The in vivo functions of ARPF2 and ARRS1 in ribosomal RNA processing and ribosome biogenesis in Arabidopsis

2020 ◽  
Vol 71 (9) ◽  
pp. 2596-2611
Author(s):  
Ilyeong Choi ◽  
Young Jeon ◽  
Youngki Yoo ◽  
Hyun-Soo Cho ◽  
Hyun-Sook Pai
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Critical Role ◽  
Ribosomal Subunit ◽  
Ribosome Profiling ◽  
Production Factor ◽  
Rrna Synthesis ◽  
Binary Complex ◽  
Rrna Processing ◽  
Loss Of Function

Abstract Yeast Rpf2 plays a critical role in the incorporation of 5S rRNA into pre-ribosomes by forming a binary complex with Rrs1. The protein characteristics and overexpression phenotypes of Arabidopsis Ribosome Production Factor 2 (ARPF2) and Arabidopsis Regulator of Ribosome Synthesis 1 (ARRS1) have been previously studied. Here, we analyze loss-of-function phenotypes of ARPF2 and ARRS1 using virus-induced gene silencing to determine their functions in pre-rRNA processing and ribosome biogenesis. ARPF2 silencing in Arabidopsis led to pleiotropic developmental defects. RNA gel blot analysis and circular reverse transcription–PCR revealed that ARPF2 depletion delayed pre-rRNA processing, resulting in the accumulation of multiple processing intermediates. ARPF2 fractionated primarily with the 60S ribosomal subunit. Metabolic rRNA labeling and ribosome profiling suggested that ARPF2 deficiency mainly affected 25S rRNA synthesis and 60S ribosome biogenesis. ARPF2 and ARRS1 formed the complex that interacted with the 60S ribosomal proteins RPL5 and RPL11. ARRS1 silencing resulted in growth defects, accumulation of processing intermediates, and ribosome profiling similar to those of ARPF2-silenced plants. Moreover, depletion of ARPF2 and ARRS1 caused nucleolar stress. ARPF2-deficient plants excessively accumulated anthocyanin and reactive oxygen species. Collectively, these results suggest that the ARPF2–ARRS1 complex plays a crucial role in plant growth and development by modulating ribosome biogenesis.

scholarly journals Impaired ribosome biogenesis in Diamond-Blackfan anemia

Blood ◽  
2006 ◽  
Vol 109 (3) ◽  
pp. 1275-1283 ◽  
Author(s):  
Valérie Choesmel ◽  
Daniel Bacqueville ◽  
Jacques Rouquette ◽  
Jacqueline Noaillac-Depeyre ◽  
Sébastien Fribourg ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Ribosomal Subunit ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Gene Encoding ◽  
Ribosomal Subunits ◽  
Diamond Blackfan Anemia ◽  
Nucleolar Organization ◽  
Ribosomal Protein S19

Abstract The gene encoding the ribosomal protein S19 (RPS19) is frequently mutated in Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. The consequence of these mutations on the onset of the disease remains obscure. Here, we show that RPS19 plays an essential role in biogenesis of the 40S small ribosomal subunit in human cells. Knockdown of RPS19 expression by siRNAs impairs 18S rRNA synthesis and formation of 40S subunits and induces apoptosis in HeLa cells. Pre-rRNA processing is altered, which leads to an arrest in the maturation of precursors to the 18S rRNA. Under these conditions, pre-40S particles are not exported to the cytoplasm and accumulate in the nucleoplasm of the cells in perinuclear dots. Consistently, we find that ribosome biogenesis and nucleolar organization is altered in skin fibroblasts from DBA patients bearing mutations in the RPS19 gene. In addition, maturation of the 18S rRNA is also perturbed in cells from a patient bearing no RPS19-related mutation. These results support the hypothesis that DBA is directly related to a defect in ribosome biogenesis and indicate that yet to be discovered DBA-related genes may be involved in the synthesis of the ribosomal subunits.

scholarly journals Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Required for Small Ribosomal Subunit Synthesis

2006 ◽  
Vol 26 (4) ◽  
pp. 1183-1194 ◽  
Author(s):  
Sander Granneman ◽  
Kara A. Bernstein ◽  
Franziska Bleichert ◽  
Susan J. Baserga
Keyword(s):  
Ribosome Biogenesis ◽  
Mutational Analysis ◽  
Ribosomal Subunit ◽  
Biochemical Properties ◽  
Dominant Negative ◽  
Rrna Synthesis ◽  
Rrna Processing ◽  
Rna Helicases ◽  
Rrna Species

ABSTRACT The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis.

scholarly journals Identification of Novel RNA-Protein Contact in Complex of Ribosomal Protein S7 and 3’-Terminal Fragment of 16S rRNA in E. coli

Acta Naturae ◽  
2012 ◽  
Vol 4 (4) ◽  
pp. 65-72 ◽  
Author(s):  
A. V. Golovin ◽  
G. A. Khayrullina ◽  
B. Kraal ◽  
А. М. Kopylov
Keyword(s):  
16S Rrna ◽  
Ribosomal Protein ◽  
Self Assembly ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Regulatory Function ◽  
Binary Complex ◽  
30S Subunit ◽  
Cross Links

For prokaryotes in vitro, 16S rRNA and 20 ribosomal proteins are capable of hierarchical self- assembly yielding a 30S ribosomal subunit. The self-assembly is initiated by interactions between 16S rRNA and three key ribosomal proteins: S4, S8, and S7. These proteins also have a regulatory function in the translation of their polycistronic operons recognizing a specific region of mRNA. Therefore, studying the RNAprotein interactions within binary complexes is obligatory for understanding ribosome biogenesis. The non-conventional RNAprotein contact within the binary complex of recombinant ribosomal protein S7 and its 16S rRNA binding site (236 nucleotides) was identified. UVinduced RNAprotein cross-links revealed that S7 cross-links to nucleotide U1321 of 16S rRNA. The careful consideration of the published RNA protein cross-links for protein S7 within the 30S subunit and their correlation with the X-ray data for the 30S subunit have been performed. The RNA protein crosslink within the binary complex identified in this study is not the same as the previously found cross-links for a subunit both in a solution, and in acrystal. The structure of the binary RNAprotein complex formed at the initial steps of self-assembly of the small subunit appears to be rearranged during the formation of the final structure of the subunit.

scholarly journals Transcriptomic analysis of ribosome biogenesis and pre-rRNA processing during growth stress in Entamoeba histolytica

2021 ◽  
Author(s):  
Sarah Naiyer ◽  
Shashi Shekhar Singh ◽  
Devinder Kaur ◽  
Yatendra Pratap Singh ◽  
Amartya Mukherjee ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Single Copy ◽  
Growth Stress ◽  
Model Organisms ◽  
Rrna Synthesis ◽  
Northern Hybridization ◽  
Rrna Processing ◽  
Mitochondrial Ribosome

Ribosome biogenesis, a multi-step process involving the transcription, modification, folding and processing of rRNA is the major consumer of cellular energy. It involves the sequential assembly of ribosomal proteins (RP)s via more than 200 ribogenesis factors. Unlike model organisms where transcription of rRNA and RP genes slows down during stress, in Entamoeba histolytica, pre-rRNA synthesis continues, and unprocessed pre-rRNA accumulates. To gain insight into the vast repertoire of ribosome biogenesis factors and understand the major components playing role during stress we computationally identified the ribosome biogenesis factors in E. histolytica. Of the total ~279 S. cerevisiae proteins, we could only find 188 proteins in E. histolytica. Some of the proteins missing in E. histolytica were also missing in humans. A number of proteins represented by multiple genes in S. cerevisiae had only a single copy in E. histolytica. It was interesting to note that E. histolytica lacked mitochondrial ribosome biogenesis factors and had far less RNase components as compared to S. cerevisiae. Northern hybridization using probes from different spacer regions depicted the accumulation of unprocessed intermediates during stress. Transcriptomic studies revealed the differential regulation of a number of ribosomal factors both in serum-starved and RRP6KD conditions. The ARB1 protein involved at multiple steps of ribosome biogenesis and NEP1 and TSR3 involved in chemical modification of 18S rRNA previously shown to accumulate pre-rRNA precursors upon downregulation in S. cerevisiae and humans were included. The data reveals the importance of some of the major factors required for regulating pre-rRNA processing during stress. This is the first report on the complete repertoire of ribosome biogenesis factors in E. histolytica.

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

2003 ◽  
Vol 23 (6) ◽  
pp. 2042-2054 ◽  
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.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Structural Basis ◽  
Peptidyl Transferase ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
In Vitro Reconstitution

AbstractRibosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

scholarly journals miR-16-5p Promotes Erythroid Maturation of Erythroleukemia Cells by Regulating Ribosome Biogenesis

2021 ◽  
Vol 14 (2) ◽  
pp. 137
Author(s):  
Christos I. Papagiannopoulos ◽  
Nikoleta F. Theodoroula ◽  
Ioannis S. Vizirianakis
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Cultured Cells ◽  
Erythroid Differentiation ◽  
Underlying Mechanism ◽  
Loss Of Function ◽  
Functional Studies ◽  
Differentiated Cells ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia

miRNAs constitute a class of non-coding RNA that act as powerful epigenetic regulators in animal and plant cells. In order to identify putative tumor-suppressor miRNAs we profiled the expression of various miRNAs during differentiation of erythroleukemia cells. RNA was purified before and after differentiation induction and subjected to quantitative RT-PCR. The majority of the miRNAs tested were found upregulated in differentiated cells with miR-16-5p showing the most significant increase. Functional studies using gain- and loss-of-function constructs proposed that miR-16-5p has a role in promoting the erythroid differentiation program of murine erythroleukemia (MEL) cells. In order to identify the underlying mechanism of action, we utilized bioinformatic in-silico platforms that incorporate predictions for the genes targeted by miR-16-5p. Interestingly, ribosome constituents, as well as ribosome biogenesis factors, were overrepresented among the miR-16-5p predicted gene targets. Accordingly, biochemical experiments showed that, indeed, miR-16-5p could modulate the levels of independent ribosomal proteins, and the overall ribosomal levels in cultured cells. In conclusion, miR-16-5p is identified as a differentiation-promoting agent in erythroleukemia cells, demonstrating antiproliferative activity, likely as a result of its ability to target the ribosomal machinery and restore any imbalanced activity imposed by the malignancy and the blockade of differentiation.

scholarly journals Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

2000 ◽  
Vol 20 (15) ◽  
pp. 5516-5528 ◽  
Author(s):  
Žaklina Strezoska ◽  
Dimitri G. Pestov ◽  
Lester F. Lau
Keyword(s):  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Weight Fraction ◽  
Rnase A ◽  
Mutant Form ◽  
Rrna Processing ◽  
Ribonucleoprotein Complexes ◽  
Nuclear Extracts ◽  
Mouse Tissues ◽  
Mouse Cells

ABSTRACT We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1Δ, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1Δ leads to cell growth arrest in the G1phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1Δ expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1Δ in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.

scholarly journals Neuronal ribosomes exhibit dynamic and context-dependent exchange of ribosomal proteins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Claudia M. Fusco ◽  
Kristina Desch ◽  
Aline R. Dörrbaum ◽  
Mantian Wang ◽  
Anja Staab ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosome Profiling ◽  
Metabolic Labeling ◽  
Local Protein Synthesis ◽  
Translational Machinery ◽  
Neuronal Synapses ◽  
Cellular Environment ◽  
Neuronal Processes ◽  
Local Protein

AbstractOwing to their morphological complexity and dense network connections, neurons modify their proteomes locally, using mRNAs and ribosomes present in the neuropil (tissue enriched for dendrites and axons). Although ribosome biogenesis largely takes place in the nucleus and perinuclear region, neuronal ribosomal protein (RP) mRNAs have been frequently detected remotely, in dendrites and axons. Here, using imaging and ribosome profiling, we directly detected the RP mRNAs and their translation in the neuropil. Combining brief metabolic labeling with mass spectrometry, we found that a group of RPs rapidly associated with translating ribosomes in the cytoplasm and that this incorporation was independent of canonical ribosome biogenesis. Moreover, the incorporation probability of some RPs was regulated by location (neurites vs. cell bodies) and changes in the cellular environment (following oxidative stress). Our results suggest new mechanisms for the local activation, repair and/or specialization of the translational machinery within neuronal processes, potentially allowing neuronal synapses a rapid means to regulate local protein synthesis.

scholarly journals Nucleolin loss-of-function leads to aberrant FGF signaling and craniofacial anomalies

2021 ◽  
Author(s):  
Soma Dash ◽  
Paul Trainor
Keyword(s):  
Ribosome Biogenesis ◽  
Craniofacial Development ◽  
Rrna Synthesis ◽  
Growth Factor Signaling ◽  
Craniofacial Anomalies ◽  
Fgf Signaling ◽  
Loss Of Function ◽  
Tissue Specific ◽  
Rrna Transcription ◽  
Exogenous Addition

rRNA transcription and ribosome biogenesis are global processes required for growth and proliferation of all cells, yet perturbation of these processes in vertebrates leads to tissue-specific defects termed ribosomopathies. Mutations in rRNA transcription and processing proteins often lead to craniofacial anomalies, however the cellular and molecular reasons for this are poorly understood. Therefore, we examined the function of the most abundant nucleolar phosphoprotein, Nucleolin (Ncl), in vertebrate development. We discovered that Nucleolin is dynamically expressed during embryonic development with high enrichment in the craniofacial tissues. Consistent with this pattern of expression, ncl homozygous mutant (ncl-/-) zebrafish present with craniofacial anomalies such as mandibulofacial hypoplasia. We observe that ncl-/- mutants exhibit decreased rRNA synthesis and p53-dependent neuroepithelial cell death. In addition, the half-life of fgf8a mRNA is reduced in ncl-/- mutants, which perturbs Fgf signaling, resulting in misregulation of Sox9a mediated chondrogenesis and Runx2 mediated osteogenesis. Exogenous addition of human recombinant FGF8 to the mutant zebrafish significantly rescues the cranioskeletal phenotype, suggesting that Nucleolin regulates osteochondroprogenitor differentiation during craniofacial development by post-transcriptionally regulating Fgf signaling. Our work has therefore uncovered a novel tissue-specific function for Nucleolin in rRNA transcription and growth factor signaling during embryonic craniofacial development.

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