Both endonucleolytic and exonucleolytic cleavage mediate ITS1 removal during human ribosomal RNA processing

Human ribosome production is up-regulated during tumorogenesis and is defective in many genetic diseases (ribosomopathies). We have undertaken a detailed analysis of human precursor ribosomal RNA (pre-rRNA) processing because surprisingly little is known about this important pathway. Processing in internal transcribed spacer 1 (ITS1) is a key step that separates the rRNA components of the large and small ribosomal subunits. We report that this was initiated by endonuclease cleavage, which required large subunit biogenesis factors. This was followed by 3′ to 5′ exonucleolytic processing by RRP6 and the exosome, an enzyme complex not previously linked to ITS1 removal. In contrast, RNA interference–mediated knockdown of the endoribonuclease MRP did not result in a clear defect in ITS1 processing. Despite the apparently high evolutionary conservation of the pre-rRNA processing pathway and ribosome synthesis factors, each of these features of human ITS1 processing is distinct from those in budding yeast. These results also provide significant insight into the links between ribosomopathies and ribosome production in human cells.     

Nucleolar stress in Diamond Blackfan anemia pathophysiology

Diamond Blackfan anemia is a red cell hypoplasia that typically presents within the first year of life. Most cases of Diamond Blackfan anemia are caused by ribosome assembly defects linked to haploinsufficiency for structural proteins of either ribosomal subunit. Nucleolar stress associated with abortive ribosome assembly leads to p53 activation via the interaction of free ribosomal proteins with HDM2, a negative regulator of p53. Significant challenges remain in linking this nucleolar stress signaling pathway to the clinical features of Diamond Blackfan anemia. Defining aspects of disease presentation may relate to developmental and physiological triggers that work in conjunction with nucleolar stress signaling to heighten the p53 response in the developing erythron after birth. The growing number of ribosomopathies provides additional challenges for linking molecular mechanisms with clinical phenotypes. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.     

Eukaryotic ribosome quality control system: a potential therapeutic target for human diseases

Protein homeostasis is well accepted as the prerequisite for proper operation of various life activities. As the main apparatus of protein translation, ribosomes play an indispensable role in the maintenance of protein homeostasis. Nevertheless, upon stimulation of various internal and external factors, malfunction of ribosomes may be evident with the excessive production of aberrant proteins, accumulation of which can result in deleterious effects on cellular fate and even cell death. Ribosomopathies are characterized as a series of diseases caused by abnormalities of ribosomal compositions and functions. Correspondingly, cell evolves several ribosome quality control mechanisms in maintaining the quantity and quality of intracellular ribosomes, namely ribosome quality control system (RQCS). Of note, RQCS can tightly monitor the entire process from ribosome biogenesis to its degradation, with the capacity of coping with ribosomal dysfunction, including misassembled ribosomes and incorrectly synthesized ribosomal proteins. In the current literature review, we mainly introduce the RQCS and elaborate on the underlying pathogenesis of several ribosomopathies. With the in-depth understanding of ribosomal dysfunction and molecular basis of RQCS, therapeutic strategy by specifically targeting RQCS remains a promising option in treating patients with ribosomopathies and other ribosome-associated human diseases.     

Ribosome biogenesis protein Urb1 acts downstream of mTOR complex 1 to modulate digestive organ development in zebrafish

Ribosome biogenesis is essential for the cell growth and division. Disruptions in ribosome biogenesis result in developmental defects and a group of diseases, known as ribosomopathies. Here, we report a mutation in zebrafish urb1, which encodes an essential ribosome biogenesis protein. The urb1 cq31 mutant exhibits hypoplastic digestive organs, which is caused by impaired cell proliferation with the differentiation of digestive organ progenitors unaffected. Knockdown of mtor or raptor leads to similar hypoplastic phenotypes and reduced expression of urb1 in the digestive organs. Overexpression of Urb1 results in overgrowth of digestive organs, and can efficiently rescue the hypoplastic liver and pancreas in the mtor and raptor morphants. Reduced syntheses of free ribosomal subunits and impaired assembly of polysomes are observed in the urb1 mutant as well as in the mtor and raptor morphants, which can be rescued by the Urb1 overexpression. These data demonstrate that Urb1 plays an important role in governing ribosome biogenesis and protein synthesis downstream of mammalian/mechanistic target of rapamycin complex 1(mTORC1), thus regulating the development of digestive organs. Our study indicates the requirement of hyperactive protein synthesis for the digestive organ development.     

The cancerous translation apparatus

Deregulations in translational control are critical features of cancer initiation and progression. Activation of key oncogenic pathways promotes rapid and dramatic translational reprogramming, not simply by increasing overall protein synthesis, but also by modulating specific mRNA networks that promote cellular transformation. Additionally, ribosomopathies caused by mutations in ribosome components alter translational regulation leading to specific pathological features, including cancer susceptibility. Exciting advances in our understanding of translational control in cancer have illuminated a striking specificity innate to the translational apparatus. Characterizing this specificity will provide novel insights into how cells normally utilize translational control to modulate gene expression, how it is deregulated in cancer, and how these processes can be targeted to develop new cancer therapies.     

核糖体相关质量控制与核糖体自噬研究进展

细胞中蛋白质处于不断合成和降解的动态更新过程中,其稳态与细胞功能密切相关。细胞中存在多种蛋白质质量控制（protein quality control,PQC）机制来监测蛋白质合成和降解过程的异常,以确保蛋白质组的完整性和细胞适应性。核糖体是细胞内数量最多的细胞器,系细胞内蛋白质合成的主要场所。现已明确,核糖体相关质量控制（ribosome-associated quality control,RQC）与核糖体自噬能通过溶酶体依赖和非依赖途径调节细胞内核糖体数量及功能以维持蛋白质稳态,从而增强细胞在应激状态下的适应能力。RQC失调、核糖体自噬障碍则参与多种疾病的发生及发展过程,靶向RQC和核糖体自噬可能成为防治多种疾病的有效手段。本综述聚焦核糖体相关的PQC途径,并进一步讨论了它们在蛋白质稳态维持中的重要地位及其在人类疾病发生发展中的潜在作用。     

Ribosomal Protein Mutations Induce Autophagy through S6 Kinase Inhibition of the Insulin Pathway

Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34⁺ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies.     

Ribosome biogenesis in skeletal development and the pathogenesis of skeletal disorders

The skeleton affords a framework and structural support for vertebrates, while also facilitating movement, protecting vital organs, and providing a reservoir of minerals and cells for immune system and vascular homeostasis. The mechanical and biological functions of the skeleton are inextricably linked to the size and shape of individual bones, the diversity of which is dependent in part upon differential growth and proliferation. Perturbation of bone development, growth and proliferation, can result in congenital skeletal anomalies, which affect approximately 1 in 3000 live births [1]. Ribosome biogenesis is integral to all cell growth and proliferation through its roles in translating mRNAs and building proteins. Disruption of any steps in the process of ribosome biogenesis can lead to congenital disorders termed ribosomopathies. In this review, we discuss the role of ribosome biogenesis in skeletal development and in the pathogenesis of congenital skeletal anomalies. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.     

Access to ribosomal protein Rpl25p by the signal recognition particle is required for efficient cotranslational translocation

Targeting of proteins to the endoplasmic reticulum (ER) occurs cotranslationally necessitating the interaction of the signal recognition particle (SRP) and the translocon with the ribosome. Biochemical and structural studies implicate ribosomal protein Rpl25p as a major ribosome interaction site for both these factors. Here we characterize an RPL25GFP fusion, which behaves as a dominant mutant leading to defects in co- but not posttranslational translocation in vivo. In these cells, ribosomes still interact with ER membrane and the translocon, but are defective in binding SRP. Overexpression of SRP can restore ribosome binding of SRP, but only partially rescues growth and translocation defects. Our results indicate that Rpl25p plays a critical role in the recruitment of SRP to the ribosome.     

New insights into 5q- syndrome as a ribosomopathy

Myelodysplastic Syndromes (MDS) are a heterogeneous group of acquired clonal bone marrow disorders, characterised by ineffective haematopoiesis. The mechanisms underlying many of these blood disorders have remained elusive due to the difficulty in pinpointing specific gene mutations or haploinsufficencies, which can occur within large deleted regions. However, there is an increasing interest in the classification of some of these diseases as ribosomopathies. Indeed, studies have implicated Ribosomal Protein (RP) S14 as a strong candidate for haploinsufficiency in 5q- syndrome, a particular form of MDS. Recently, two novel mouse models have provided evidence for the involvement of both RPS14 and the p53 pathway, and specific miRNAs in 5q- syndrome. In this review we will discuss: 5q- syndrome mouse models, the possible mechanisms underlying this blood disorder with respect to the candidate genes, and comparisons with other ribosomopathies, and the involvement of the p53 pathway in these diseases.     

The Ribosome Biogenesis Protein Nol9 Is Essential for Definitive Hematopoiesis and Pancreas Morphogenesis in Zebrafish

Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9 ^{sa1022/sa1022} mutants have a defect in 28S rRNA processing. The nol9 ^{sa1022/sa1022} larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9 ^{sa1022/sa1022} larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9 ^{sa1022/sa1022} embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9 ^{sa1022/sa1022} mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate.     

Oxa1-Ribosome Complexes Coordinate the Assembly of Cytochrome c Oxidase in Mitochondria

The terminal enzyme of the respiratory chain, cytochrome c oxidase, consists of a hydrophobic reaction center formed by three mitochondrially encoded subunits with which 9–10 nuclear encoded subunits are associated. The three core subunits are synthesized on mitochondrial ribosomes and inserted into the inner membrane in a co-translational reaction facilitated by the Oxa1 insertase. Oxa1 consists of an N-terminal insertase domain and a C-terminal ribosome-binding region. Mutants lacking the C-terminal region show specific defects in co-translational insertion, suggesting that the close contact of the ribosome with the insertase promotes co-translational insertion of nascent chains. In this study, we inserted flexible linkers of 100 or 200 amino acid residues between the insertase domain and ribosome-binding region of Oxa1 of Saccharomyces cerevisiae. In the absence of the ribosome receptor Mba1, these linkers caused a length-dependent decrease in mitochondrial respiratory activity caused by diminished levels of cytochrome c oxidase. Interestingly, considerable amounts of mitochondrial translation products were still integrated into the inner membrane in these linker mutants. However, they showed severe defects in later stages of the biogenesis process, presumably during assembly into functional complexes. Our observations suggest that the close proximity of Oxa1 to ribosomes is not only used to improve membrane insertion but is also critical for the productive assembly of the subunits of the cytochrome c oxidase. This points to a role for Oxa1 in the spatial coordination of the ribosome with assembly factors that are critical for enzyme biogenesis.     

Reversion from erythromycin dependence in Escherichia coli: strains altered in ribosomal sub-unit association and ribosome assembly

A mutant of Escherichia coli dependent on erythromycin for growth spontaneously gives erythromycin-independent strains with altered or missing ribosomal proteins. strains with defects in ribosome assembly were sought and obtained from among these revertants. Two organisms in which ribosomal protein L19 is altered and absent respectively have 70S ribosomes whose dissociation into sub-units is particularly sensitive to pressures generated during centrifuging. The mutant that lacks protein L19 also accumulates ribosome precursor particles during exponential growth as do others including mutants that lack proteins S20 or L1. These strains also show unbalanced synthesis of RNA and so will be useful in investigating both the pathways and the regulation of ribosome assembly.     

The Hsp90 Cochaperones Cpr6, Cpr7, and Cns1 Interact with the Intact Ribosome

The abundant molecular chaperone Hsp90 is essential for the folding and stabilization of hundreds of distinct client proteins. Hsp90 is assisted by multiple cochaperones that modulate Hsp90''s ATPase activity and/or promote client interaction, but the in vivo functions of many of these cochaperones are largely unknown. We found that Cpr6, Cpr7, and Cns1 interact with the intact ribosome and that Saccharomyces cerevisiae lacking CPR7 or containing mutations in CNS1 exhibited sensitivity to the translation inhibitor hygromycin. Cpr6 contains a peptidyl-prolyl isomerase (PPIase) domain and a tetratricopeptide repeat (TPR) domain flanked by charged regions. Truncation or alteration of basic residues near the carboxy terminus of Cpr6 disrupted ribosome interaction. Cns1 contains an amino-terminal TPR domain and a poorly characterized carboxy-terminal domain. The isolated carboxy-terminal domain was able to interact with the ribosome. Although loss of CPR6 does not cause noticeable growth defects, overexpression of CPR6 results in enhanced growth defects in cells expressing the temperature-sensitive cns1-G90D mutation (the G-to-D change at position 90 encoded by cns1). Cpr6 mutants that exhibit reduced ribosome interaction failed to cause growth defects, indicating that ribosome interaction is required for in vivo functions of Cpr6. Together, these results represent a novel link between the Hsp90 molecular-chaperone machine and protein synthesis.