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.

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 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 Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Structural Basis ◽  
Catalytic Core ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Translation Systems

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (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. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, 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 define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

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

2004 ◽  
Vol 24 (12) ◽  
pp. 5534-5547 ◽  
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.

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

2007 ◽  
Vol 177 (4) ◽  
pp. 573-578 ◽  
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.

scholarly journals Regulation of Ribosomal Protein OperonsrplM-rpsI,rpmB-rpmG, andrplU-rpmAat the Transcriptional and Translational Levels

2016 ◽  
Vol 198 (18) ◽  
pp. 2494-2502 ◽  
Author(s):  
Leonid V. Aseev ◽  
Ludmila S. Koledinskaya ◽  
Irina V. Boni
Keyword(s):  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
General Rule ◽  
Single Copy ◽  
Translation Initiation Region ◽  
Content Type ◽  
Regulatory Pathways ◽  
E Coli

ABSTRACTIt is widely assumed that in the best-characterized model bacteriumEscherichia coli, transcription units encoding ribosomal proteins (r-proteins) and regulation of their expression have been already well defined. However, transcription start sites for severalE. colir-protein operons have been established only very recently, so that information concerning the regulation of these operons at the transcriptional or posttranscriptional level is still missing. This paper describes for the first time thein vivoregulation of three r-protein operons,rplM-rpsI,rpmB-rpmG, andrplU-rpmA. The results demonstrate that transcription of all three operons is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation, in parallel with the rRNA operons. By using single-copy translational fusions with the chromosomallacZgene, we show here that at the translation level only one of these operons,rplM-rpsI, is regulated by the mechanism of autogenous repression involving the 5′ untranslated region (UTR) of the operon mRNA, whilerpmB-rpmGandrplU-rpmAare not subject to this type of regulation. This may imply that translational feedback control is not a general rule for modulating the expression ofE. colir-protein operons. Finally, we report that L13, a primary protein in 50S ribosomal subunit assembly, serves as a repressor ofrplM-rpsIexpressionin vivo, acting at a target within therplMtranslation initiation region. Thus, L13 represents a novel example of regulatory r-proteins in bacteria.IMPORTANCEIt is important to obtain a deeper understanding of the regulatory mechanisms responsible for coordinated and balanced synthesis of ribosomal components. In this paper, we highlight the major role of a stringent response in regulating transcription of three previously unexplored r-protein operons, and we show that only one of them is subject to feedback regulation at the translational level. Improved knowledge of the regulatory pathways controlling ribosome biogenesis may promote the development of novel antibacterial agents.

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

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.

scholarly journals Pol I Transcription and Pre-rRNA Processing Are Coordinated in a Transcription-dependent Manner in Mammalian Cells

2007 ◽  
Vol 18 (2) ◽  
pp. 394-403 ◽  
Author(s):  
K. Kopp ◽  
J. Z. Gasiorowski ◽  
D. Chen ◽  
R. Gilmore ◽  
J. T. Norton ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Ribosome Biogenesis ◽  
Rrna Synthesis ◽  
Dependent Manner ◽  
Rrna Processing ◽  
Rdna Transcription ◽  
Pol I ◽  
Active Transcription ◽  
Key Steps ◽  
Processing Factors

Pre-rRNA synthesis and processing are key steps in ribosome biogenesis. Although recent evidence in yeast suggests that these two processes are coupled, the nature of their association is unclear. In this report, we analyze the coordination between rDNA transcription and pre-rRNA processing in mammalian cells. We found that pol I transcription factor UBF interacts with pre-rRNA processing factors as analyzed by immunoprecipitations, and the association depends on active rRNA synthesis. In addition, injections of plasmids containing the human rDNA promoter and varying lengths of 18S rDNA into HeLa nuclei show that pol I transcription machinery can be recruited to rDNA promoters regardless of the product that is transcribed, whereas subgroups of pre-rRNA processing factors are recruited to plasmids only when specific pre-rRNA fragments are produced. Our observations suggest a model for sequential recruitment of pol I transcription factors and pre-rRNA processing factors to elongating pre-rRNA on an as-needed basis rather than corecruitment to sites of active transcription.

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

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.

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

2021 ◽  
Author(s):  
Majeed Bakari-Soale ◽  
Nonso Josephat Ikenge ◽  
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

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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