mRNA Translocation Research Articles

N6-Methyladenosine Modification and Its Regulation of Respiratory Viruses.

N6-methyladenosine (m6A) is a ubiquitous RNA modification in eukaryotes. It plays important roles in the translocation, stabilization and translation of mRNA. Many recent studies have shown that the dysregulation of m6A modification is connected with diseases caused by pathogenic viruses, and studies on the role of m6A in virus-host interactions have shown that m6A plays a wide range of regulatory roles in the life cycle of viruses. Respiratory viruses are common pathogens that can impose a large disease burden on young children and elderly people. Here, we review the effects of m6A modification on respiratory virus replication and life cycle and host immunity against viruses.

MR1 expression and surface translocation in airway epithelial cells from COPD or smoker lungs is altered following exposure to IFNγ or MAIT cells.

Abstract MAIT cells, a highly prevalent subset of T cells restricted by the MHC Class I-like molecule MR1, play a key role in early response to bacterial infection. Chronic obstructive pulmonary disease (COPD) pathogenesis is associated with airway inflammation, increased infiltration by CD8+ T lymphocytes, and infection-driven exacerbations. Cigarette smoke (CS), a leading cause of COPD, leads to impaired immune function of airway epithelial cells (AECs) and decreases surface expression of classical MHC Class I molecules. However, the impact of CS and COPD on MR1 expression and airway MAIT cell function is unknown. We have demonstrated that exposure to CS alters MAIT cell responses to primary AECs from healthy, COPD, or smoker lungs. Here, we examined whether CS impacts expression of MR1 mRNA or surface translocation of MR1 in these primary AEC populations as a possible mechanism for this dysregulation. Short-term treatment of AECs with CS or infection with S. pneumoniae (SP) did not alter MR1 expression in any of the donor AEC groups. However, incubating CS-treated or SP-infected AECs with MAIT cells increased MR1 expression. This upregulation may be mediated through IFNγ signaling pathways, as treatment with IFNγ alone resulted in MR1 upregulation. Our data further suggest MAIT cell- and IFNγ-mediated MR1 upregulation is altered in COPD cells. Additionally, we observed increased baseline surface expression of MR1 protein in smoker AECs and reduced surface translocation upon addition of exogenous ligand. COPD- and CS-mediated dysregulation of MR1 expression could impact MAIT cell function in airways, resulting in inflammation or reduced early response to bacteria, contributing to lower airway colonization and COPD exacerbations.

High-Resolution DNA Dual-Rulers Reveal a New Intermediate State in Ribosomal Frameshifting.

Ribosomal frameshifting is an important pathway used by many viruses for protein synthesis that involves mRNA translocation of various numbers of nucleotides. Resolving the mRNA positions with subnucleotide precision will provide critical mechanistic information that is difficult to obtain with current techniques. We report a method of high‐resolution DNA rulers with subnucleotide precision and the discovery of new frameshifting intermediate states on mRNA containing a GA7G motif. Two intermediate states were observed with the aid of fusidic acid, one at the “0” reading frame and the other near the “−1” reading frame, in contrast to the “−2” and “−1” frameshifting products found in the absence of the antibiotic. We termed the new near‐“−1” intermediate the Post(−1*) state because it was shifted by approximately half a nucleotide compared to the normal “−1” reading frame at the 5’‐end. This indicates a ribosome conformation that is different from the conventional model of three reading frames. Our work reveals uniquely precise mRNA motions and subtle conformational changes that will complement structural and fluorescence studies.

Nanogold induces anti-inflammation against oxidative stress induced in human neural stem cells exposed to amyloid-beta peptide

Alzheimer's disease (AD) is a neurodegenerative disorder with progressive memory loss resulting in dementia. Amyloid-beta (Aβ) peptides play a critical role in the pathogenesis of the disease by promoting inflammation and oxidative stress, leading to neurodegeneration in the brains of AD patients. Numerous in vitro 3D cell culture models are useful mimics for understanding cellular changes that occur during AD under in vivo conditions. The 3D Bioprinter developed at the CELLINK INKREDIBLE was used in this study to directly investigate the influence of 3D conditions on human neural stem cells (hNSCs) exposed to Aβ. The development of anti-AD drugs is usually difficult, mainly due to a lack of therapeutic efficacy and enhanced serious side effects. Gold nanoparticles (AuNPs) demonstrate benefits in the treatment of several diseases, including AD, and may provide a novel therapeutic approach for AD patients. However, the neuroprotective mechanisms by which AuNPs exert these beneficial effects in hNSCs treated with Aβ are still not well understood. Therefore, we tested the hypothesis that AuNPs protect against Aβ-induced inflammation and oxidative stress in hNSCs under 3D conditions. Here, we showed that AuNPs improved the viability of hNSCs exposed to Aβ, which was correlated with the reduction in the expression of inflammatory cytokines, such as TNF-α and IL-1β. In addition, AuNPs rescued the levels of the transcripts of inhibitory kappa B kinase (IKK) in Aβ-treated hNSCs. The Aβ-mediated increases in mRNA, protein, and nuclear translocation levels of NF-κB (p65), a key transcription factor involved in inflammatory responses, were all significantly abrogated following co-treatment of hNSCs with AuNPs. In addition, treatment with AuNPs significantly restored iNOS and COX-2 levels in Aβ-treated hNSCs. Importantly, hNSCs co-treated with AuNPs were significantly protected from Aβ-induced oxidative stress, as detected using the DCFH-DA and DHE staining assays. Furthermore, hNSCs co-treated with AuNPs were significantly protected from the Aβ-induced reduction in the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2 downstream antioxidant target genes (SOD-1, SOD-2, Gpx1, GSH, Catalase, and HO-1). Moreover, AuNPs reduced the aggregates and increased the proteasome activity and the expression of HSP27 and HSP70 genes in Aβ-treated hNSCs. Taken together, these findings provide the first evidence extending our understanding of the molecular mechanisms under 3D scaffold conditions by which AuNPs reverse the inflammation and oxidative stress-induced in hNSCs exposed to Aβ. These findings may facilitate the development of novel treatments for AD.

M6A RNA Methylation Regulators Act as Potential Prognostic Biomarkers in Lung Adenocarcinoma

N6-methyladenosine [m(6)A/m6A] methylation is one of the most common RNA modifications in eukaryotic cell mRNA and plays an important regulatory role in mRNA metabolism, splicing, translocation, stability, and translation. Previous studies have demonstrated that the m6A modification is highly associated with tumor cell proliferation, migration, and invasion. In the present study, five m6A regulatory factors have been revealed, namely heterogeneous nuclear ribonucleoprotein A2/B1(HNRNPA2B1), heterogeneous nuclear ribonucleoprotein C (HNRNPC), Vir like m6A methyltransferase associated protein (KIAA1429/VIRMA), RNA binding motif protein 15 (RBM15) and methyltransferase like 3 (METTL3), which are closely related to the overall survival (OS) of patients with lung adenocarcinoma (LUAD). These five m6A regulatory factors exhibited potential prognostic value for the 1, 3, and 5-years survival outcomes of LUAD patients. Our findings revealed that several signaling pathways, such as cell cycle, DNA replication, RNA degradation, RNA polymerase, nucleotide excision repair and basal transcription factors, are activated in the high-risk group of LUAD patients.

Anti-inflammatory activity of the water extract of Chloranthus serratus roots in LPS-stimulated RAW264.7 cells mediated by the Nrf2/HO-1, MAPK and NF-κB signaling pathways

Ethnopharmacological relevanceChloranthus serratus is a traditional Chinese medicine for treating arthritis and bruises. Aim of the studyTo investigate the dose-effect relationship and molecular mechanisms of the water extract of C. serratus roots (WECR) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Materials and methodsThe cell viability was detected by CCK-8 method. One-step method, DCFH-DA fluorescence probe method and immunofluorescence method were used to detect nitric oxide (NO), reactive oxygen species (ROS) and p65 nuclear transcription, respectively. Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) were detected by enzyme linked immunosorbent assay. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA were detected by quantitative real-time PCR. Western blotting was taken to determine the contents of the relevant proteins in the nuclear transcription factor E2 related factor 2/heme oxygenase-1 (Nrf2/HO-1), mitogen-activated protein kinases (MAPK) and nuclear factor-kappa B (NF-κB) pathways. ResultsThe concentrations of 3, 30 and 300 μg/mL were optimized as low, medium and high concentrations of the WECR, respectively, and 1 μg/mL was selected as the optimal concentration of LPS to activate macrophages. The dose of the positive drug dexamethasone was 0.13 mg/mL. The WECR could not only inhibit LPS-induced cell differentiation and the overexpression of NO, IL-6, TNF-α, PGE2 and ROS but also promote the expression of Nrf2 and HO-1, and down-regulate the phosphorylation levels of ERK, JNK, p38 and p65. After the WECR treatment, the expression levels of iNOS and COX-2 mRNA and nuclear translocation of p65 were all inhibited. ConclusionsThe WECR exerts its anti-inflammatory activity by inhibiting the MAPK and NF-κB pathways, activating the Nrf2/HO-1 pathway and down-regulating inflammatory factor levels in a dose-dependent manner.

Mutations in domain IV of elongation factor EF-G confer -1 frameshifting.

A recent crystal structure of a ribosome complex undergoing partial translocation in the absence of elongation factor EF-G showed disruption of codon–anticodon pairing and slippage of the reading frame by −1, directly implicating EF-G in preservation of the translational reading frame. Among mutations identified in a random screen for dominant-lethal mutations of EF-G were a cluster of six that map to the tip of domain IV, which has been shown to contact the codon–anticodon duplex in trapped translocation intermediates. In vitro synthesis of a full-length protein using these mutant EF-Gs revealed dramatically increased −1 frameshifting, providing new evidence for a role for domain IV of EF-G in maintaining the reading frame. These mutations also caused decreased rates of mRNA translocation and rotational movement of the head and body domains of the 30S ribosomal subunit during translocation. Our results are in general agreement with recent findings from Rodnina and coworkers based on in vitro translation of an oligopeptide using EF-Gs containing mutations at two positions in domain IV, who found an inverse correlation between the degree of frameshifting and rates of translocation. Four of our six mutations are substitutions at positions that interact with the translocating tRNA, in each case contacting the RNA backbone of the anticodon loop. We suggest that EF-G helps to preserve the translational reading frame by preventing uncoupled movement of the tRNA through these contacts; a further possibility is that these interactions may stabilize a conformation of the anticodon that favors base-pairing with its codon.

Structural insights into mammalian mitochondrial translation elongation catalyzed by mtEFG1.

Mitochondria are eukaryotic organelles of bacterial origin where respiration takes place to produce cellular chemical energy. These reactions are catalyzed by the respiratory chain complexes located in the inner mitochondrial membrane. Notably, key components of the respiratory chain complexes are encoded on the mitochondrial chromosome and their expression relies on a dedicated mitochondrial translation machinery. Defects in the mitochondrial gene expression machinery lead to a variety of diseases in humans mostly affecting tissues with high energy demand such as the nervous system, the heart, or the muscles. The mitochondrial translation system has substantially diverged from its bacterial ancestor, including alterations in the mitoribosomal architecture, multiple changes to the set of translation factors and striking reductions in otherwise conserved tRNA elements. Although a number of structures of mitochondrial ribosomes from different species have been determined, our mechanistic understanding of the mitochondrial translation cycle remains largely unexplored. Here, we present two cryo-EM reconstructions of human mitochondrial elongation factor G1 bound to the mammalian mitochondrial ribosome at two different steps of the tRNA translocation reaction during translation elongation. Our structures explain the mechanism of tRNA and mRNA translocation on the mitoribosome, the regulation of mtEFG1 activity by the ribosomal GTPase-associated center, and the basis of decreased susceptibility of mtEFG1 to the commonly used antibiotic fusidic acid.

Extending the Spacing between the Shine–Dalgarno Sequence and P-Site Codon Reduces the Rate of mRNA Translocation

By forming base-pairing interactions with the 3' end of 16S rRNA, mRNA Shine-Dalgarno (SD) sequences positioned upstream of open reading frames facilitate translation initiation. During the elongation phase of protein synthesis, intragenic SD-like sequences stimulate ribosome frameshifting and may also slow down ribosome movement along mRNA. Here, we show that the length of the spacer between the SD sequence and P-site codon strongly affects the rate of ribosome translocation. Increasing the spacer length beyond 6 nt destabilizes mRNA-tRNA-ribosome interactions and results in a 5- to 10-fold reduction of the translocation rate. These observations suggest that during translation, the spacer between the SD sequence and P-site codon undergoes structural rearrangements, which slow down mRNA translocation and promote mRNA frameshifting.

Exosome‐associated ELAVL1 adversely affects cardiac macrophage‐fibroblast crosstalk in diabetes

BackgroundMacrophage dysfunction, inflammation and excessive fibroblast activation has been implicated in the pathogenesis of diabetes‐associated heart disease. However, the paracrine mechanisms are not completely understood. We sought to unravel the influence of diabetes on exosome (Exo) transfer of Human antigen R (HuR or ELAVL1, an mRNA stabilizing protein) and its implications on cardiac fibrosis and dysfunction.Methods and ResultsWe evaluated the invitro and invivo effects of macrophage cellular‐ and exosome‐HuR or its deficiency on paracrine mechanisms of diabetic milieu‐induced inflammatory and fibrosis‐related signaling. Human failing hearts show higher HuR levels as compared to non‐failing hearts. Diabetic milieu (db/db mice) show increase in HuR expression in cardiac‐ and cultured bone marrow derived‐macrophages (BMMac). High glucose (HG, 30mM) stimulates HuR nuclear‐to‐cytoplasmic translocation and transfer of HuR mRNA and protein into exosomes. PKC‐delta inhibition blocks HG‐induced HuR translocation. Exosome from macrophages exposed to diabetic milieu (HG or db/db mice) significantly increases proinflammatory and profibrogenic responses in fibroblast (invitro) or cardiac fibrosis in mice. Exo‐HuR deficiency (HuR knockdown in macrophage) abrogates the above effects. In diabetic mice, macrophage depletion followed by reconstitution with HuR‐deficient‐BMMac decreases ANG II‐induced cardiac fibrosis and improves LV dysfunction as compared to BMMacWT administered mice.ConclusionsTo the best of our knowledge, this is the first study to demonstrate that diabetes‐mediated increase in BMMac‐HuR expression, its cytoplasmic translocation and exosome transfer activates fibrogenesis signaling invitro and invivo, and HuR deficiency attenuates these deleterious effects. The data suggests that HuR might be targeted to alleviate macrophage dysfunction and pathological fibrosis in diabetes mellitus.Support or Funding InformationNational Institutes of Health (NIH) grants HL116729 (to P.K.), HL138023 (to P.K. and J.Z.), and American Heart Association Grant‐in‐aid GRNT 25860041 (to P.K.) and Transformational Project Award 19TPA34850100 (to P.K.).

Engineering of Ribosome-inactivating Proteins for Improving Pharmacological Properties.

Ribosome-inactivating proteins (RIPs) are N-glycosidases, which depurinate a specific adenine residue in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. This loop is important for anchoring elongation factor (EF-G for prokaryote or eEF2 for eukaryote) in mRNA translocation. Translation is inhibited after the attack. RIPs therefore may have been applied for anti-cancer, and anti-virus and other therapeutic applications. The main obstacles of treatment with RIPs include short plasma half-life, non-selective cytotoxicity and antigenicity. This review focuses on the strategies used to improve the pharmacological properties of RIPs on human immunodeficiency virus (HIV) and cancers. Coupling with polyethylene glycol (PEG) increases plasma time and reduces antigenicity. RIPs conjugated with antibodies to form immunotoxins increase the selective toxicity to target cells. The prospects for future development on the engineering of RIPs for improving their pharmacological properties are also discussed.

Expression Profile of miRNA-17-3p and miRNA-17-5p Genes in Gastric Cancer Patients with Helicobacter pylori Infection.

The most common chronic bacterial infection is Helicobacter pylori. The connection between chronic H. pylori infection and gastric cancer is recognized. The early detection of gastric cancer improves survival. miRNAs regulate gene expression in eukaryotes by inhibiting mRNA translocation or degradation. The objective of this study was to compare the expression of miRNA-17-3p and miRNA-17-5p genes in gastric cancer patients with Helicobacter pylori infection. Herein, 30 isolates were identified as H. pylori based on urease test, and 30 and 12 cases were isolated from gastric cancer patients and non-Helicobacter pylori cases as control, respectively. A peripheral blood sample was collected from patients. Analysis of total mRNA extracts from peripheral blood samples, for gene expression changes (miRNA-17-3p and miRNA-17-5p) by quantitative real-time polymerase chain reaction (qRT-PCR), was done. As said by the results, p values showed that expression levels of miRNA-17-3p and miRNA-17-5p were significantly higher in H. pylori-positive GC patients and H. pylori-positive non-GC patients with comparing by healthy controls. So, there was no significant difference between expression levels of miRNA-17-3p and miRNA-17-5p in H. pylori-positive GC patients and H. pylori-positive non-GC patients. Considering our results, the high expression of miRNA-17-3p and miRNA-17-5p has a direct relationship with increased cell proliferation, inhibition of tumor cell apoptosis and tumor angiogenesis, in addition to miRNAs play an important role as biomarkers in helping for detection of the patient by H. pylori infection to become cancerous. Therefore, it can be used to make specific diagnostic kits and to treat patients.

Design powerful predictor for mRNA subcellular location prediction in Homo sapiens.

Messenger RNAs (mRNAs) shoulder special responsibilities that transmit genetic code from DNA to discrete locations in the cytoplasm. The locating process of mRNA might provide spatial and temporal regulation of mRNA and protein functions. The situ hybridization and quantitative transcriptomics analysis could provide detail information about mRNA subcellular localization; however, they are time consuming and expensive. It is highly desired to develop computational tools for timely and effectively predicting mRNA subcellular location. In this work, by using binomial distribution and one-way analysis of variance, the optimal nonamer composition was obtained to represent mRNA sequences. Subsequently, a predictor based on support vector machine was developed to identify the mRNA subcellular localization. In 5-fold cross-validation, results showed that the accuracy is 90.12% for Homo sapiens (H. sapiens). The predictor may provide a reference for the study of mRNA localization mechanisms and mRNA translocation strategies. An online web server was established based on our models, which is available at http://lin-group.cn/server/iLoc-mRNA/.

Structural basis of elongation factor 2 switching.

Archaebacterial and eukaryotic elongation factor 2 (EF-2) and bacterial elongation factor G (EF-G) are five domain GTPases that catalyze the ribosomal translocation of tRNA and mRNA. In the classical mechanism of activation, GTPases are switched on through GDP/GTP exchange, which is accompanied by the ordering of two flexible segments called switch I and II. However, crystal structures of EF-2 and EF-G have thus far not revealed the conformations required by the classical mechanism. Here, we describe crystal structures of Methanoperedens nitroreducens EF-2 (MnEF-2) and MnEF-2-H595N bound to GMPPCP (GppCp) and magnesium displaying previously unreported compact conformations. Domain III forms interfaces with the other four domains and the overall conformations resemble that of SNU114, the eukaryotic spliceosomal GTPase. The gamma phosphate of GMPPCP is detected through interactions with switch I and a P-loop structural element. Switch II is highly ordered whereas switch I shows a variable degree of ordering. The ordered state results in a tight interdomain arrangement of domains I-III and the formation of a portion of a predicted monovalent cation site involving the P-loop and switch I. The side chain of an essential histidine residue in switch II is placed in the inactive conformation observed for the “on” state of elongation factor EF-Tu. The compact conformations of MnEF-2 and MnEF-2-H595N suggest an “on” ribosome-free conformational state.

Anti-Inflammatory Effects of Shenfu Injection against Acute Lung Injury through Inhibiting HMGB1-NF-κB Pathway in a Rat Model of Endotoxin Shock.

Shenfu injection (SFI), a Chinese herbal medicine with substances extracted from Ginseng Radix et Rhizoma Rubra and Aconiti Lateralis Radix Praeparata, is widely used as an anti-inflammatory reagent to treat endotoxin shock in China. However, the mechanism of SFI in endotoxin shock remains to be illuminated. High mobility group box 1 (HMGB1), a vital inflammatory factor in the late stage of endotoxin shock, may stimulate multiple signalling cascades, including κB (NF-κB), a nuclear transcription factor, as well as tumour necrosis factor (TNF)-α and interleukin (IL)-1β, among others in the overexpression of downstream proinflammatory cytokines. An investigation into the effects of SFI on the inhibition of the HMGB1-NF-κB pathway revealed the contribution of SFI to acute lung injury (ALI) in a rat model of endotoxin shock. To assess the anti-inflammatory activity of SFI, 5 ml/kg, 10 ml/kg, or 15 ml/kg of SFI was administered to different groups of rats following an injection of LPS, and the mean arterial pressure (MAP) at 5 h and the survival rate at 72 h were measured. 24 h after LPS injection, we observed pathological changes in the lung tissue and measured the mRNA expression, production, translocation, and secretion of HMGB1, as well as the expression of the NF-κB signal pathway-related proteins inhibitor of NF-κB (IκB)-α, P50, and P65. We also evaluated the regulation of SFI on the secretion of inflammatory factors including interleukin-1 beta (IL-1β) and TNF-α. SFI effectively prevented the drop in MAP, relieved lung tissue damage, and increased the survival rate in the endotoxin shock model in dose-dependent manner. SFI inhibited the transcription, expression, translocation, and secretion of HMGB1, increased the expression of toll-like receptor (TLR4), increased the production of IκB-α, and decreased the levels of P65, P50, and TNF-α in the lung tissue of endotoxin shock rats in a dose-dependent manner. Furthermore, SFI decreased the secretion of proinflammatory cytokines TNF-α and IL-1β. In summary, SFI improves the survival rate of endotoxin shock, perhaps through inhibiting the HMGB1-NF-κB pathway and thus preventing cytokine storm.

Imaging within single NPCs reveals NXF1's role in mRNA export on the cytoplasmic side of the pore.

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein-protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export.

Advances in m 6A modification and its regulation of viral replication

N 6-methyladenosine (m 6A) is a prevalent modification of RNA in eukaryotes and plays an important role in the process of mRNA translocation, stabilization and translation. m 6A exerts different influences on the viral replication cycle, and both viral replication and host immune response to the virus are affected by m 6A. In this review, we summarize recent studies on the mechanism of m 6A modification and its effects on viral replication and host immune response, in order to provide a reference for epigenetic regulation in the viral life cycle.

ADAR1 promotes robust hypoxia signaling via distinct regulation of multiple HIF-1α-inhibiting factors.

Adenosine deaminase acting on RNA (ADAR)-catalyzed adenosine-to-inosine RNA editing is potentially dysregulated in neoplastic progression. However, how this transcriptome recoding process is functionally correlated with tumorigenesis remains largely elusive. Our analyses of RNA editome datasets identify hypoxia-related genes as A-to-I editing targets. In particular, two negative regulators of HIF-1A-the natural antisense transcript HIF1A-AS2 and the ubiquitin ligase scaffold LIMD1-are directly but differentially modulated by ADAR1. We show that HIF1A-AS2 antagonizes the expression of HIF-1A in the immediate-early phase of hypoxic challenge, likely through a convergent transcription competition in cis ADAR1 in turn suppresses transcriptional progression of the antisense gene. In contrast, ADAR1 affects LIMD1 expression post-transcriptionally, by interfering with the cytoplasmic translocation of LIMD1 mRNA and thus protein translation. This multi-tier regulation coordinated by ADAR1 promotes robust and timely accumulation of HIF-1α upon oxygen depletion and reinforces target gene induction and downstream angiogenesis. Our results pinpoint ADAR1-HIF-1α axis as a hitherto unrecognized key regulator in hypoxia.

Spontaneous ribosomal translocation of mRNA and tRNAs into a chimeric hybrid state

The elongation factor G (EF-G)-catalyzed translocation of mRNA and tRNA through the ribosome is essential for vacating the ribosomal A site for the next incoming aminoacyl-tRNA, while precisely maintaining the translational reading frame. Here, the 3.2-Å crystal structure of a ribosome translocation intermediate complex containing mRNA and two tRNAs, formed in the absence of EF-G or GTP, provides insight into the respective roles of EF-G and the ribosome in translocation. Unexpectedly, the head domain of the 30S subunit is rotated by 21°, creating a ribosomal conformation closely resembling the two-tRNA chimeric hybrid state that was previously observed only in the presence of bound EF-G. The two tRNAs have moved spontaneously from their A/A and P/P binding states into ap/P and pe/E states, in which their anticodon loops are bound between the 30S body domain and its rotated head domain, while their acceptor ends have moved fully into the 50S P and E sites, respectively. Remarkably, the A-site tRNA translocates fully into the classical P-site position. Although the mRNA also undergoes movement, codon-anticodon interaction is disrupted in the absence of EF-G, resulting in slippage of the translational reading frame. We conclude that, although movement of both tRNAs and mRNA (along with rotation of the 30S head domain) can occur in the absence of EF-G and GTP, EF-G is essential for enforcing coupled movement of the tRNAs and their mRNA codons to maintain the reading frame.

Polyadenylation sites and their characteristics in the genome of channel catfish (Ictalurus punctatus) as revealed by using RNA-Seq data

Polyadenylation plays important roles in gene expression regulation in eukaryotes, which typically involves cleavage and poly(A) tail addition at the polyadenylation site (PAS) of the pre-mature mRNA. Many eukaryotic genes contain more than one PASs, termed as alternative polyadenylation (APA). As a crucial post-transcriptional regulation, polyadenylation affects various aspects of RNA metabolism such as mRNA stability, translocation, and translation. However, polyadenylation has been rarely studied in teleosts. Here we conducted polyadenylation analysis in channel catfish, a commercially important aquaculture species around the world. Using RNA-Seq data, we identified 20,320 PASs which were classified into 14,500 clusters by merging adjacent PASs. Most of the PASs were found in 3′ UTRs, followed by intron regions based on the annotation of channel catfish reference genome. No apparent difference in PAS distribution was observed between the sense and antisense strand of the channel catfish genome. The sequence analysis of nucleotide composition and motif around PASs yielded a highly similar profile among various organisms, suggesting the conservation and importance of polyadenylation in evolution. Using APA genes with more than two PASs, gene ontology enrichment revealed genes particularly involved in RNA binding. Reactome pathway analysis showed the enrichment of the innate immune system, especially neutrophil degranulation.