Ribosomal Protein Genes Research Articles

Zero valent iron improved methane production and specifically reduced aminoglycoside and tetracycline resistance genes in anaerobic digestion.

It is unadvisable to discuss the antibiotic resistance genes (ARGs) reduction in anaerobic digestion (AD) system neglecting its main purpose-methane production. The methane production improvement coupling with antibiotic resistance genes (ARGs) reduction in anaerobic digestion (AD) by zero valent iron (ZVI) were simultaneously investigated. Whether the role of ZVI on the ARGs fate was random or specific was clarified through the high-throughput qPCR (HT-qPCR). Results indicated that ZVI improved methane production and ARGs reduction by 23.9% and 25.0%, respectively. The improved methane production was associated with chemical reaction and variances of microbial community caused by ZVI, where DIET between Petrimonas, Clostridium and Syntrophomonas, Methanosarcina was established along with ACAS being enriched. ZVI specifically, not randomly, facilitated the reduction of aminoglycoside resistance genes of antibiotic inactivation and tetracycline resistance genes of ribosomal protection proteins. The specifical reduction could be attributed to enzyme activity inhibition and intracellular ionic disturbance caused by higher amounts of ZVI, although most of ARGs fate could be well explained by microbial community which contributed the most to ARGs dynamics as a whole. ZVI-based AD was a promising way for the improvement of methane production coupling ARGs reduction.

Identification of 16SrXXX-A Phytoplasma Associated with Salt cedar (Tamarix chinensis Lour.) witches'-broom (SCWB) in Southern Xinjiang of China.

Salt cedar is an ornamental shrub/moderate tree species native to Asia and East Europe, and grows in salt-alkali soil, desert and other dry areas, which plays an important role in wind prevention and sand fixation as well as maintaining ecological balance. Salt cedar witches'-broom (SCWB), which was extremely pernicious to Salt cedar. It was first observed and reported in Xi'an, China in 2005 (Zhao et al.2005). Witches' broom symptoms were observed on 20 out of 150 (13.3%) salt plants surveyed from the Alar region and 10 out of 86 (11.6%) plants from the Akesu region in southern of Xinjiang in May 2020. The damaged plants compared with asymptomatic plants (Fig.1A), the major symptoms included branches clustered, intersegment shorten and coarsen, giving rise to the formation of clusters (Fig.1B). Total plant DNA was extracted from phloem tissues with asymptomatic symptoms and phloem tissues with witches'-broom symptoms by a CTAB-based DNA extraction method (Green et al.1999). The 16S rRNA gene and the phytoplasma universal primers P1/P7 and rpF1/rpR1 of the rp (ribosomal protein) gene were used for Polymerase chain reaction (PCR) amplification by using the extracted plant total DNA as the template. The PCR product was used as the template and the R16F2n/R16R2 prmer was used for nested PCR amplification of the 16S rRNA gene after the amplification was completed. The results show that no product was obtained in asymptomatic plants. When DNA samples from witches'-broom symptomatic plants were used as templates, fragments with lengths 1219 bp and 1174 bp, corresponding to 16S rRNA gene and rp gene, were obtained. 16S rRNA gene was sequenced and deposited in GenBank under accession number MW447513. BLAST analysis revealed that the partial 16S rRNA sequence of the phytoplasma associated with P. aphylla witches' broom showed highest sequence identity (99.67%) to salt cedar witches' broom phytoplasma, 'Candidatus Phytoplasma tamaricis' (Accession Number: FJ432664). Phylogenetic and molecular evolutionary analyses were conducted using MEGA-X (Kumar et al., 2018). Results showed taht the SCWB and 16S rXXX group's'Candidatus Phytoplasma tamaricis', (GenBank accession: FJ432664) have the highest affinity (Fig.2A). A virtual restriction fragment length polymorphism(RFLP) was done to determinethe subgroup ( Zhao et al. 2009). The 16S rDNA sequence from the Tamarix chinensis plant showed 99.3% similarity with that of the "Candidatus Phytoplasma tamaricis" reference strain (GenBank accession: FJ432664), suggesting that the phytoplasma in this study belongs to "Candidatus Phytoplasma tamaricis"-related strain. Therefore, it can be stated that SCWB belongs to the 16S rXXX group. The partial rp sequences only shared 84.74% sequence similarity with that of 'Candidatus Phytoplasma prunorum' (MG383523) of Apple proliferation group, a known subgroup 16S rX. Blast analysis based on the partial rp sequences showed that it shares less than 90% similarity with that of any known phytoplasma (Fig 2B), we suspect that this is due to a lack of sequenced rp gene sequences for the 16S rXXX group. To our knowledge, this is the first report of Salt Cedar Witches' Broom phytoplasma in Xinjiang province, China. As a consequence, we guess the SCWB phytoplasma rp gene belongs to 16S rXXX-rp group, which is also the first report about the 16SrXXX-rp group. Because SCWB1 is the only strain in the 16S rXXX group, and it is the representative strain of the 16S rXXX-A subgroup (Zhao et al. 2009). So, the SCWB disease we found in southern Xinjiang belongs to the 16S rXXX-A subgroup.

Identification of the Upregulation of MRPL13 as a Novel Prognostic Marker Associated with Overall Survival Time and Immunotherapy Response in Breast Cancer.

Mitochondrial ribosomal protein (MRPL) genes have been reported to participate in many cellular processes, such as cell proliferation, apoptosis, and cell cycle. Meanwhile, the occurrence rate of breast cancer (BRCA) in China steadily increased. Exploring the prognostic value of MRPL genes in BRCA could provide novel biomarkers for BRCA. In this study, to identify prognosis-related genes in breast cancer, the P value and the hazard ratio (HR) of all genes are analyzed with TCGA database. We revealed higher expression level of CEL, PGK1, WNT3A, USP41, LINC02037, PCMT1, LRP11, MCTS1, TCP1, TMEM31, STK4-AS1, STXBP5, LOC100287036, SLC16A2, MRPL13, DERL1, and TARS was correlated to shorter OS time in BRCA. However, higher expression level of JCHAIN, KLRB1, and TNFRSF14 was correlated to longer OS time in BRCA. The further analysis demonstrated MRPL13 was overexpressed in BRCA. Subtype analysis showed that MRPL13 was overexpressed in luminal, HER2-positive BRCA, and TNBC samples and was highest in TNBC samples. Moreover, we revealed higher expression of MRPL13 was significantly correlated to shorter OS time and higher TMB levels in BRCA. Pan-cancer analysis further revealed the prognostic value of MRPL13 in human cancers. MRPL13 expression was significantly increased in multiple human cancers, such as bladder cancer, colon cancer, liver cancer, and prostate cancer. Pan-cancer TMB and overall survival time showed dysregulation of MRPL13 is significantly related to the OS and TMB levels in various cancers. These results further proved that MRPL13 may be a pan-cancer biomarker for predicting prognosis and the response to immunotherapy.

Whole Genome Sequencing of Diamond Blackfan Anemia Syndrome Patients Detects Mutations That Alter mRNA Splicing

Diamond Blackfan anemia syndrome (DBAS) is a rare, heritable bone marrow failure syndrome characterized by severe macrocytic anemia, congenital anomalies and predisposition to cancer, most often diagnosed during infancy. More than 98% of DBAS patients with a molecular diagnosis have mutations in a gene encoding one of the ~80 ribosomal proteins (RP) leading to haploinsufficiency. A molecular diagnosis in a patient with DBAS is critical for a definitive diagnosis, the identification of compatible related transplant donors, and developing reproductive strategies for families. Targeted sequencing of RP genes, single nucleotide polymorphism comparative genome hybridization (SNP array) to detect >30 kb deletions (Farrar et al. Blood. 2011) and exome sequencing (WES) (Ulrisch et al. Am J Hum Genet. 2018) has identified RP mutations in ~80% of patients, leaving ~20% of patients with DBAS without a molecular diagnosis.Targeted sequencing and WES focus on only coding sequences. We hypothesized that remaining 20% of DBAS mutations were in the non-coding regions of RP genes, such as promoters or introns. To test this hypothesis, we collected DNA with informed consent for whole genome sequencing (WGS) analysis from 14 patients with no molecular diagnosis after targeted sequencing, SNP array or WES. On average, we aligned ~3.2x10 7 paired end reads of 250 base pairs for each patient (~65X coverage). We focused our analysis on the sequences in and around the RP genes. To identify deletions, we used a suite of detection tools: DELLY, GRIDSS, MANTA, and LUMPY. More than 90% of deletions identified by any 2 of these tools were confirmed by PCR.We identified 5 deletions in the introns of RP genes, ranging from 11 to 467 base pairs in length, which we hypothesized disrupted splicing of the nascent RNA transcript. To test this, we created minigenes in which we replaced exon 2 of a gamma globin gene with either the WT or mutant RP exon. All wild type exons spliced normally. A 467 base pair deletion in RPL27 exon 3 was sufficient to prevent the correct splicing of that intron. Examination of the eCLIP data for RNA binding proteins revealed that spliceosome complex proteins (including SF3B1, SF3B4 and EFTUD2) and Dead-box RNA helicases bind in the deleted region. A 28 base pair deletion in exon 3 of RPL6 removes a polypyrimidine tract that is a critical part of the 3' splice junction consensus sequence, which we presume is also deleterious. The other 3 intronic deletions did not disrupt splicing.We also identified 2 causative point mutations. A point mutation 5 bases into intron 1 of the RPS26 gene changes a base in the 5' splice donor consensus sequence, which activated a cryptic splice donor in the 5' untranslated region. This aberrant splice removes the ATG initiation codon causing an untranslatable RNA. In another patient, we identified a mutation in exon 1 of the RPS27 gene, judged to be a benign amino acid change. This mutation disrupted splicing.by activating a cryptic splice donor site in the 5' untranslated region which removes the ATG initiation codon and causes a frame shift.We were referred two patients with possible duplications of the RPL35a gene. To identify duplications, we employed MinION long read single molecule sequencing. We had an average read length of ~ 6-10kb with the longest read being 1.3Mb. Overall coverage was >85X. We used minimap2 to align the reads to the reference human genome and used SNIFFLES to call the variants. One patient was the parent of DBAS-affected patient with no history of anemia. In this patient, we identified a duplication of 400 kb that included the entire RPL35a region along with genes on either side. We conclude that this duplication is not likely to cause DBA. The second patient was diagnosed with DBAS. In this patient, we identified a duplication of 4 kb including exons 1 and 2 of RPL35a We conclude that this duplication disrupts the RPL35a gene and is a likely cause of DBA.Whole genome sequencing of 15 DBAS patients identified 5 likely causative mutations in RP genes, confirming that most genetically undiagnosed cases of DBAS will involve known genes encoding RP. We conclude that the pipeline for obtaining a molecular diagnosis for DBAS from targeted sequencing, SNP array, and exome sequencing to whole genome sequencing. DisclosuresVlachos: Novartis: Membership on an entity's Board of Directors or advisory committees. Lipton: Celgene: Membership on an entity's Board of Directors or advisory committees.

A Novel RPS19-Edited Hematopoietic Stem Cell Model of Diamond-Blackfan Anemia for Development of Lentiviral Vector Gene Therapy

Diamond-Blackfan anemia (DBA) is a congenital hypoplastic anemia that typically manifests in infancy as macrocytic anemia with reticulocytopenia. About 80% of DBA cases are caused by heterozygous loss-of-function mutations or deletions in one of 23 ribosomal proteins (RP) genes, with RPS19 being affected in ~25% of patients. Current therapies are suboptimal, and it is difficult to obtain DBA patient hematopoietic stem and progenitor cells (HSPCs) in sufficient quantity for preclinical development of new therapies. To address this gap, we used CRISPR/Cas9-edited healthy donor CD34 + HSPCs to create a novel model of RPS19-mutated DBA and used this model to develop an efficient RPS19-encoding lentiviral vector (LV) for gene therapy.Healthy donor CD34 + HSPCs were electroporated with ribonucleoprotein (RNP) complex consisting of Cas9 and guide RNAs (gRNAs) targeting RPS19 or the AAVS1 locus as a negative control, then grown in medium to support erythroid differentiation. All gRNAs analyzed generated high-frequency on-target insertion-deletion (indels) mutations (Fig. A). RPS19 indels specifically declined over time, suggesting that RPS19 disruption impairs cell proliferation and/or survival. To rescue the defect, we constructed a third-generation, self-inactivating LV expressing RPS19+GFP (RPS19/GFP LV). Transduction was optimized using poloxamer and prostaglandin E2 and linear transduction efficiency was noted even at high multiplicity of infection (MOI) (Fig. B). An MOI of 20 was used for subsequent experiments. In methylcellulose medium, RPS19 RNP-treated cells generated 72% fewer burst forming unit-erythroid (BFU-E) colonies compared to AAVS1 RNP-treated control cells. RPS19/GFP LVs with three different promoters (EF1α short, EF1α long and MND) partially restored BFU-E formation similarly (Fig. C); the EF1α short promoter was chosen for subsequent experiments due its track record for clinical use. We down-titrated the RNP concentration to generate a total indel frequency of ~25%, which resulted in approximately equal frequencies of RPS19 +/+ and RPS19 +/- BFU-E colonies. RPS19 -/- colonies were detected only after edited HSPCs were rescued by RPS19/GFP LV, due to lethality of this genotype (Fig. D). Transfection of CD34 + HSPCs with RPS19 RNP caused a 49% reduction in cell number after 14 days of liquid culture in erythroid differentiation medium compared to control HSPCs; this was corrected by treatment with RPS19/GFP LV (Fig. E). RPS19 RNP treatment of CD34 + HSPCs had no effect on the expansion of cells grown under myeloid differentiation conditions (Fig. F).We analyzed RNP-treated CD34 + cells further by transducing them with RPS19/GFP LV or control LV encoding GFPalone, transplanting them into immunodeficient NSGW mice and analyzing human donor cell progeny in mouse bone marrow after 16 weeks. In cells treated with AAVS1 RNP and GFP LV, the indel frequency dropped from 27.2±1.5% (SD) at 72 hours after editing (input) to 15.5±4.4% at 16 weeks post-transplant (43% reduction) (Fig. G). In HSPCs treated with RPS19 RNP and GFP LV the indel frequency dropped from 20.9±3.1% in input cells to 1.8±0.9% after 16 weeks (Fig. G) (92% reduction). In contrast, the indel frequency of donor HSPCs treated with RPS19 RNP and RPS19/GFP LV dropped from 23.6±2.7% in input cells to 8.4±1.6% (64% reduction), which represents a 5-fold increase in indel frequency compared to treatment with control GFP LV (p< 0.01)(Fig. G). In flow cytometry-purified, donor HSPC-derived myeloid, B-lymphocyte, HSPC and erythroid lineages at 16 weeks after xenotransplantation, the mean indel rates were 1.3% to 2.5% in cells derived from HSPCs treated with RPS19 RNP and GFP LV. Indel rates ranged from 6.9% to 9.2% in the progeny of input HSPCs that were rescued by RPS19/GFP LV, representing a 4-6-fold increase compared to transduction with control GFP LV (p<0.01) (Fig. H).In summary, our studies show that Cas9-mediated disruption of RPS19 in CD34 + HSPCs causes a selective erythroid defect in RPS19 +/- cells, recapitulating the canonical DBA defect. Additionally, deficient bone marrow repopulation by RPS19 +/- cells suggests an HSC defect, consistent with pancytopenia that is observed in many older DBA patients. The optimized RPS19 LV transduces HSPCs at high efficiency and alleviates both defects, supporting its potential utility for DBA therapy. [Display omitted] DisclosuresYen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Weiss: Beam Therapeutics: Current holder of stock options in a privately-held company; Forma Therapeutics: Consultancy; Novartis: Consultancy; Cellarity Inc.: Consultancy.

Recruitment of MLL1 Complex Is Essential for SETBP1 to Induce Myeloid Transformation

Abnormal activation of SETBP1 due to overexpression or missense mutations occurs frequently in various myeloid neoplasms and associates with poor prognosis. Direct activation of Hoxa9/Hoxa10/Myb transcription by SETBP1 and its missense mutants is essential for their transforming capability; however, the underlying mechanisms for such activation remain elusive. We found that knockdown of Mll1 in mouse myeloid progenitors immortalized by SETBP1 or its missense mutant SETBP1(D/N) caused significant reduction in the mRNA levels of Hoxa9/Hoxa10/Myb, suggesting that Mll1 is critical for their transcriptional activation induced by SETBP1 and its missense mutants. Physical association of MLL1 with SETBP1/SETBP1(D/N) was readily detected by co-immunoprecipitation in nuclear extracts of these cells, further suggesting that they may form a complex in myeloid cells to activate transcription. This complex formation is likely mediated by direct interactions between SETBP1/SETBP1(D/N) and MLL1 as both SETBP1 and SETBP1(D/N) are capable of interacting with multiple regions of MLL1 in binding assays using proteins synthesized by in vitro transcription and translation. To better understand the extent of SETBP1/SETBP1(D/N)-MLL1 interaction in regulating gene transcription, we carried out both ChIP-seq and RNA-seq analysis in mouse Lin -Sca-1 +c-Kit + (LSK) cells transduced by pMYs retrovirus expressing SETBP1 or SETBP1(D/N) or empty pMYs virus. These analyses revealed extensive overlap in genomic occupancy for MLL1 and SETBP1/SETBP1(D/N) and their cooperation in activating many oncogenic transcription factor genes in addition to Hoxa9/Hoxa10/Myb, including additional HoxA genes (Hoxa1, Hoxa3, Hoxa5, Hoxa6, and Hoxa7), Myc, Eya1, Mef2c, Meis1, Sox4, Mecom, and Lmo2. A large group of ribosomal protein genes were also found to be directly activated by MLL1 and SETBP1/SETBP1(D/N), identifying ribosomal biogenesis as another significant pathway induced by their cooperation. To further assess the requirement for MLL1 in SETBP1-induced transformation using a genetic approach, we also generated SETBP1/SETBP1(D/N)-induced immortalized myeloid progenitors and AMLs using LSK cells from Mll1 conditional knockout mice. Mll1 deletion in immortalized progenitors significantly decreased SETBP1/SETBP1(D/N)-induced transcriptional activation and their colony-forming potential. More importantly, Mll1 deletion significantly extended the survival of mice transplanted with SETBP1/SETBP1(D/N)-induced AMLs, indicating that Mll1 is essential for the maintenance of such leukemias in vivo. We further found that pharmacological inhibition of MLL1 complex using a WDR5 inhibitor OICR-9429 efficiently abrogated SETBP1/SETBP1(D/N)-induced transcriptional activation and transformation. Thus, MLL1 complex plays a critical role in Setbp1-induced transcriptional activation and transformation and represents a promising target for treating myeloid neoplasms with SETBP1 activation. DisclosuresMaciejewski: Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

ORPER: A Workflow for Constrained SSU rRNA Phylogenies.

The continuous increase in sequenced genomes in public repositories makes the choice of interesting bacterial strains for future sequencing projects ever more complicated, as it is difficult to estimate the redundancy between these strains and the already available genomes. Therefore, we developed the Nextflow workflow “ORPER”, for “ORganism PlacER”, containerized in Singularity, which allows the determination the phylogenetic position of a collection of organisms in the genomic landscape. ORPER constrains the phylogenetic placement of SSU (16S) rRNA sequences in a multilocus reference tree based on ribosomal protein genes extracted from public genomes. We demonstrate the utility of ORPER on the Cyanobacteria phylum, by placing 152 strains of the BCCM/ULC collection.

The role of uspE in virulence and biofilm formation by Histophilus somni

UspE is a global regulator in Escherichia coli. To study the function of Histophilus somni uspE, strain 2336::TnuspE was identified from a bank of mutants generated with EZ::Tn5™<KAN-2> Tnp Transposome™ that were biofilm deficient. The 2336::TnuspE mutant was highly attenuated in mice, the electrophoretic profile of its lipooligosaccharide (LOS) indicated the LOS was truncated, and the mutant was significantly more serum-sensitive compared to the wildtype strain. In addition to forming a deficient biofilm, exopolysaccharide (EPS) production was also compromised, but the electrophoretic profile of outer membrane proteins was not altered. RNA sequence analysis revealed that the transcription levels of some stress response chaperones, transport proteins, and a large number of ribosomal protein genes in 2336::TnuspE were significantly differentially regulated compared to strain 2336. Therefore, uspE may differentially function in direct and indirect expression of H. somni genes, but its attenuation may be linked to poor biofilm formation and rapid clearance of the bacteria resulting from a compromised LOS structure. Our results support that uspE is a global stress regulatory gene in H. somni.

The Glucocorticoid Receptor Polymorphism Landscape in Patients With Diamond Blackfan Anemia Reveals an Association Between Two Clinically Relevant Single Nucleotide Polymorphisms and Time to Diagnosis.

NR3C1, the gene encoding the glucocorticoid receptor, is polymorphic presenting numerous single nucleotide polymorphisms (SNPs) some of which are emerging as leading cause in the variability of manifestation and/or response to glucocorticoids in human diseases. Since 60–80% of patients with Diamond Blackfan anemia (DBA), an inherited pure red cell aplasia induced by mutations in ribosomal protein genes became transfusion independent upon treatment with glucocorticoids, we investigated whether clinically relevant NR3C1 SNPs are associated with disease manifestation in DBA. The eight SNPs rs10482605, rs10482616, rs7701443, rs6189/rs6190, rs860457, rs6198, rs6196, and rs33388/rs33389 were investigated in a cohort of 91 European DBA patients. Results were compared with those observed in healthy volunteers (n=37) or present in public genome databases of Italian and European populations. Although, cases vs. control analyses suggest that the frequency of some of the minor alleles is significantly altered in DBA patients with respect to healthy controls or to the Italian or other European registries, lack of consistency among the associations across different sets suggests that overall the frequency of these SNPs in DBA is not different from that of the general population. Demographic data (47 females and 31 males) and driver mutations (44 S and 29 L genes and eight no-known mutation) are known for 81 patients while glucocorticoid response is known, respectively, for 81 (36 responsive and 45 non-responsive) and age of disease onsets for 79 (55 before and 24 after 4months of age) patients. Neither gender nor leading mutations were associated with the minor alleles or with disease manifestation. In addition, none of the SNPs met the threshold in the response vs. non-responsive groups. However, two SNPs (rs6196 and rs860457) were enriched in patients manifesting the disease before 4months of age. Although the exact biomechanistical consequences of these SNPs are unknown, the fact that their configuration is consistent with that of regulatory regions suggests that they regulate changes in glucocorticoid response during ontogeny. This hypothesis was supported by phosphoproteomic profiling of erythroid cells expanded ex vivo indicating that glucocorticoids activate a ribosomal signature in cells from cord blood but not in those from adult blood, possibly providing a compensatory mechanism to the driving mutations observed in DBA before birth.

Research Progress on Pathogenesis of Congenital Pure Red Cell Aplasia---Review

Congenital pure red cell aplasia, also known as Diamond-Blackfan anemia (DBA), is a hereditary disease characterized by pure red cell aplasia and congenital malformation. Its main clinical features are anemia, dysplasia, and tumor susceptibility. Ribosomal protein (RP) gene mutation is the main pathogenesis of DBA. The most common type of gene mutation is RPS19 gene mutation. Heterozygous mutations in as many as 19 RP genes and other non-RP genes mutations have been identified in DBA. This review summarized briedfly the latest research advances in the pathogenesis of DBA.

Dried seahorse in traditional medicine: A narrative review

Seahorses are classified as members of Syngnathidae family, which includes pipefishes, pipehorses, and seadragons. China, including Hong Kong, uses 250 tons of seahorses as Traditional Chinese Medicine (TCM) every year. It is popular in Traditional Medicines (TM) especially TMC and its derivatives. The TCM formulations of dried seahorse strengthens the kidney and enhances immunity to treat the aging process. Base on the molecular biology analysis, S6 ribosomal protein gene, S7 ribosomal protein gene, and the S20 ribosomal protein gene have been identified in dried seahorses, which help to reduce the cough symptoms. The present mini-review discusses the background of the use of dried seahorses, the TCM theory, the TCM formulations, the molecular biology, and analysis of its usage in traditional medicine.

Nitrosative stress induces downregulation of ribosomal protein genes via MYCT1 in vascular smooth muscle cells.

In our previous genomic studies in human intracranial aneurysms, we observed downregulations in the expression of a number of ribosomal protein genes and the c-Myc-related gene MYC target 1 (MYCT1). So far there is no information about the roles of MYCT1 in vascular cells. Our study aims to investigate the functional roles of MYCT1 in vascular smooth muscle cells (SMCs). Primary SMCs were isolated from rat thoracic aorta and cultured in vitro. The mRNA and protein expressions were determined by real-time PCR and western blot respectively. Apoptosis was detected by measuring caspase 3/7 activity. Collagen production was determined with ELISA. Using PCR, we validated our previous genomic data showing that the expressions of MYCT1 and ribosomal protein genes were decreased in human aneurysm tissues. In vascular SMCs, we showed that nitrosative stress downregulated the expression of both MYCT1 and ribosomal proteins. Knockdown of MYCT1 mimicked the effects of nitrosative stress on ribosomal protein expressions, whereas overexpression of MYCT1 blunted the effects of nitrosative stress. MYCT1-dependent downregulation of ribosomal proteins compromised the protein translational capacity of the cells for collagen production. Moreover, the endogenously expressed MYCT1 in vascular SMCs was involved in maintaining normal cellular functions including survival, proliferation and migration. MYCT1-dependent gene regulation may, at least partly, explain the downregulated expressions of ribosomal proteins observed in human intracranial aneurysms. It is suggested that MYCT1 may represent a novel molecular target for counteracting the decreased activity of aneurysmal SMCs for tissue repairmen/regeneration.

Electronic cigarette smoke reduces ribosomal protein gene expression to impair protein synthesis in primary human airway epithelial cells

The widespread use of electronic cigarettes (e-cig) is a serious public health concern; however, mechanisms by which e-cig impair the function of airway epithelial cells—the direct target of e-cig smoke—are not fully understood. Here we report transcriptomic changes, including decreased expression of many ribosomal genes, in airway epithelial cells in response to e-cig exposure. Using RNA-seq we identify over 200 differentially expressed genes in air–liquid interface cultured primary normal human bronchial epithelial (NHBE) exposed to e-cig smoke solution from commercial e-cig cartridges. In particular, exposure to e-cig smoke solution inhibits biological pathways involving ribosomes and protein biogenesis in NHBE cells. Consistent with this effect, expression of corresponding ribosomal proteins and subsequent protein biogenesis are reduced in the cells exposed to e-cig. Gas chromatography/mass spectrometry (GC/MS) analysis identified the presence of five flavoring chemicals designated as ‘high priority’ in regard to respiratory health, and methylglyoxal in e-cig smoke solution. Together, our findings reveal the potential detrimental effect of e-cig smoke on ribosomes and the associated protein biogenesis in airway epithelium. Our study calls for further investigation into how these changes in the airway epithelium contribute to the current epidemic of lung injuries in e-cig users.

Ribosome Proteins Represented by RPL27A Mark the Development and Metastasis of Triple-Negative Breast Cancer in Mouse and Human.

Triple-negative breast cancer (TNBC) is known to have a poor prognosis and limited treatment options. The lack of targeted therapies and poor prognosis of patients with TNBC have made it urgent to discover novel critical diagnosis and therapeutic targets in the TNBC field. Here, in the current study, we integrated the single-cell RNA-sequencing (scRNA-seq) data from four normal mouse mammary tissues and four mouse breast tumors. Comparative analysis was conducted to identify the gene profiles of normal epithelial cells and cancer cells at different models. Surprisingly, two ribosomal protein genes, Rpl27a and Rpl15, were significantly upregulated in the cancer cells in all the TNBC models. Next, we accessed the scRNA-seq data from human primary and metastatic TNBC tissues, and comparative analysis revealed gene profiles of human primary and metastatic TNBC cancer cells. Ribosomal protein genes, represented by RPL27A and RPL15, showed significantly upregulated expression in metastatic TNBC cancer cells. Pathway analysis on the upregulated genes of the metastatic TNBC cancer cells identified the key regulators and signaling pathways that were driving the metastasis of the TNBC cancer cells. Specifically, EIF2 signaling was significantly activated, and major member genes of this signaling pathway were upregulated. In vitro study revealed that targeting RPL27A or EIF2 signaling in a TNBC cell line, MDA-MB-231, significantly reduced cell migration and invasion. Altogether, these data suggested that the RPL27A gene is conducting critical functions in TNBC cancer development and metastasis and is a potential therapeutic target for TNBC.

Insights into molecular structure, genome evolution and phylogenetic implication through mitochondrial genome sequence of Gleditsia sinensis

Gleditsia sinensis is an endemic species widely distributed in China with high economic and medicinal value. To explore the genomic evolution and phylogenetic relationships of G. sinensis, the complete mitochondrial (mt) genome of G. sinensis was sequenced and assembled, which was firstly reported in Gleditsia. The mt genome was circular and 594,121 bp in length, including 37 protein-coding genes (PCGs), 19 transfer RNA (tRNA) genes and 3 ribosomal RNA (rRNA) genes. The overall base composition of the G. sinensis mt genome was 27.4% for A, 27.4% for T, 22.6% for G, 22.7% for C. The comparative analysis of PCGs in Fabaceae species showed that most of the ribosomal protein genes and succinate dehydrogenase genes were lost. In addition, we found that the rps4 gene was only lost in G. sinensis, whereas it was retained in other Fabaceae species. The phylogenetic analysis based on shared PCGs of 24 species (22 Fabaceae and 2 Solanaceae) showed that G. sinensis is evolutionarily closer to Senna species. In general, this research will provide valuable information for the evolution of G. sinensis and provide insight into the phylogenetic relationships within the family Fabaceae.

H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

Cytosolic GDH1 degradation restricts protein synthesis to sustain tumor cell survival following amino acid deprivation.

The mTORC1 pathway plays key roles in regulating various biological processes, including sensing amino acid deprivation and driving expression of ribosomal protein (RP)-coding genes. In this study, we observed that depletion of glutamate dehydrogenase 1 (GDH1), an enzyme that converts glutamate to α-ketoglutarate (αKG), confers resistance to amino acid deprivation on kidney renal clear cell carcinoma (KIRC) cells. Mechanistically, under conditions of adequate nutrition, GDH1 maintains RP gene expression in a manner dependent on its enzymatic activity. Following amino acid deprivation or mTORC1 inhibition, GDH1 translocates from mitochondria to the cytoplasm, where it becomes ubiquitinated and degraded via the E3 ligase RNF213. GDH1 degradation reduces intracellular αKG levels by more than half and decreases the activity of αKG-dependent lysine demethylases (KDMs). Reduced KDM activity in turn leads to increased histone H3 lysine 9 and 27 methylation, further suppressing RP gene expression and preserving nutrition to support cell survival. In summary, our study exemplifies an economical and efficient strategy of solid tumor cells for coping with amino acid deficiency, which might in the future be targeted to block renal carcinoma progression.

Yorkie ensures robust tissue growth in Drosophila ribosomal protein mutants.

Heterozygosity of ribosomal protein genes causes a variety of developmental abnormalities in humans, which are collectively known as ribosomopathies, yet the underlying mechanisms remain elusive. Here, we analyzed Drosophila Minute (M)/+ mutants, a group of mutants heterozygous for ribosomal protein genes that exhibit a characteristic thin-bristle phenotype. We found that, although M/+ flies develop essentially normal wings, simultaneous deletion of one copy of the Hippo pathway effector yki resulted in severe wing growth defects. These defects were caused by JNK-mediated cell death in the wing pouch via Eiger/TNF signaling. The JNK activation in M/+, yki/+ wing discs required the caspase Dronc, which is normally blocked by DIAP1. Notably, heterozygosity of yki reduced DIAP1 expression in the wing pouch, leading to elevation of Dronc activity. Dronc and JNK formed a positive-feedback loop that amplifies Dronc activation, leading to apoptosis. Our observations suggest a mechanism of robust tissue growth whereby tissues with reduced ribosomal protein prevent ectopic apoptosis via Yki activity.

Transcriptional control of ribosome biogenesis in yeast: links to growth and stress signals.

Ribosome biogenesis requires prodigious transcriptional output in rapidly growing yeast cells and is highly regulated in response to both growth and stress signals. This minireview focuses on recent developments in our understanding of this regulatory process, with an emphasis on the 138 ribosomal protein genes (RPGs) themselves and a group of >200 ribosome biogenesis (RiBi) genes whose products contribute to assembly but are not part of the ribosome. Expression of most RPGs depends upon Rap1, a pioneer transcription factor (TF) required for the binding of a pair of RPG-specific TFs called Fhl1 and Ifh1. RPG expression is correlated with Ifh1 promoter binding, whereas Rap1 and Fhl1 remain promoter-associated upon stress-induced down regulation. A TF called Sfp1 has also been implicated in RPG regulation, though recent work reveals that its primary function is in activation of RiBi and other growth-related genes. Sfp1 plays an important regulatory role at a small number of RPGs where Rap1–Fhl1–Ifh1 action is subsidiary or non-existent. In addition, nearly half of all RPGs are bound by Hmo1, which either stabilizes or re-configures Fhl1–Ifh1 binding. Recent studies identified the proline rotamase Fpr1, known primarily for its role in rapamycin-mediated inhibition of the TORC1 kinase, as an additional TF at RPG promoters. Fpr1 also affects Fhl1–Ifh1 binding, either independently or in cooperation with Hmo1. Finally, a major recent development was the discovery of a protein homeostasis mechanism driven by unassembled ribosomal proteins, referred to as the Ribosome Assembly Stress Response (RASTR), that controls RPG transcription through the reversible condensation of Ifh1.

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and invivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how invivo tissue-specific phenotypes emerge in ribosomopathies.