Eukaryotic Ribosome Assembly.

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.

Functions of ribosomal proteins in assembly of eukaryotic ribosomes in vivo.

The proteome of cells is synthesized by ribosomes, complex ribonucleoproteins that in eukaryotes contain 79-80 proteins and four ribosomal RNAs (rRNAs) more than 5,400 nucleotides long. How these molecules assemble together and how their assembly is regulated in concert with the growth and proliferation of cells remain important unanswered questions. Here, we review recently emerging principles to understand how eukaryotic ribosomal proteins drive ribosome assembly in vivo. Most ribosomal proteins assemble with rRNA cotranscriptionally; their association with nascent particles is strengthened as assembly proceeds. Each subunit is assembled hierarchically by sequential stabilization of their subdomains. The active sites of both subunits are constructed last, perhaps to prevent premature engagement of immature ribosomes with active subunits. Late-assembly intermediates undergo quality-control checks for proper function. Mutations in ribosomal proteins that affect mostly late steps lead to ribosomopathies, diseases that include a spectrum of cell type-specific disorders that often transition from hypoproliferative to hyperproliferative growth.

Ribosome biogenesis factors-from names to functions.

The assembly of ribosomal subunits is a highly orchestrated process that involves a huge cohort of accessory factors. Most eukaryotic ribosome biogenesis factors were first identified by genetic screens and proteomic approaches of pre-ribosomal particles in Saccharomyces cerevisiae. Later, research on human ribosome synthesis not only demonstrated that the requirement for many of these factors is conserved in evolution, but also revealed the involvement of additional players, reflecting a more complex assembly pathway in mammalian cells. Yet, it remained a challenge for the field to assign a function to many of the identified factors and to reveal their molecular mode of action. Over the past decade, structural, biochemical, and cellular studies have largely filled this gap in knowledge and led to a detailed understanding of the molecular role that many of the players have during the stepwise process of ribosome maturation. Such detailed knowledge of the function of ribosome biogenesis factors will be key to further understand and better treat diseases linked to disturbed ribosome assembly, including ribosomopathies, as well as different types of cancer.

The novel pre-rRNA detection workflow "Riboprobing" allows simple identification of undescribed RNA species.

Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes, both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with the transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs, which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, northern blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with primer extension, both approaches often use radioactivity and are time-consuming and costly. Here, we present "Riboprobing," a linker ligation-based workflow followed by reverse transcription and PCR for easy and fast detection and characterization of pre-rRNA species and their 5' as well as 3' ends. Using standard molecular biology laboratory equipment, "Riboprobing" allows reliable discrimination of pre-rRNA species not resolved by northern blot (e.g., 27SA2, 27SA3, and 27SB pre-rRNA). The method can successfully be used for the analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that lacks most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5'ETS (external transcribed spacer) for the assembly process.

Naphthoquinone-induced arylation inhibits Sirtuin 7 activity.

Natural or synthetic naphthoquinones have been identified to interfere with biological systems and, in particular, exhibit anticancer properties. As redox cyclers, they generate reactive oxygen species in cells and, as electrophiles, they react with nucleophiles, mainly thiols, and form covalent adducts. To further decipher the molecular mechanism of action of naphthoquinones in human cells, we analyzed their effects in HeLa cells. First, we demonstrated that the naphthoquinones menadione and plumbagin inhibited the nucleolar NAD+-dependent deacetylase Sirtuin 7 in vitro. As assessed by their inhibition of rDNA transcription, pre-rRNA processing and formation of etoposide-induced 53BP1 foci, menadione and plumbagin also inhibited Sirtuin 7 catalytic activity in vivo. Second, we established that when sulfhydryl arylation by menadione or plumbagin was prevented by the thiol reducing agent N-acetyl-L-cysteine, the inhibition of Sirtuin 7 catalytic activity was also blocked. Finally, we discuss how inhibition of Sirtuin 7 might be crucial in defining menadione or plumbagin as anti-tumor agents that can be used in combination with other anti-tumor strategies.

Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish.

Dnttip2 is one of the components of the small subunit (SSU) processome. In yeast, depletion of dnttip2 leads to an inefficient processing of pre-rRNA and a decrease in synthesis of the mature 18S rRNA. However, the biological roles of Dnttip2 in higher organisms are poorly defined. In this study, we demonstrate that dnttip2 is a maternal gene in zebrafish. Depletion of Dnttip2 leads to embryonic lethal with severe digestive organs hypoplasia. The loss of function of Dnttip2 also leads to partial defects in cleavage at the A0-site and E-site during 18S rRNA processing. In conclusion, Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish.

Yeast Dxo1 is required for 25S rRNA maturation and acts as a transcriptome-wide distributive exonuclease.

The Dxo1/Rai1/DXO family of decapping and exonuclease enzymes can catalyze the in vitro removal of chemically diverse 5' ends from RNA. Specifically, these enzymes act poorly on RNAs with a canonical 7mGpppN cap, but instead prefer RNAs with a triphosphate, monophosphate, hydroxyl, or nonconventional cap. In each case, these enzymes generate an RNA with a 5' monophosphate, which is then thought to be further degraded by Rat1/Xrn1 5' exoribonucleases. For most Dxo1/Rai1/DXO family members, it is not known which of these activities is most important in vivo. Here we describe the in vivo function of the poorly characterized cytoplasmic family member, yeast Dxo1. Using RNA-seq of 5' monophosphate ends, we show that Dxo1 can act as a distributive exonuclease, removing a few nucleotides from endonuclease or decapping products. We also show that Dxo1 is required for the final 5' end processing of 25S rRNA, and that this is the primary role of Dxo1. While Dxo1/Rai1/DXO members were expected to act upstream of Rat1/Xrn1, this order is reversed in 25S rRNA processing, with Dxo1 acting downstream from Rat1. Such a hand-off from a processive to a distributive exonuclease may be a general phenomenon in the precise maturation of RNA ends.

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing.

In eukaryotes, the nucleolus is the site of ribosome biosynthesis, an essential process in all cells. While human ribosome assembly is largely evolutionarily conserved, many of the regulatory details underlying its control and function have not yet been well-defined. The nucleolar protein RSL24D1 was originally identified as a factor important for 60S ribosomal subunit biogenesis. In addition, the PeBoW (BOP1-PES1-WDR12) complex has been well-defined as required for pre-28S rRNA processing and cell proliferation. In this study, we show that RSL24D1 depletion impairs both pre-ribosomal RNA (pre-rRNA) transcription and mature 28S rRNA production, leading to decreased protein synthesis and p53 stabilization in human cells. Surprisingly, each of the PeBoW complex members is also required for pre-rRNA transcription. We demonstrate that RSL24D1 and WDR12 co-immunoprecipitate with the RNA polymerase I subunit, RPA194, and regulate its steady state levels. These results uncover the dual role of RSL24D1 and the PeBoW complex in multiple steps of ribosome biogenesis, and provide evidence implicating large ribosomal subunit biogenesis factors in pre-rRNA transcription control.

Diamond-Blackfan anemia, the archetype of ribosomopathy: How distinct is it from the other constitutional ribosomopathies?

Diamond-Blackfan anemia (DBA) was the first ribosomopathy described in humans. DBA is a congenital hypoplastic anemia, characterized by macrocytic aregenerative anemia, manifesting by differentiation blockage between the BFU-e/CFU-e developmental erythroid progenitor stages. In 50 % of the DBA cases, various malformations are noted. Strikingly, for a hematological disease with a relative erythroid tropism, DBA is due to ribosomal haploinsufficiency in 24 different ribosomal protein (RP) genes. A few other genes have been described in DBA-like disorders, but they do not fit into the classical DBA phenotype (Sankaran et al., 2012; van Dooijeweert et al., 2022; Toki et al., 2018; Kim et al., 2017 [1-4]). Haploinsufficiency in a RP gene leads to defective ribosomal RNA (rRNA) maturation, which is a hallmark of DBA. However, the mechanistic understandings of the erythroid tropism defect in DBA are still to be fully defined. Erythroid defect in DBA has been recently been linked in a non-exclusive manner to a number of mechanisms that include: 1) a defect in translation, in particular for the GATA1 erythroid gene; 2) a deficit of HSP70, the GATA1 chaperone, and 3) free heme toxicity. In addition, p53 activation in response to ribosomal stress is involved in DBA pathophysiology. The DBA phenotype may thus result from the combined contributions of various actors, which may explain the heterogenous phenotypes observed in DBA patients, even within the same family.

Crossing boundaries of light microscopy resolution discerns novel assemblies in the nucleolus.

The nucleolus is the largest membraneless organelle and nuclear body in mammalian cells. It is primarily involved in the biogenesis of ribosomes, essential macromolecular machines responsible for synthesizing all proteins required by the cell. The assembly of ribosomes is evolutionarily conserved and accounts for the most energy-consuming cellular process needed for cell growth, proliferation, and homeostasis. Despite the significance of this process, the substructural mechanistic principles of the nucleolar function in preribosome biogenesis have only recently begun to emerge. Here, we provide a new perspective using advanced super-resolution microscopy and single-molecule MINFLUX nanoscopy on the mechanistic principles governing ribosomal RNA-seeded nucleolar formation and the resulting tripartite suborganization of the nucleolus driven, in part, by liquid-liquid phase separation. With recent advances in the cryogenic electron microscopy (cryoEM) structural analysis of ribosome biogenesis intermediates, we highlight the current understanding of the step-wise assembly of preribosomal subunits in the nucleolus. Finally, we address how novel anticancer drug candidates target early steps in ribosome biogenesis to exploit these essential dependencies for growth arrest and tumor control.

Defective kernel 58 encodes an Rrp15p domain-containing protein essential to ribosome biogenesis and seed development in maize.

Ribosome biogenesis is a highly dynamic and orchestrated process facilitated by hundreds of ribosomal biogenesis factors and small nucleolar RNAs. While many of the advances are derived from studies in yeast, ribosome biogenesis remains largely unknown in plants despite its importance to plant growth and development. Through characterizing the maize (Zea mays) defective kernel and embryo-lethal mutant dek58, we show that DEK58 encodes an Rrp15p domain-containing protein with 15.3% identity to yeast Rrp15. Over-expression of DEK58 rescues the mutant phenotype. DEK58 is localized in the nucleolus. Ribosome profiling and RNA gel blot analyses show that the absence of DEK58 reduces ribosome assembly and impedes pre-rRNA processing, accompanied by the accumulation of nearly all the pre-rRNA processing intermediates and the production of an aberrant processing product P-25S*. DEK58 interacts with ZmSSF1, a maize homolog of the yeast Ssf1 in the 60S processome. DEK58 and ZmSSF1 interact with ZmCK2α, a putative component of the yeast UTP-C complex involved in the small ribosomal subunit processome. These results demonstrate that DEK58 is essential to seed development in maize. It functions in the early stage of pre-rRNA processing in ribosome biogenesis, possibly through interacting with ZmSSF1 and ZmCK2α in maize.

Rate of transcription elongation and sequence-specific pausing by RNA polymerase I directly influence rRNA processing.

One of the first steps in ribosome biogenesis is transcription of the ribosomal DNA by RNA polymerase I (Pol I). Processing of the resultant rRNA begins cotranscriptionally, and perturbation of Pol I transcription elongation results in defective rRNA processing. Mechanistic insight regarding the link between transcription elongation and ribosome assembly is lacking because of limited in vivo methods to assay Pol I transcription. Here, we use native elongating transcript sequencing (NET-Seq) with a strain of Saccharomyces cerevisiae containing a point mutation in Pol I, rpa190-F1205H, which results in impaired rRNA processing and ribosome assembly. We previously demonstrated that this mutation caused a mild reduction in the transcription elongation rate of Pol I in vitro; however, transcription elongation by the mutant has not been characterized in vivo. Here, our findings demonstrate that the mutant Pol I has an increased pause propensity during processive transcription elongation both in vitro and in vivo. NET-Seq reveals that rpa190-F1205H Pol I displays alternative pause site preferences in vivo. Specifically, the mutant is sensitized to A/G residues in the RNA:DNA hybrid and at the last incorporated nucleotide position. Furthermore, both NET-Seq and EM analysis of Miller chromatin spreads reveal pileups of rpa190-F1205H Pol I throughout the ribosomal DNA, particularly at the 5' end of the 35S gene. This combination of in vitro and in vivo analyses of a Pol I mutant provides novel insights into Pol I elongation properties and indicates how these properties are crucial for efficient cotranscriptional rRNA processing and ribosome assembly.

The nucleolar protein NOL12 is required for processing of large ribosomal subunit rRNA precursors in Arabidopsis.

BACKGROUND: NOL12 5'-3' exoribonucleases, conserved among eukaryotes, play important roles in pre-rRNA processing, ribosome assembly and export. The most well-described yeast counterpart, Rrp17, is required for maturation of 5.8 and 25S rRNAs, whereas human hNOL12 is crucial for the separation of the large (LSU) and small (SSU) ribosome subunit rRNA precursors. RESULTS: In this study we demonstrate that plant AtNOL12 is also involved in rRNA biogenesis, specifically in the processing of the LSU rRNA precursor, 27S pre-rRNA. Importantly, the absence of AtNOL12 alters the expression of many ribosomal protein and ribosome biogenesis genes. These changes could potentially exacerbate rRNA biogenesis defects, or, conversely, they might stem from the disturbed ribosome assembly caused by delayed pre-rRNA processing. Moreover, exposure of the nol12 mutant to stress factors, including heat and pathogen Pseudomonas syringae, enhances the observed molecular phenotypes, linking pre-rRNA processing to stress response pathways. The aberrant rRNA processing, dependent on AtNOL12, could impact ribosome function, as suggested by improved mutant resistance to ribosome-targeting antibiotics. CONCLUSION: Despite extensive studies, the pre-rRNA processing pathway in plants remains insufficiently characterized. Our investigation reveals the involvement of AtNOL12 in the maturation of rRNA precursors, correlating this process to stress response in Arabidopsis. These findings contribute to a more comprehensive understanding of plant ribosome biogenesis.

Tumor suppressor PNRC1 blocks rRNA maturation by recruiting the decapping complex to the nucleolus.

Focal deletions occur frequently in the cancer genome. However, the putative tumor-suppressive genes residing within these regions have been difficult to pinpoint. To robustly identify these genes, we implemented a computational approach based on non-negative matrix factorization, NMF, and interrogated the TCGA dataset. This analysis revealed a metagene signature including a small subset of genes showing pervasive hemizygous deletions, reduced expression in cancer patient samples, and nucleolar function. Amid the genes belonging to this signature, we have identified PNRC1, a nuclear receptor coactivator. We found that PNRC1 interacts with the cytoplasmic DCP1α/DCP2 decapping machinery and hauls it inside the nucleolus. PNRC1-dependent nucleolar translocation of the decapping complex is associated with a decrease in the 5'-capped U3 and U8 snoRNA fractions, hampering ribosomal RNA maturation. As a result, PNRC1 ablates the enhanced proliferation triggered by established oncogenes such as RAS and MYC These observations uncover a previously undescribed mechanism of tumor suppression, whereby the cytoplasmic decapping machinery is hauled within nucleoli, tightly regulating ribosomal RNA maturation.

MTR4 adaptor PICT1 functions in two distinct steps during pre-rRNA processing.

Ribosome biogenesis proceeds with the successive cleavage and trimming of the large 47S rRNA precursor, where the RNA exosome plays major roles in concert with the Ski2-like RNA helicase, MTR4. The recent finding of a consensus amino acid sequence, the arch-interacting motif (AIM), for binding to the arch domain in MTR4 suggests that recruitment of the RNA processing machinery to the maturing pre-rRNA at appropriate places and timings is mediated by several adaptor proteins possessing the AIM sequence. In yeast Saccharomyces cerevisiae, Nop53 plays such a role in the maturation of the 3'-end of 5.8S rRNA. Here, we investigated the functions of PICT1 (also known as GLTSCR2 or NOP53), a mammalian ortholog of Nop53, during ribosome biogenesis in human cells. PICT1 interacted with MTR4 and exosome in an AIM-dependent manner. Overexpression of PICT1 mutants defecting AIM sequence and siRNA-mediated depletion of PICT1 showed that PICT1 is involved in two distinct pre-rRNA processing steps during the generation of 60S ribosomes; first step is the early cleavage of 32S intermediate RNA, while the second step is the late maturation of 12S precursor into 5.8S rRNA. The recruitment of MTR4 and RNA exosome via the AIM sequence was required only during the late processing step. Although, the depletion of MTR4 and PICT1 induced stabilization of the tumor suppressor p53 protein in cancer cell lines, the depletion of the exosome catalytic subunits, RRP6 and DIS3, did not exert such an effect. These results suggest that recruitment of the RNA processing machinery to the 3'-end of pre-5.8S rRNA may be involved in the induction of the nucleolar stress response, but the pre-rRNA processing capabilities themselves were not involved in this process.

Wide mutational analysis to ascertain the functional roles of eL33 in ribosome biogenesis and translation initiation.

An extensive mutational analysis of RPL33A, encoding the yeast ribosomal protein L33A (eL33) allowed us to identify several novel rpl33a mutants with different translational phenotypes. Most of the rpl33a mutants are defective in the processing of 35S and 27S pre-rRNA precursors and the production of mature rRNAs, exhibiting reductions in the amounts of ribosomal subunits and altered polysome profiles. Some of the rpl33a mutants exhibit a Gcd- phenotype of constitutive derepression of GCN4 translation and strong slow growth phenotypes at several temperatures. Interestingly, some of the later mutants also show a detectable increase in the UUG/AUG translation initiation ratio that can be suppressed by eIF1 overexpression, suggesting a requirement for eL33 and a correct 60S/40S subunit ratio for the proper recognition of the AUG start codon. In addition to producing differential reductions in the rates of pre-rRNA maturation and perhaps in r-protein assembly, most of the point rpl33a mutations alter specific molecular interactions of eL33 with the rRNAs and other r-proteins in the 60S structure. Thus, rpl33a mutations cause distinctive effects on the abundance and/or functionality of 60S subunits, leading to more or less pronounced defects in the rates and fidelity of mRNA translation.

Functional regions in the 5' external transcribed spacer of yeast pre-rRNA.

Ribosomal subunits are assembled on a precursor rRNA that includes four spacers in addition to mature rRNA sequences. The 5' external transcribed spacer (5' ETS) is the most prominent one that recruits U3 snoRNA and a plethora of proteins during the early assembly of 90S small subunit preribosomes. Here, we have conducted a comprehensive mutational analysis of 5' ETS by monitoring the processing and assembly of a plasmid-expressed pre-18S RNA. Remarkably, nearly half of the 5' ETS sequences, when depleted individually, are dispensable for 18S rRNA processing. The dispensable elements largely bind at the surface of the 90S structure. Defective assembly of 5' ETS completely blocks the last stage of 90S formation yet has little effect on the early assembly of 5' and central domains of 18S rRNA. Our study reveals the functional regions of 5' ETS and provides new insight into the assembly hierarchy of 90S preribosomes.

Rcl1 suppresses tumor progression of hepatocellular carcinoma: a comprehensive analysis of bioinformatics and in vitro experiments.

BACKGROUND: RNA 3'-terminal phosphate cyclase-like protein (Rcl1) is involved in pre-rRNA processing, but its implication in cancers remains unclear. METHODS: RCL1 expressions in 21 malignancies was examinated through GEPIA website portal. Clinical implication data related to RCL1 level in Hepatocellular Carcinoma (HCC) samples were downloaded through TCGA, ICGC, GEO databases. Survival analysis and gene function enrichment analyses were performed through R software. The correlation between RCL1 expression and tumor immune infiltration was assessed via the TIMER2.0 database. The effects of Rcl1 overexpression or knockdown on cell growth and metastasis was evaluated by CCK8, transwell, and cell cycle assays. RESULTS: RCL1 expression is commonly down-regulated in HCC. The lower expression of RCL1 is associated with higher tumor stage, higher AFP level, vascular invasion, and poor prognosis. RCL1 expression has a significant correlation with immune cells infiltration in HCC, especially myeloid-derived suppressor cell (MDSC). Moreover, it was further identified that Rcl1 expression was reduced in HCC cell lines and negatively correlated with invasion of HCC cell lines. Immunofluorescence (IF) analysis revealed that the level of Rcl1 expression in the cytoplasm of HCC cells is significantly lower than that in the cytoplasm of L-02 cell. Moreover, both gain- and loss-of-function studies demonstrated that Rcl1 inhibited the growth and metastasis of HCC cells and regulated cell cycle progression in vitro. CONCLUSIONS: Rcl1 may serve as a novel tumor suppressor in HCC, and its biological effect needs further study.

Upon heat stress processing of ribosomal RNA precursors into mature rRNAs is compromised after cleavage at primary P site in Arabidopsis thaliana.

Transcription and processing of 45S rRNAs in the nucleolus are keystones of ribosome biogenesis. While these processes are severely impacted by stress conditions in multiple species, primarily upon heat exposure, we lack information about the molecular mechanisms allowing sessile organisms without a temperature-control system, like plants, to cope with such circumstances. We show that heat stress disturbs nucleolar structure, inhibits pre-rRNA processing and provokes imbalanced ribosome profiles in Arabidopsis thaliana plants. Notably, the accuracy of transcription initiation and cleavage at the primary P site in the 5'ETS (5' External Transcribed Spacer) are not affected but the levels of primary 45S and 35S transcripts are, respectively, increased and reduced. In contrast, precursors of 18S, 5.8S and 25S RNAs are rapidly undetectable upon heat stress. Remarkably, nucleolar structure, pre-rRNAs from major ITS1 processing pathway and ribosome profiles are restored after returning to optimal conditions, shedding light on the extreme plasticity of nucleolar functions in plant cells. Further genetic and molecular analysis to identify molecular clues implicated in these nucleolar responses indicate that cleavage rate at P site and nucleolin protein expression can act as a checkpoint control towards a productive pre-rRNA processing pathway.

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

Ribosome biogenesis, a multi-step process involving transcription, modification, folding and processing of rRNA, is the major consumer of cellular energy. It involves 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. Northern hybridization from different spacer regions depicted the accumulation of unprocessed intermediates during stress. To gain insight into the vast repertoire of ribosome biogenesis factors and understand the major components playing role during stress we computationally identified ribosome biogenesis factors in E. histolytica. Of the ∼279 Saccharomyces 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 a single copy in E. histolytica. Interestingly E. histolytica lacked mitochondrial ribosome biogenesis factors and had far less RNase components compared to S. cerevisiae. Transcriptomic studies revealed the differential regulation of ribosomal factors both in serum starved and RRP6 down-regulation conditions. These included the NEP1 and TSR3 proteins that chemically modify 18S-rRNA. Pre-rRNA precursors accumulate upon downregulation of the latter proteins in S. cerevisiae and humans. These data reveal the major factors that regulate pre-rRNA processing during stress in E. histolytica and provide the first complete repertoire of ribosome biogenesis factors in this early-branching protist.