Kinetoplastid Specific RNA-Protein Interactions in Trypanosoma cruzi Ribosome Biogenesis

RNA binding proteins (RBP) play essential roles in the highly conserved and coordinated process of ribosome biogenesis. Our laboratory has previously characterized two essential and abundant RBPs, P34 and P37, in Trypanosoma brucei which are required for several critical steps in ribosome biogenesis. The genes for these proteins have only been identified in kinetoplastid organisms but not in the host genome. We have identified a homolog of the TbP34 and TbP37 in a T. cruzi strain (termed TcP37/NRBD). Although the N-terminal APK-rich domain and RNA recognition motifs are conserved, the C-terminal region which contains putative nuclear and nucleolar localization signals in TbP34 and TbP37 is almost entirely missing from TcP37/NRBD. We have shown that TcP37/NRBD is expressed in T. cruzi epimastigotes at the level of mature mRNA and protein. Despite the loss of the C-terminal domain, TcP37/NRBD is present in the nucleus, including the nucleolus, and the cytoplasm. TcP37/NRBD interacts directly with Tc 5S rRNA, but does not associate with polyadenylated RNA. TcP37/NRBD also associates in vivo and in vitro with large ribosomal protein TcL5 and, unlike the case of T. brucei, this association is strongly enhanced by the presence of 5S rRNA, suggesting that the loss of the C-terminal domain of TcP37/NRBD may alter the interactions within the complex. These results indicate that the unique preribosomal complex comprised of L5, 5S rRNA, and the trypanosome-specific TcP37/NRBD or TbP34 and TbP37 is functionally conserved in trypanosomes despite the differences in the C-termini of the trypanosome-specific protein components.     

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.     

Genetic Analysis of the Ribosome Biogenesis Factor Ltv1 of Saccharomyces cerevisiae

Ribosome biogenesis has been studied extensively in the yeast Saccharomyces cerevisiae. Yeast Ltv1 is a conserved 40S-associated biogenesis factor that has been proposed to function in small subunit nuclear export. Here we show that Ltv1 has a canonical leucine-rich nuclear export signal (NES) at its extreme C terminus that is both necessary for Crm1 interaction and Ltv1 export. The C terminus of Ltv1 can substitute for the NES in the 60S-export adapter Nmd3, demonstrating that it is a functional NES. Overexpression of an Ltv1 lacking its NES (Ltv1∆C13) was strongly dominant negative and resulted in the nuclear accumulation of RpS3-GFP; however, export of the pre-40S was not affected. In addition, expression of endogenous levels of Ltv1∆C protein complemented both the slow-growth phenotype and the 40S biogenesis defect of an ltv1 deletion mutant. Thus, if Ltv1 is a nuclear export adapter for the pre-40S subunit, its function must be fully redundant with additional export factors. The dominant negative phenotype of Ltv1∆NES overexpression was suppressed by co-overexpressing RpS3 and its chaperone, Yar1, or by deletion of the RpS3-binding site in Ltv1∆NES, suggesting that titration of RpS3 by Ltv1∆NES is deleterious in yeast. The dominant-negative phenotype did not correlate with a decrease in 40S levels but rather with a reduction in the polysome-to-monosome ratio, indicating reduced rates of translation. We suggest that titration of RpS3 by excess nuclear Ltv1 interferes with 40S function or with a nonribosomal function of RpS3.     

40S Ribosome Biogenesis Co-Factors Are Essential for Gametophyte and Embryo Development

Ribosome biogenesis is well described in Saccharomyces cerevisiae. In contrast only very little information is available on this pathway in plants. This study presents the characterization of five putative protein co-factors of ribosome biogenesis in Arabidopsis thaliana, namely Rrp5, Pwp2, Nob1, Enp1 and Noc4. The characterization of the proteins in respect to localization, enzymatic activity and association with pre-ribosomal complexes is shown. Additionally, analyses of T-DNA insertion mutants aimed to reveal an involvement of the plant co-factors in ribosome biogenesis. The investigated proteins localize mainly to the nucleolus or the nucleus, and atEnp1 and atNob1 co-migrate with 40S pre-ribosomal complexes. The analysis of T-DNA insertion lines revealed that all proteins are essential in Arabidopsis thaliana and mutant plants show alterations of rRNA intermediate abundance already in the heterozygous state. The most significant alteration was observed in the NOB1 T-DNA insertion line where the P-A3 fragment, a 23S-like rRNA precursor, accumulated. The transmission of the T-DNA through the male and female gametophyte was strongly inhibited indicating a high importance of ribosome co-factor genes in the haploid stages of plant development. Additionally impaired embryogenesis was observed in some mutant plant lines. All results support an involvement of the analyzed proteins in ribosome biogenesis but differences in rRNA processing, gametophyte and embryo development suggested an alternative regulation in plants.     

血管内皮生长因子及其受体在斑马鱼胚胎血管发育中的作用

血管内皮生长因子(vascular endothelial growth factor,VEGF)是一种多功能的细胞因子,其主要作用是促进血管内皮细胞增殖和增加血管通透性,是肿瘤及正常组织血管生成的中心调控因素。以VEGF为靶点的肿瘤血管靶向性治疗成为近几年肿瘤治疗的新途径。斑马鱼作为一种重要的模式生物,被广泛用于胚胎的分子发育机制、疾病模型的构建以及药物筛选等研究中。文章对斑马鱼作为心血管系统研究模型的优势及其血管研究方法做一阐述,重点对斑马鱼VEGF及其受体的最新研究进展做了介绍,并展望了其发展前景。     

RNA Mimicry by the Fap7 Adenylate Kinase in Ribosome Biogenesis

During biogenesis of the 40S and 60S ribosomal subunits, the pre-40S particles are exported to the cytoplasm prior to final cleavage of the 20S pre-rRNA to mature 18S rRNA. Amongst the factors involved in this maturation step, Fap7 is unusual, as it both interacts with ribosomal protein Rps14 and harbors adenylate kinase activity, a function not usually associated with ribonucleoprotein assembly. Human hFap7 also regulates Cajal body assembly and cell cycle progression via the p53–MDM2 pathway. This work presents the functional and structural characterization of the Fap7–Rps14 complex. We report that Fap7 association blocks the RNA binding surface of Rps14 and, conversely, Rps14 binding inhibits adenylate kinase activity of Fap7. In addition, the affinity of Fap7 for Rps14 is higher with bound ADP, whereas ATP hydrolysis dissociates the complex. These results suggest that Fap7 chaperones Rps14 assembly into pre-40S particles via RNA mimicry in an ATP-dependent manner. Incorporation of Rps14 by Fap7 leads to a structural rearrangement of the platform domain necessary for the pre-rRNA to acquire a cleavage competent conformation.     

Rational Extension of the Ribosome Biogenesis Pathway Using Network-Guided Genetics

Biogenesis of ribosomes is an essential cellular process conserved across all eukaryotes and is known to require >170 genes for the assembly, modification, and trafficking of ribosome components through multiple cellular compartments. Despite intensive study, this pathway likely involves many additional genes. Here, we employ network-guided genetics—an approach for associating candidate genes with biological processes that capitalizes on recent advances in functional genomic and proteomic studies—to computationally identify additional ribosomal biogenesis genes. We experimentally evaluated >100 candidate yeast genes in a battery of assays, confirming involvement of at least 15 new genes, including previously uncharacterized genes (YDL063C, YIL091C, YOR287C, YOR006C/TSR3, YOL022C/TSR4). We associate the new genes with specific aspects of ribosomal subunit maturation, ribosomal particle association, and ribosomal subunit nuclear export, and we identify genes specifically required for the processing of 5S, 7S, 20S, 27S, and 35S rRNAs. These results reveal new connections between ribosome biogenesis and mRNA splicing and add >10% new genes—most with human orthologs—to the biogenesis pathway, significantly extending our understanding of a universally conserved eukaryotic process.     

Ribosome Biogenesis in African Trypanosomes Requires Conserved and Trypanosome-Specific Factors

Large ribosomal subunit protein L5 is responsible for the stability and trafficking of 5S rRNA to the site of eukaryotic ribosomal assembly. In Trypanosoma brucei, in addition to L5, trypanosome-specific proteins P34 and P37 also participate in this process. These two essential proteins form a novel preribosomal particle through interactions with both the ribosomal protein L5 and 5S rRNA. We have generated a procyclic L5 RNA interference cell line and found that L5 itself is a protein essential for trypanosome growth, despite the presence of other 5S rRNA binding proteins. Loss of L5 decreases the levels of all large-subunit rRNAs, 25/28S, 5.8S, and 5S rRNAs, but does not alter small-subunit 18S rRNA. Depletion of L5 specifically reduced the levels of the other large ribosomal proteins, L3 and L11, whereas the steady-state levels of the mRNA for these proteins were increased. L5-knockdown cells showed an increase in the 40S ribosomal subunit and a loss of the 60S ribosomal subunits, 80S monosomes, and polysomes. In addition, L5 was involved in the processing and maturation of precursor rRNAs. Analysis of polysomal fractions revealed that unprocessed rRNA intermediates accumulate in the ribosome when L5 is depleted. Although we previously found that the loss of P34 and P37 does not result in a change in the levels of L5, the loss of L5 resulted in an increase of P34 and P37 proteins, suggesting the presence of a compensatory feedback loop. This study demonstrates that ribosomal protein L5 has conserved functions, in addition to nonconserved trypanosome-specific features, which could be targeted for drug intervention.     

Correlation between Ribosome Biogenesis and the Magnitude of Hypertrophy in Overloaded Skeletal Muscle

External loads applied to skeletal muscle cause increases in the protein translation rate, which leads to muscle hypertrophy. Although some studies have demonstrated that increases in the capacity and efficiency of translation are involved in this process, it remains unclear how these two factors are related to the magnitude of muscle hypertrophy. The present study aimed to clarify the roles played by the capacity and efficiency of translation in muscle hypertrophy. We used an improved synergist ablation in which the magnitude of compensatory hypertrophy could be controlled by partial removal of synergist muscles. Male rats were assigned to four groups in which the plantaris muscle was unilaterally subjected to weak (WK), moderate (MO), middle (MI), and strong (ST) overloading by four types of synergist ablation. Fourteen days after surgery, the weight of the plantaris muscle per body weight increased by 8%, 22%, 32% and 45%, in the WK, MO, MI and ST groups, respectively. Five days after surgery, 18+28S rRNA content (an indicator of translational capacity) increased with increasing overload, with increases of 1.8-fold (MO), 2.2-fold (MI), and 2.5-fold (ST), respectively, relative to non-overloaded muscle (NL) in the WK group. rRNA content showed a strong correlation with relative muscle weight measured 14 days after surgery (r = 0.98). The phosphorylated form of p70S6K (a positive regulator of translational efficiency) showed a marked increase in the MO group, but no further increase was observed with further increase in overload (increases of 22.6-fold (MO), 17.4-fold (MI), and 18.2-fold (ST), respectively, relative to NL in the WK group). These results indicate that increases in ribosome biogenesis at the early phase of overloading are strongly dependent on the amount of overloading, and may play an important role in increasing the translational capacity for further gain of muscular size.     

The Drug Diazaborine Blocks Ribosome Biogenesis by Inhibiting the AAA-ATPase Drg1

The drug diazaborine is the only known inhibitor of ribosome biogenesis and specifically blocks large subunit formation in eukaryotic cells. However, the target of this drug and the mechanism of inhibition were unknown. Here we identify the AAA-ATPase Drg1 as a target of diazaborine. Inhibitor binding into the second AAA domain of Drg1 requires ATP loading and results in inhibition of ATP hydrolysis in this site. As a consequence the physiological activity of Drg1, i.e. the release of Rlp24 from pre-60S particles, is blocked, and further progression of cytoplasmic preribosome maturation is prevented. Our results identify the first target of an inhibitor of ribosome biogenesis and provide the mechanism of inhibition of a key step in large ribosomal subunit formation.