Eukaryotic Ribosome Biogenesis Research Articles

Identification of Key mRNAs and lncRNAs in Neonatal Sepsis by Gene Expression Profiling.

Neonatal sepsis is one of the most prevalent causes of death of the neonates. However, the mechanisms underlying neonatal sepsis remained unclear. The present study identified a total of 1128 upregulated mRNAs and 1008 downregulated mRNAs, 28 upregulated lncRNAs, and 61 downregulated lncRNAs in neonatal sepsis. Then, we constructed PPI networks to identify key regulators in neonatal sepsis, including ITGAM, ITGAX, TLR4, ITGB2, SRC, ELANE, RPLP0, RPS28, RPL26, and RPL27. lncRNA coexpression analysis showed HS.294603, LOC391811, C12ORF47, LOC729021, HS.546375, HNRPA1L-2, LOC158345, and HS.495041 played important roles in the progression of neonatal sepsis. Bioinformatics analysis showed DEGs were involved in the regulation cellular extravasation, acute inflammatory response, macrophage activation of NF-kappa B signaling pathway, TNF signaling pathway, HIF-1 signaling pathway, Toll-like receptor signaling pathway, and ribosome, RNA transport, and spliceosome. lncRNAs were involved in regulating ribosome, T cell receptor signaling pathway, RNA degradation, insulin resistance, ribosome biogenesis in eukaryotes, and hematopoietic cell lineage. We thought this study provided useful information for identifying novel therapeutic markers for neonatal sepsis.

Transcriptome analysis of liver provides insight into metabolic and translation changes under hypoxia and reoxygenation stress in silver sillago (Sillago sihama)

Hypoxia can lead to adverse effects on growth, reproduction, behavioral activities and survival in fish, and is one of the most critical factors in the aquatic environment. The liver is an important target organ for reducing toxin accumulation and hypoxia in fish. In this study, silver sillago (Sillago sihama) was exposed to normoxia (dissolved oxygen, DO = 8.0 mg/L), hypoxia for 1 h (hypoxia 1 h, DO = 1.5 mg/L), hypoxia for 4 h (hypoxia 4 h, DO = 1.5 mg/L) and reoxygenation for 4 h after hypoxia 4 h (reoxygenation 4 h, DO = 8.0 mg/L). Results showed that the expression of 506, 1721, and 1230 differentially expressed genes (DEGs) (|log2(fold change) > 1.0| and padj < 0.05) were identified at hypoxia 1 h, hypoxia 4 h, and reoxygenation 4 h in the liver, respectively. The enrichment analysis showed that the DEGs were significantly enriched in metabolic and translation changes pathways, including mapk signaling pathway, p53 signaling pathway, fatty acid metabolism, protein export, ribosome biogenesis in eukaryotes. The DEGs of 17 genes validated the RNA-seq results by quantitative real-time PCR (qRT-PCR). This study provides a comprehensive understanding of the transcriptional changes that occur in different hypoxia and insights into the mechanisms of hypoxia adaptation of the liver in S. sihama.

From Snapshots to Flipbook-Resolving the Dynamics of Ribosome Biogenesis with Chemical Probes.

The synthesis of ribosomes is one of the central and most resource demanding processes in each living cell. As ribosome biogenesis is tightly linked with the regulation of the cell cycle, perturbation of ribosome formation can trigger severe diseases, including cancer. Eukaryotic ribosome biogenesis starts in the nucleolus with pre-rRNA transcription and the initial assembly steps, continues in the nucleoplasm and is finished in the cytoplasm. From start to end, this process is highly dynamic and finished within few minutes. Despite the tremendous progress made during the last decade, the coordination of the individual maturation steps is hard to unravel by a conventional methodology. In recent years small molecular compounds were identified that specifically block either rDNA transcription or distinct steps within the maturation pathway. As these inhibitors diffuse into the cell rapidly and block their target proteins within seconds, they represent excellent tools to investigate ribosome biogenesis. Here we review how the inhibitors affect ribosome biogenesis and discuss how these effects can be interpreted by taking the complex self-regulatory mechanisms of the pathway into account. With this we want to highlight the potential of low molecular weight inhibitors to approach the dynamic nature of the ribosome biogenesis pathway.

Good Vibrations: Structural Remodeling of Maturing Yeast Pre-40S Ribosomal Particles Followed by Cryo-Electron Microscopy.

Assembly of eukaryotic ribosomal subunits is a very complex and sequential process that starts in the nucleolus and finishes in the cytoplasm with the formation of functional ribosomes. Over the past few years, characterization of the many molecular events underlying eukaryotic ribosome biogenesis has been drastically improved by the “resolution revolution” of cryo-electron microscopy (cryo-EM). However, if very early maturation events have been well characterized for both yeast ribosomal subunits, little is known regarding the final maturation steps occurring to the small (40S) ribosomal subunit. To try to bridge this gap, we have used proteomics together with cryo-EM and single particle analysis to characterize yeast pre-40S particles containing the ribosome biogenesis factor Tsr1. Our analyses lead us to refine the timing of the early pre-40S particle maturation steps. Furthermore, we suggest that after an early and structurally stable stage, the beak and platform domains of pre-40S particles enter a “vibrating” or “wriggling” stage, that might be involved in the final maturation of 18S rRNA as well as the fitting of late ribosomal proteins into their mature position.

De novo transcriptome assembly and sex-biased gene expression in the gonads of Amur catfish (Silurus asotus)

Amur catfish is extensively distributed and cultured in Asian countries. Despite of economic importance, the genomic information of this species remains limited. A reference transcriptome of Amur catfish was assembled and the sex-biased gene expression in the gonads was characterized using RNA-sequencing. The assembled transcriptome of Amur catfish consisted of 74,840 transcripts. The N50, mean length and max length of transcripts are 1970, 1235 and 16,748 bp. Putative sex-specific transcripts were identified and sex-specific expression of the representative genes was verified by RT-PCR. Differential expression analysis identified 5401 ovary-biased and 5618 testis-biased genes. The ovary-biased genes were mainly enriched in pathways such as RNA transport and ribosome biogenesis in eukaryotes. The testis-biased genes were enriched in calcium signaling and cytokine-cytokine receptor interaction, etc. Our data provide a valuable genomic resource for further investigating the genetic basis of sex determination, sex differentiation and sexual dimorphism of catfish.

Auxiliary domains of the HrpB bacterial DExH-box helicase shape its RNA preferences

ABSTRACTRNA helicases are fundamental players in RNA metabolism: they remodel RNA secondary structures and arrange ribonucleoprotein complexes. While DExH-box RNA helicases function in ribosome biogenesis and splicing in eukaryotes, information is scarce about bacterial homologs. HrpB is the only bacterial DExH-box protein whose structure is solved. Besides the catalytic core, HrpB possesses three accessory domains, conserved in all DExH-box helicases, plus a unique C-terminal extension (CTE). The function of these auxiliary domains remains unknown. Here, we characterize genetically and biochemically Pseudomonas aeruginosa HrpB homolog. We reveal that the auxiliary domains shape HrpB RNA preferences, affecting RNA species recognition and catalytic activity. We show that, among several types of RNAs, the single-stranded poly(A) and the highly structured MS2 RNA strongly stimulate HrpB ATPase activity. In addition, deleting the CTE affects only stimulation by structured RNAs like MS2 and rRNAs, while deletion of accessory domains results in gain of poly(U)-dependent activity. Finally, using hydrogen-deuterium exchange, we dissect the molecular details of HrpB interaction with poly(A) and MS2 RNAs. The catalytic core interacts with both RNAs, triggering a conformational change that reorients HrpB. Regions within the accessory domains and CTE are, instead, specifically responsive to MS2. Altogether, we demonstrate that in bacteria, like in eukaryotes, DExH-box helicase auxiliary domains are indispensable for RNA handling.

Physiological and transcriptomicresponses of Lanzhou Lily (Lilium davidii, var. unicolor) to cold stress.

Low temperature induces changes in plants at physiological and molecular levels, thus affecting growth and development. The Lanzhou lily (Lilium davidii, var. unicolor) is an important medicinal plant with high economic value. However, the molecular mechanisms underlying its photosynthetic and antioxidation responses to low temperature still remain poorly understood. This study subjected the Lanzhou lily to the two temperatures of 20°C (control) and 4°C (low temperature) for 24 h. Physiological parameters related to membrane integrity, photosynthesis, antioxidant system, and differentially expressed genes were investigated. Compared with control, low temperature increased the relative electrical conductivity by 43.2%, while it decreased net photosynthesis rate, ratio of variable to maximal fluorescence, and catalase activity by 47.3%, 10.1%, and 11.1%, respectively. In addition, low temperature significantly increased the content of soluble protein, soluble sugar, and proline, as well as the activity of superoxide dismutase and peroxidase. Comparative transcriptome profiling showed that a total of 238,109 differentially expressed genes were detected. Among these, 3,566 were significantly upregulated while 2,982 were significantly downregulated in response to low temperature. Gene Ontology enrichment analysis indicated that in response to low temperature, the mostly significantly enriched differentially expressed genes were mainly involved in phosphorylation, membrane and protein kinase activity, as well as photosynthesis, light harvesting, light reaction, and alpha,alpha-trehalose-phosphate synthase activity. Kyoto Encyclopedia of Genes and Genomes enrichment analysis also indicated that the most significantly enriched pathways involved ribosome biogenesis in eukaryotes, phenylalanine metabolism, circadian rhythm, porphyrin and chlorophyll metabolism, photosynthesis of antenna proteins, photosynthesis, and carbon fixation in photosynthetic organisms. Moreover, the expression patterns of 10 randomly selected differentially expressed genes confirmed the RNA-Seq results. These results expand the understanding of the physiological and molecular mechanisms underlying the response of the Lanzhou lily to low temperature stress.

Transcriptome Analysis Reveals the Effects of Chinese Chive (Allium tuberosum R.) Extract on Fusarium oxysporum f. sp. radicis-lycopersici Spore Germination.

Fusarium oxysporum f. sp. radicis-lycopersici (Forl) causes Fusarium crown and root rot of tomato, leading to severe yield losses. Chinese chive and the Chinese chive extract reportedly have antifungal effects. In this study, Chinese chive extract treatments inhibited Forl spore germination, with an EC50 of 0.40gml-1 in vitro. Furthermore, the mechanism underlying the fungicidal effects of the Chinese chive extract was analyzed by RNA sequencing. A total of 1252 differentially expressed genes (DEGs) were detected, of which 396 were upregulated and 856 were downregulated. The DEGs were related to starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, galactose metabolism, fatty acid metabolism, sphingolipid metabolism, glycerophospholipid metabolism, peroxisomes, ribosome biogenesis in eukaryotes, mismatch repair, and the phosphatidylinositol signaling system, implying these pathways contribute to the fungicidal activity of the Chinese chive extract. The qRT-PCR results verified the accuracy of the RNA sequencing data. Thus, the Chinese chive extract can inhibit Forl spore germination by affecting spore nutrient metabolism.

Independent prognostic implications of RRM2 in lung adenocarcinoma.

Background: Ribonucleoside-diphosphate reductase subunit M2 (RRM2) is the catalytic subunit of ribonucleotide reductase and modulates the enzymatic activity, which is essential for DNA replication and repair. However, the role of RRM2 in lung adenocarcinoma (LUAD) remains unclear.Methods: In this study, we explored the expression pattern and prognostic value of RRM2 in LUAD across TCGA, GEO, Oncomine, UALCAN, PrognoScan, and Kaplan-Meier Plotter, and confirmed its independent prognostic value via Cox analyses. LinkedOmics and GEPIA2 were applied to investigate co-expression and functional networks associated with RRM2. Besides, we used TIMER to assess the correlation between RRM2 and the main six types of tumor-infiltrating immune cells. Lastly, the correlations between immune signatures of immunomodulators, chemokines, and 28 tumor-infiltrating lymphocytes (TILs) and RRM2 were examined by tumor purity-corrected partial Spearman's rank correlation coefficient through TIMER portal.Results: RRM2 was found upregulated in tumor tissues in TCGA-LUAD, and validated in multiple independent cohorts. Moreover, whether in TCGA or other cohorts, high RRM2 expression was found to be associated with poor survival. Cox analyses showed that high RRM2 expression was an independent risk factor for overall survival, disease-specific survival, and progression-free survival of LUAD. Functional network analysis suggested that RRM2 regulates RNA transport, oocyte meiosis, spliceosome, ribosome biogenesis in eukaryotes, and cellular senescence signaling through pathways involving multiple cancer-related kinases and E2F family. Also, RRM2 expression correlated with infiltrating levels of B cells, CD4+ T cells, and neutrophils. Subsequent analysis found that B cells and dendritic cells could predict the outcome of LUAD. B cells were identified as an independent risk factor among six types of immune cells through Cox analyses. At last, the correlation analysis showed RRM2 correlated with 67.68% (624/922) of the immune signatures we performed.Conclusion: Our research showed that RRM2 could independently predict the prognosis of LUAD and was associated with immune infiltration. In particular, the tight relationship between RRM2 and B cell marker genes are the potential epicenter of the immune response and one of the critical factors affecting the prognosis. Our findings laid the foundation for further research on the immunomodulatory role of RRM2 in LUAD.

Shaping the Nascent Ribosome: AAA-ATPases in Eukaryotic Ribosome Biogenesis.

AAA-ATPases are molecular engines evolutionarily optimized for the remodeling of proteins and macromolecular assemblies. Three AAA-ATPases are currently known to be involved in the remodeling of the eukaryotic ribosome, a megadalton range ribonucleoprotein complex responsible for the translation of mRNAs into proteins. The correct assembly of the ribosome is performed by a plethora of additional and transiently acting pre-ribosome maturation factors that act in a timely and spatially orchestrated manner. Minimal disorder of the assembly cascade prohibits the formation of functional ribosomes and results in defects in proliferation and growth. Rix7, Rea1, and Drg1, which are well conserved across eukaryotes, are involved in different maturation steps of pre-60S ribosomal particles. These AAA-ATPases provide energy for the efficient removal of specific assembly factors from pre-60S particles after they have fulfilled their function in the maturation cascade. Recent structural and functional insights have provided the first glimpse into the molecular mechanism of target recognition and remodeling by Rix7, Rea1, and Drg1. Here we summarize current knowledge on the AAA-ATPases involved in eukaryotic ribosome biogenesis. We highlight the latest insights into their mechanism of mechano-chemical complex remodeling driven by advanced cryo-EM structures and the use of highly specific AAA inhibitors.

Inhibiting eukaryotic ribosome biogenesis: Mining new tools for basic research and medical applications.

The formation of new ribosomes is a fundamental cellular process for each living cell and is tightly interwoven with cell cycle control and proliferation. Minimal disturbances of this pathway can result in ribosomopathies including an increased risk for certain cancer types. Thus, targeting ribosome biogenesis is an emerging strategy in cancer therapy. However, due to its complex nature, we are only at the beginning to understand the dynamics of the ribosome biogenesis pathway. One arising approach that will help us to embrace the tight timely cascade of events that is needed to form a new ribosome is the use of targeted chemical inhibition. However, only very few specific chemical inhibitors of the ribosome biogenesis pathway have been identified so far. Here we review our recently published screen to identify novel inhibitors of the ribosome biogenesis pathway in yeast (Awad et al., 2019, BMC Biology). These inhibitors can provide novel tools for basic research and can serve as starting-points to develop new chemotherapeutics.

Tsr4 Is a Cytoplasmic Chaperone for the Ribosomal Protein Rps2 in Saccharomyces cerevisiae.

Eukaryotic ribosome biogenesis requires the action of approximately 200 trans-acting factors and the incorporation of 79 ribosomal proteins (RPs). The delivery of RPs to preribosomes is a major challenge for the cell because RPs are often highly basic and contain intrinsically disordered regions prone to nonspecific interactions and aggregation. To counteract this, eukaryotes developed dedicated chaperones for certain RPs that promote their solubility and expression, often by binding eukaryote-specific extensions of the RPs. Rps2 (uS5) is a universally conserved RP that assembles into nuclear pre-40S subunits. However, a chaperone for Rps2 had not been identified. Our laboratory previously characterized Tsr4 as a 40S biogenesis factor of unknown function. Here, we report that Tsr4 cotranslationally associates with Rps2. Rps2 harbors a eukaryote-specific N-terminal extension that is critical for its interaction with Tsr4. Moreover, Tsr4 perturbation resulted in decreased Rps2 levels and phenocopied Rps2 depletion. Despite Rps2 joining nuclear pre-40S particles, Tsr4 appears to be restricted to the cytoplasm. Thus, we conclude that Tsr4 is a cytoplasmic chaperone dedicated to Rps2.

Transcriptome Analysis of Low-Temperature-Induced Breaking of Garlic Aerial Bulb Dormancy.

The long history of asexual reproduction of garlic using garlic cloves has resulted in virus accumulation and genetic depression. Propagation of garlic seedlings by tissue culture can both eliminate viruses and improve breeding efficiency. Aerial bulbs are the first-choice materials for breeding virus-free garlic seedlings under external conditions, but they show dormancy just like garlic bulbs. However, low temperatures can quickly break dormancy. In this research, we used a high-throughput sequencing method to sequence aerial bulbs during dormancy and after low-temperature-induced breaking of dormancy to screen out the key differentially expressed genes (DEGs) associated with low temperature and to provide a theoretical basis for exploring the molecular mechanism of low-temperature-induced breaking of aerial bulb dormancy. The high-throughput transcriptome sequencing results showed that 6,675 DEGs were upregulated and 36,596 DEGs were downregulated in the aerial bulbs given low-temperature treatment. Then, 19,507 DEGs were assigned KEGG annotations, among which most DEGs were annotated to the metabolism pathway (11,817 genes, accounting for 60.58%), followed by the genetic information processing pathway (4,521 genes, accounting for 23.18%). The DEGs were mostly concentrated in pathways such as protein processing in the endoplasmic reticulum, plant-pathogen interaction, plant hormone signal transduction, and ribosome biogenesis in eukaryotes, with significant enrichment. The key DEGs related to calcium signaling, hormonal signaling, and transcription factors were screened out, including CaM, CDPK, and CML in accessory pathways of calcium signaling; GA20ox, GAI1, and GA2ox in accessory pathways of hormonal signaling; and transcription factor genes such as MYB, AP2/ERF, bHLH, MADS, and bZIP. qRT-PCR verification results were consistent with the sequencing results, indicating that the transcriptome sequencing data were accurate and reliable. Our results provide a theoretical basis for breaking the dormancy of aerial bulbs with low-temperature treatment to produce virus-free seedlings and increase the output and quality of garlic.

Trypanosoma brucei Homologue of Regulator of Ribosome Synthesis 1 (Rrs1) Has Direct Interactions with Essential Trypanosome-Specific Proteins.

Studies in eukaryotic ribosome biogenesis have largely been performed in yeast, where they have described a highly complex process involving numerous protein and RNA components. Due to the complexity and crucial nature of this process, a number of checkpoints are necessary to ensure that only properly assembled ribosomes are released into the cytoplasm. Assembly of the 5S ribonucleoprotein (RNP) complex is one of these checkpoints for late-stage 60S subunit maturation. Studies in Saccharomyces cerevisiae have identified the 5S rRNA and four proteins, L5, L11, Rpf2, and Rrs1, as comprising the ribosome-associated 5S RNP. Work from our laboratory has shown that in the eukaryotic pathogen Trypanosoma brucei, the 5S RNP includes trypanosome-specific proteins P34/P37, as well as homologues of L5, Rpf2, and 5S rRNA. In this study, we examine a homologue of Rrs1 and identify it as an additional member of the T. brucei 5S RNP. Using RNA interference, we show that TbRrs1 is essential for the survival of T. brucei and has an important role in ribosome subunit formation and, together with TbRpf2, plays a role in 25/28S and 5.8S rRNA processing. We further show that TbRrs1 is a member of the T. brucei 5S RNP through the identification of novel direct interactions with P34/P37 and 5S rRNA as well as with TbL5 and TbRpf2. These unique characteristics of TbRrs1 highlight the importance of studying ribosome biogenesis in the context of diverse organisms and identify interactions that could be targeted for future drug development.IMPORTANCETrypanosoma brucei is a parasite responsible for human and animal African trypanosomiasis. Current treatments for these diseases have numerous problems, and the development of novel chemotherapeutics can be achieved by identifying targets that are parasite specific and part of essential processes. Ribosome biogenesis is the process of generating translation-competent ribosomes and is critical for survival in all organisms. Work from our laboratory has shown that the formation of the 5S RNP, a crucial checkpoint in ribosome biogenesis, requires trypanosome-specific proteins P34/P37 and homologues of Rpf2 and L5 which possess parasite-specific characteristics. In this study, we characterize TbRrs1, an additional member of the T. brucei 5S RNP, and show that it is essential for parasite survival and has unique interactions with P34/P37 and 5S rRNA. This expands our understanding of the 5S RNP in T. brucei and identifies new targets for future drug development.

Thermophile 90S Pre-ribosome Structures Reveal the Reverse Order of Co-transcriptional 18S rRNA Subdomain Integration.

Eukaryotic ribosome biogenesis involves RNA folding and processing that depend on assembly factors and small nucleolar RNAs (snoRNAs). The 90S (SSU-processome) is the earliest pre-ribosome structurally analyzed, which was suggested to assemble stepwise along the growing pre-rRNA from 5' > 3', but this directionality may not be accurate. Here, by analyzing the structure of a series of 90S assembly intermediates from Chaetomium thermophilum, we discover a reverse order of 18S rRNA subdomain incorporation. Large parts of the 18S rRNA 3' and central domains assemble first into the 90S before the 5' domain is integrated. This final incorporation depends on a contact between a heterotrimer Enp2-Bfr2-Lcp5 recruited to the flexible 5' domain and Kre33, which reconstitutes the Kre33-Enp-Brf2-Lcp5 module on the compacted 90S. Keeping the 5' domain temporarily segregated from the 90S scaffold could provide extra time to complete the multifaceted 5' domain folding, which depends on a distinct set of snoRNAs and processing factors.

Conformational switches control early maturation of the eukaryotic small ribosomal subunit.

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5' external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5' external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5' external transcribed spacer ribonucleoprotein - provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

Stress response regulator FoSkn7 participates in the pathogenicity of Fusariumoxysporum f. sp. cubense race 4 by conferring resistance to exogenous oxidative stress

An ortholog of the response regulator protein Skn7 in Fusariumoxysporum f. sp. cubense race 4 (Foc4) was identified here and named FoSkn7. Mutants lacking FoSkn7 displayed higher sensitivity than the wild-type strain to oxidative stress. In addition, the FoSkn7-deletion mutant caused an oxidative burst in banana seedlings at early stages of infection and had reduced virulence on Giant Cavendish bananas (Musa spp.). These results showed that FoSkn7 confers resistance to exogenous oxidative stress. To understand the underlying mechanism of this resistance to exogenous oxidative stress, we used RNA-seq to identify genes that are differentially regulated between the FoSkn7-deletion mutant and the wild-type strain. Among 104 upregulated and 566 downregulated genes with a two-fold change cutoff, 26 downregulated genes were related to stimulus response and antioxidant activity. KEGG enrichment analysis showed that 150 differentially expressed genes were enriched in 67 metabolic pathways, mainly including ribosomes, ribosome biogenesis in eukaryotes, RNA polymerase, peroxisome, glutamate metabolism, fatty acid degradation, and protein processing in the endoplasmic reticulum. These results suggest that FoSkn7, as an important stress response regulator of Foc4, confers resistance to exogenous oxidative stress by regulating the expression of several stress-response- and antioxidant-related genes and metabolic pathways to promote the virulence of Foc4.

Molecular mechanism of the RNA helicase DHX37 and its activation by UTP14A in ribosome biogenesis.

Eukaryotic ribosome biogenesis is a highly orchestrated process involving numerous assembly factors including ATP-dependent RNA helicases. The DEAH helicase DHX37 (Dhr1 in yeast) is activated by the ribosome biogenesis factor UTP14A to facilitate maturation of the small ribosomal subunit. We report the crystal structure of DHX37 in complex with single-stranded RNA, revealing a canonical DEAH ATPase/helicase architecture complemented by a structurally unique carboxy-terminal domain (CTD). Structural comparisons of the nucleotide-free DHX37-RNA complex with DEAH helicases bound to RNA and ATP analogs reveal conformational changes resulting in a register shift in the bound RNA, suggesting a mechanism for ATP-dependent 3′–5′ RNA translocation. We further show that a conserved sequence motif in UTP14A interacts with and activates DHX37 by stimulating its ATPase activity and enhancing RNA binding. In turn, the CTD of DHX37 is required, but not sufficient, for interaction with UTP14A in vitro and is essential for ribosome biogenesis in vivo. Together, these results shed light on the mechanism of DHX37 and the function of UTP14A in controlling its recruitment and activity during ribosome biogenesis.

Exposure to triclosan changes the expression of microRNA in male juvenile zebrafish (Danio rerio)

Triclosan (TCS) is a broad-spectrum antibacterial agent which is widely used in various personal care products and cosmetics. It has been found that TCS affects endocrine, immune, nervous, reproductive, and developmental system. Although microRNAs (miRNAs) act a pivotal part in lots of metabolic activities, whether and how they are related to the process of TCS-induced toxicity is unknown. In the present study, TCS induced changes in miRNAs and target gene expression in male zebrafish (Danio rerio) brain, and the potential mechanism was studied. Male juvenile zebrafish were exposed to 0 and 68 μg/L TCS for 42 d. miRNA was isolated from the brain pool of the zebrafish and the expression profiles of 255 known zebrafish miRNAs were analysed by using Affymetrix miRNA 4.0 microarrays. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assay the expression of 5 differentially expressed miRNAs in the microarray data and some related-genes in brains. The GO term analysis revealed that miRNAs significantly affected by TCS exposure were mainly involved in translation, transcription, DNA-templated, protein transport, and motor neuron axon guidance biological process. Pathway analysis showed that target genes of 5 differentially expressed miRNAs prominently participate in basal transcription factors, purine metabolism, and ribosome biogenesis in eukaryotes. In addition, key genes in purine metabolism pathway and oxidative stress related-genes were significantly changed. These findings offer novel insight into the mechanisms of epigenetic regulation in TCS-induced toxicity in male zebrafish, and distinguish novel miRNA biomarkers for exposure to TCS.

The Npa1p complex chaperones the assembly of the earliest eukaryotic large ribosomal subunit precursor.

The early steps of the production of the large ribosomal subunit are probably the least understood stages of eukaryotic ribosome biogenesis. The first specific precursor to the yeast large ribosomal subunit, the first pre-60S particle, contains 30 assembly factors (AFs), including 8 RNA helicases. These helicases, presumed to drive conformational rearrangements, usually lack substrate specificity in vitro. The mechanisms by which they are targeted to their correct substrate within pre-ribosomal particles and their precise molecular roles remain largely unknown. We demonstrate that the Dbp6p helicase, essential for the normal accumulation of the first pre-60S pre-ribosomal particle in S. cerevisiae, associates with a complex of four AFs, namely Npa1p, Npa2p, Nop8p and Rsa3p, prior to their incorporation into the 90S pre-ribosomal particles. By tandem affinity purifications using yeast extracts depleted of one component of the complex, we show that Npa1p forms the backbone of the complex. We provide evidence that Npa1p and Npa2p directly bind Dbp6p and we demonstrate that Npa1p is essential for the insertion of the Dbp6p helicase within 90S pre-ribosomal particles. In addition, by an in vivo cross-linking analysis (CRAC), we map Npa1p rRNA binding sites on 25S rRNA adjacent to the root helices of the first and last secondary structure domains of 25S rRNA. This finding supports the notion that Npa1p and Dbp6p function in the formation and/or clustering of root helices of large subunit rRNAs which creates the core of the large ribosomal subunit RNA structure. Npa1p also crosslinks to snoRNAs involved in decoding center and peptidyl transferase center modifications and in the immediate vicinity of the binding sites of these snoRNAs on 25S rRNA. Our data suggest that the Dbp6p helicase and the Npa1p complex play key roles in the compaction of the central core of 25S rRNA and the control of snoRNA-pre-rRNA interactions.