan Parasite ◽

Growth Defect ◽

Large Ribosomal Subunit ◽

Severe Growth Defect ◽

Severe Growth

ABSTRACT In the protozoan parasite Trypanosoma brucei, the large rRNA, which is a single 3.4- to 5-kb species in most organisms, is further processed to form six distinct RNAs, two larger than 1 kb (LSU1 and LSU2) and four smaller than 220 bp. The small rRNA SR1 separates the two large RNAs, while the remaining small RNAs are clustered at the 3′ end of the precursor rRNA. One would predict that T. brucei possesses specific components to carry out these added processing events. We show here that the trypanosomatid-specific nucleolar phosphoprotein NOPP44/46 is involved in this further processing. Cells depleted of NOPP44/46 by RNA interference had a severe growth defect and demonstrated a defect in large-ribosomal-subunit biogenesis. Concurrent with this defect, a significant decrease in processing intermediates, particularly for SR1, was seen. In addition, we saw an accumulation of aberrant processing intermediates caused by cleavage within either LSU1 or LSU2. Though it is required for large-subunit biogenesis, we show that NOPP44/46 is not incorporated into the nascent particle. Thus, NOPP44/46 is an unusual protein in that it is both nonconserved and required for ribosome biogenesis.

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Intersection of the Kap123p-Mediated Nuclear Import and Ribosome Export Pathways

Molecular and Cellular Biology ◽

10.1128/mcb.23.6.2042-2054.2003 ◽

2003 ◽

Vol 23 (6) ◽

pp. 2042-2054 ◽

Cited By ~ 49

Author(s):

Y. Sydorskyy ◽

D. J. Dilworth ◽

E. C. Yi ◽

D. R. Goodlett ◽

R. W. Wozniak ◽

...

Keyword(s):

Nuclear Import ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Genetic Interaction ◽

Ribosomal Subunit ◽

Nucleocytoplasmic Transport ◽

Rrna Processing ◽

Ribosomal Subunits ◽

Double Mutant Strain ◽

Ribosome Export

ABSTRACT Kap123p is a yeast β-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a ΔRAI1-induced decrease in the overall biogenesis efficiency.

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Control points in eucaryotic ribosome biogenesis

Biochemistry and Cell Biology ◽

10.1139/o91-002 ◽

1991 ◽

Vol 69 (1) ◽

pp. 5-22 ◽

Cited By ~ 56

Author(s):

D. E. Larson ◽

P. Zahradka ◽

B. H. Sells

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Ribosomal Proteins ◽

Ribosome Biogenesis ◽

Rna Polymerase Iii ◽

Ribosomal Subunits ◽

Rrna Species ◽

Polymerase I ◽

Ribosomal Precursor ◽

Precursor Molecules

Ribosome biogenesis in eucaryotic cells involves the coordinated synthesis of four rRNA species, transcribed by RNA polymerase I (18S, 28S, 5.8S) and RNA polymerase III (5S), and approximately 80 ribosomal proteins translated from mRNAs synthesized by RNA polymerase II. Assembly of the ribosomal subunits in the nucleolus, the site of 45S rRNA precursor gene transcription, requires the movement of 5S rRNA and ribosomal proteins from the nucleoplasm and cytoplasm, respectively, to this structure. To integrate these events and ensure the balanced production of individual ribosomal components, different strategies have been developed by eucaryotic organisms in response to a variety of physiological changes. This review presents an overview of the mechanisms modulating the production of ribosomal precursor molecules and the rate of ribosome biogenesis in various biological systems.Key words: rRNA, ribosomal proteins, nucleolus, ribosome.

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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.

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