rRNA Expression Research Articles

BMP-7 enhances ribosomal RNA levels in chondrocytes via BAPX1-NKX3.2

Purpose: During osteoarthritis (OA) progression the articular chondrocyte undergoes a phenotypic switch in which the chondrocyte acquires a catabolic and hypertrophy-like state. Bone morphogenetic protein (BMP)-7 (or OP-1) is known for its anti-catabolic and pro-anabolic properties in cartilage repair and in OA chondrocytes. In its anabolic state the chondrocyte’s metabolism and protein synthesis are up-regulated. In order to meet this higher demand of protein synthesis, it is expected that the translational capacity of the chondrocyte is increased after exposure to BMP-7. The cellular availability of maturated ribosomal RNAs (rRNA) is rate-limiting in the assembly of ribosomes and previously it has been shown that BMP-7 treatment resulted in increased expression levels of bagpipe homeobox homolog 1 (BAPX-1/NKX3.2). We therefore hypothesize that BMP-7 enhances the translational capacity of articular chondrocytes via BAPX-1/NKX3.2-dependent synthesis of rRNAs. Methods: OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty. SW1353 cells and OA HACs were exposed to BMP-7 (1 nM) and expression levels of rRNAs (18S, 5.8S, 28S) rRNA processing snoRNAs (RMRP and U3), a crucial co-factor in rRNA transcription (UBF-1) and BAPX-1/NKX3.2 were determined by RT-qPCR (and immunoblotting for BAPX-1/NKX3.2). BAPX-1/NKX3.2 overexpression and knockdown were achieved via transfection of a FLAG-BAPX-1/NKX3.2 plasmid or a BAPX-1/NKX3.2 siRNA duplex. Human OA cartilage explants from total knee arthroplasty were exposed to BMP-7 (1 nM) and gene expression levels of rRNAs were measured via qPCR. Alterations in rRNA gene transcription were determined by transfecting SW1353 cells and OA HACs with a 47S promoter reporter plasmid (Cypridina luciferase) to determine transcriptional activity of the 47S pre-rRNA gene. Translational capacity of the cells was directly determined via puromycilation assays. Results: BMP-7 treatment resulted in increased 18S and 5.8S rRNA levels, increased UBF-1, RMRP and U3 expression. This correlated with increased BAPX-1/NKX3.2 mRNA and protein expression. Overexpression of BAPX-1/NKX3.2 resulted in increased rRNA expression levels and the reciprocal knockdown of BAPX-1/NKX3.2 resulted in decreased rRNA expression levels. Exposure of OA cartilage explants to BMP-7 confirmed our in vitro data (increase of 18S, 5.8S, UBF-1, RMRP, U3 and BAPX-1 expression levels). Treatment of chondrocytic cells with BMP-7 increased 47S pre-rRNA transcription as well as overall translational capacity. Conclusions: Here we show that BMP-7 induces increased cellular levels of maturated rRNAs, with concomitant induction of factors involved in the transcription and maturation of rRNAs. This process is directly influenced by BAPX-1/NKX3.2 in similar ways as BMP-7. In line with these results, 47S pre-rRNA transcription is increased after exposure to BMP-7, as well as the overall translational capacity. In future research the functionality of increased transcriptional activity of the 47S pre-rRNA gene will be determined by quantification of ribosome levels after BMP-7 exposure. Our data provide important novel insight into the mechanism behind the anabolic properties of BMP-7 and may provide a new molecular cue to target the chondrocyte phenotype in OA.

A recurrent de novo missense mutation in UBTF causes developmental neuroregression.

UBTF (upstream binding transcription factor) exists as two isoforms; UBTF1 regulates rRNA transcription by RNA polymerase 1, whereas UBTF2 regulates mRNA transcription by RNA polymerase 2. Herein, we describe 4 patients with very similar patterns of neuroregression due to recurrent de novo mutations in UBTF (GRCh37/hg19, NC_000017.10: g.42290219C > T, NM_014233.3: c.628G > A) resulting in the same amino acid change in both UBTF1 and UBTF2 (p.Glu210Lys [p.E210K]). Disease onset in our cohort was at 2.5 to 3 years and characterized by slow progression of global motor, cognitive and behavioral dysfunction. Notable early features included hypotonia with a floppy gait, high-pitched dysarthria and hyperactivity. Later features included aphasia, dystonia, and spasticity. Speech and ambulatory ability were lost by the early teens. Magnetic resonance imaging showed progressive generalized cerebral atrophy (supratentorial > infratentorial) with involvement of both gray and white matter. Patient fibroblasts showed normal levels of UBTF transcripts, increased expression of pre-rRNA and 18S rRNA, nucleolar abnormalities, markedly increased numbers of DNA breaks, defective cell-cycle progression, and apoptosis. Expression of mutant human UBTF1 in Drosophila neurons was lethal. Although no loss-of-function variants are reported in the Exome Aggregation Consortium (ExAC) database and Ubtf-/- is early embryonic lethal in mice, Ubtf+/- mice displayed only mild motor and behavioral dysfunction in adulthood. Our data underscore the importance of including UBTF E210K in the differential diagnosis of neuroregression and suggest that mainly gain-of-function mechanisms contribute to the pathogenesis of the UBTF E210K neuroregression syndrome.

A recurrent de novo missense mutation in UBTF causes developmental neuroregression.

UBTF (upstream binding transcription factor) exists as two isoforms; UBTF1 regulates rRNA transcription by RNA polymerase 1, whereas UBTF2 regulates mRNA transcription by RNA polymerase 2. Herein, we describe 4 patients with very similar patterns of neuroregression due to recurrent de novo mutations in UBTF (GRCh37/hg19, NC_000017.10: g.42290219C > T, NM_014233.3: c.628G > A) resulting in the same amino acid change in both UBTF1 and UBTF2 (p.Glu210Lys [p.E210K]). Disease onset in our cohort was at 2.5 to 3 years and characterized by slow progression of global motor, cognitive and behavioral dysfunction. Notable early features included hypotonia with a floppy gait, high-pitched dysarthria and hyperactivity. Later features included aphasia, dystonia, and spasticity. Speech and ambulatory ability were lost by the early teens. Magnetic resonance imaging showed progressive generalized cerebral atrophy (supratentorial > infratentorial) with involvement of both gray and white matter. Patient fibroblasts showed normal levels of UBTF transcripts, increased expression of pre-rRNA and 18S rRNA, nucleolar abnormalities, markedly increased numbers of DNA breaks, defective cell-cycle progression, and apoptosis. Expression of mutant human UBTF1 in Drosophila neurons was lethal. Although no loss-of-function variants are reported in the Exome Aggregation Consortium (ExAC) database and Ubtf−/− is early embryonic lethal in mice, Ubtf+/− mice displayed only mild motor and behavioral dysfunction in adulthood. Our data underscore the importance of including UBTF E210K in the differential diagnosis of neuroregression and suggest that mainly gain-of-function mechanisms contribute to the pathogenesis of the UBTF E210K neuroregression syndrome.

PWP1 promotes nutrient-responsive expression of 5S ribosomal RNA.

ABSTRACTPWP1 is a chromatin binding protein with an important role in animal growth control downstream of mTOR-mediated nutrient sensing. PWP1 has been shown to control tissue growth by promoting the transcription of 5.8S, 18S and 28S ribosomal RNAs (rRNAs) by RNA polymerase I (Pol I). Concomitantly with Pol I, RNA Polymerase III (Pol III) contributes to ribosome biogenesis by transcribing 5S rRNA in the nucleoplasm. Pol III activity is also closely controlled by nutrient-dependent signaling, however, how the activities of Pol I and Pol III are coordinated in response to nutrient-derived signals remains insufficiently understood. Experiments in Drosophila larvae and human cells reported here show that PWP1 associates with the chromatin at the 5S rDNA loci and is needed for nutrient-induced expression of 5S rRNA. Similar to the Pol I target rDNAs, PWP1 epigenetically maintains 5S rDNA in a transcription competent state. Thus, as a common regulator of Pol I and Pol III, PWP1 might contribute to coordinated control of ribosomal gene expression in response to nutrition.This article has an associated First Person interview with the first author of the paper.

Isolation of Scardovia wiggsiae using real-time polymerase chain reaction from the saliva of children with early childhood caries.

This study aimed to quantitatively assess the levels of Scardovia wiggsiae in caries-free and early childhood caries (ECC)- and severe ECC (SECC)-affected children using real-time polymerase chain reaction (RT-PCR). Forty-five children aged <71 months were randomly recruited from the Outpatient Clinic at the Department of Pedodontics and Preventive Dentistry at Subharti Dental College and Hospital, Meerut, India. Fifteen children suffering from ECC, 15 with SECC, and 15 children without ECC were enrolled in the study. About 1-2 mL of unstimulated saliva was collected and subjected to microbial analysis using RT-PCR. The SECC group (n = 15) was found to have significantly higher mean relative 16s rRNA expression of S. wiggsiae (3.67) than both ECC (n = 15) and controls (n = 15) (1.69 and 0.85, respectively). S. wiggsiae was detected in 86.7% of the SECC and 60% ECC group and was detected negligibly in the control (caries free) group. The correlation of decayed, missing, or filled surface levels with 16s rRNA levels showed significant positive correlation with 16S rRNA in both ECC and SECC patients. Salivary levels of S. wiggsiae were significantly associated with ECC in children. S. wiggsiae represents a new frontier in the microbial etiology of ECC. This may lead to the development of new antimicrobial agents targeted to this organism and improve the treatment of ECC.

MicroRNA-542 Promotes Mitochondrial Dysfunction and SMAD Activity and Is Elevated in Intensive Care Unit-acquired Weakness.

Loss of skeletal muscle mass and function is a common consequence of critical illness and a range of chronic diseases, but the mechanisms by which this occurs are unclear. To identify microRNAs (miRNAs) that were increased in the quadriceps of patients with muscle wasting and to determine the molecular pathways by which they contributed to muscle dysfunction. miRNA-542-3p/5p (miR-542-3p/5p) were quantified in the quadriceps of patients with chronic obstructive pulmonary disease and intensive care unit-acquired weakness (ICUAW). The effect of miR-542-3p/5p was determined on mitochondrial function and transforming growth factor-β signaling in vitro and in vivo. miR-542-3p/5p were elevated in patients with chronic obstructive pulmonary disease but more markedly in patients with ICUAW. In vitro, miR-542-3p suppressed the expression of the mitochondrial ribosomal protein MRPS10 and reduced 12S ribosomal RNA (rRNA) expression, suggesting mitochondrial ribosomal stress. miR-542-5p increased nuclear phospho-SMAD2/3 and suppressed expression of SMAD7, SMURF1, and PPP2CA, proteins that inhibit or reduce SMAD2/3 phosphorylation, suggesting that miR-542-5p increased transforming growth factor-β signaling. In mice, miR-542 overexpression caused muscle wasting, and reduced mitochondrial function, 12S rRNA expression, and SMAD7 expression, consistent with the effects of the miRNAs in vitro. Similarly, in patients with ICUAW, the expression of 12S rRNA and of the inhibitors of SMAD2/3 phosphorylation were reduced, indicative of mitochondrial ribosomal stress and increased transforming growth factor-β signaling. In patients undergoing aortic surgery, preoperative levels of miR-542-3p/5p were positively correlated with muscle loss after surgery. Elevated miR-542-3p/5p may cause muscle atrophy in intensive care unit patients through the promotion of mitochondrial dysfunction and activation of SMAD2/3 phosphorylation.

Robertsonian translocation 13/14 associated with rRNA genes overexpression and intellectual disability

BackgroundThe Robertsonian translocations inherited from parents with a normal phenotype are often discovered through children with pathogenesis. The exact causes of pathologies in children with clinical manifestations are often unknown and vary greatly in the reported cases: uniparental disomy, de novo rearrangements, changes in methylation patterns and gene expression, including ribosomal genes. Aim of the studyMolecular-cytogenetic investigation of a clinical case of intellectual disability. Material and methodsGTG-banding, Ag-NOR staining, fluorescent in situ hybridization, PCR, real-time PCR. ResultsWe describe a family case of a translocation rob (13; 14) and elevated rRNA expression in the proband with developmental delay and in his phenotypically normal mother. We show the loss of the p-arms of original chromosomes and the absence of NORs on the derived chromosome. The whole-chromosome uniparental disomy is excluded. ConclusionThe translocated chromosome in the proband was most likely inherited from the mother and did not come about de novo with normal chromosomes 13 and 14 being obtained from the father. The cause of the pathogenesis in the proband still remains unknown. We hypothesize that it could be caused by impaired imprinting manifesting in altered methylation levels of loci on the derivative chromosome.

Arsenic Contamination in Agricultural Soil Reduces Metabolic Activity of Total and Free-Living Nitrogen-Fixing Bacteria as Revealed by Real-Time qPCR

ABSTRACTLaboratory microcosms were used to assess the impact of arsenic (As) contamination on agricultural soil bacterial activity focusing free-living nitrogen fixers for three months. Periodically collected microcosm samples were analyzed by RT–qPCR following extraction of total RNA and cDNA preparation for assessing the metabolically active bacterial population. RT–qPCR data showed the gradual increase of 16S rRNA and nifH gene expression, and relative activity of diazotrophs in the enriched soil bacterial consortia under short time As exposure up to 20 ppm and 10 ppm, respectively. A similar trend of these variables was also noticed but up to 1 ppm As when incubating the bulk soil for the same duration. Reduced bacterial activity was noticed at higher concentration of As although in short time exposure. Extending the As exposure time, the bacterial activities in both enriched consortia and bulk soil were decreased. Although, the relative activity of diazotrophs in enriched consortia was increased in presence of 10 ppm As, the same was decreased in bulk soil when exposed to >1 ppm As for long time indicating susceptibility of nitrogen fixer to As contamination in soil. PCA of the data obtained also indicated a negative correlation between As concentrations and diazotrophic activity.

PYGOPUS2 expression in prostatic adenocarcinoma is a potential risk stratification marker for PSA progression following radical prostatectomy

AimsProstate cancer (PrCa) is the most frequently diagnosed non-cutaneous cancer in men. Without clear pathological indicators of disease trajectory at diagnosis, management of PrCa is challenging, given its wide-ranging manifestation...

Alteration of rRNA gene copy number and expression in patients with intellectual disability and heteromorphic acrocentric chromosomes

BackgroundIntellectual disability (ID) is an important medical and social problem that can be caused by different genetic and environmental factors. One such factor could be rDNA amplification and changes in rRNA expression and maturation. Aim of the studyThe aim of the present study was to investigate rRNA levels in patients with heteromorphism of the p-arms of acrocentric chromosomes bearing nucleolus organizer regions compared to a healthy control group. Material and methodsFrequencies of p-arms enlargements in patients with ID and in healthy people were analyzed by G-banding screening. rRNA gene copy numbers on affected acrocentric chromosomes in peripheral blood lymphocytes were evaluated in ID patients and healthy bearers using FISH, and in immortalized lymphocytes of one patient – using FISH and real time PCR. Simultaneously, levels of 18S, 28S and 5,8S rRNA in both groups by means of qRT-PCR were investigated. ResultsNo difference in acrocentric chromosome heteromorphism frequency in patients versus the healthy group were found. However, we found an amplification of rDNA, a significant elevation in 28S and 5.8S rRNA expression and changes in the 28S/18S rRNA ratio in ID patients compared to healthy controls. At the same time, FISH appeared to be not reliable enough for copy number evaluation, but RT-PCR showed rDNA copy changes in heteromorphic cells compared to normal. ConclusionOur findings indicate a loss of the correct regulation of rDNA activity and processing after amplification. This could disturb the ribosomal apparatus and thus lead to intellectual disability via at least two mechanisms.

Small nucleoli are a cellular hallmark of longevity

Animal lifespan is regulated by conserved metabolic signalling pathways and specific transcription factors, but whether these pathways affect common downstream mechanisms remains largely elusive. Here we show that NCL-1/TRIM2/Brat tumour suppressor extends lifespan and limits nucleolar size in the major C. elegans longevity pathways, as part of a convergent mechanism focused on the nucleolus. Long-lived animals representing distinct longevity pathways exhibit small nucleoli, and decreased expression of rRNA, ribosomal proteins, and the nucleolar protein fibrillarin, dependent on NCL-1. Knockdown of fibrillarin also reduces nucleolar size and extends lifespan. Among wildtype C. elegans, individual nucleolar size varies, but is highly predictive for longevity. Long-lived dietary restricted fruit flies and insulin-like-peptide mutants exhibit small nucleoli and fibrillarin expression, as do long-lived dietary restricted and IRS1 knockout mice. Furthermore, human muscle biopsies from individuals who underwent modest dietary restriction coupled with exercise also display small nucleoli. We suggest that small nucleoli are a cellular hallmark of longevity and metabolic health conserved across taxa.

Human papillomavirus type 8 E7 protein binds nuclear myosin 1c and downregulates the expression of pre-rRNA.

Our aim was to search for new cellular binding partners for the E6 and E7 oncogenes of beta human papillomaviruses (HPV), whose direct role in skin carcinogenesis has not been thoroughly investigated. By employing glutathione S-transferase pulldown and coimmunoprecipitation, we identified nuclear myosin 1c as a binding partner of HPV 8 E7 protein. As nuclear myosin 1c is an essential component of the RNA polymerase I transcription complex, we studied the effects of HPV 8 E7 protein expression on ribosomal RNA (rRNA) expression. Here we show that the activity of RNA polymerase I is decreased and that pre-rRNA expression is consequently reduced due to HPV 8 E7 expression. However, the expression levels of mature cytoplasmic 18S and 28S rRNA are retained. We propose that by relieving their resources from the energy-consuming process of rRNA transcription, HPV 8 E7 expressing cells might support more efficient virus replication in the differentiating epithelium.

Pre-45s rRNA promotes colon cancer and is associated with poor survival of CRC patients

One characteristic of cancer cells is the abnormally high rate of cell metabolism to sustain their enhanced proliferation. However, the behind mechanism of this phenomenon is still elusive. Here we find that enhanced precursor 45s ribosomal RNA (pre-45s rRNA) is one of the core mechanisms in promoting the pathogenesis of colorectal cancer (CRC). Pre-45s rRNA expression is significantly higher in primary CRC tumor tissues samples and cancer cell lines compared with the non-tumorous colon tissues, and is associated with tumor sizes. Knockdown of pre-45s rRNA inhibits G1/S cell-cycle transition by stabilizing p53 through inducing murine double minute 2 (MDM2) and ribosomal protein L11 (RpL11) interaction. In addition, we revealed that high rate of cancer cell metabolism triggers the passive release of calcium ion from endoplasmic reticulum to the cytoplasm. The elevated calcium ion in the cytoplasm activates the signaling cascade of calcium/calmodulin-dependent protein kinase II, ribosomal S6 kinase (S6K) and ribosomal S6K (CaMKII-S6K-UBF). The activated UBF promotes the transcription of rDNA, which therefore increases pre-45s rRNA. Disruption of CaMKII-S6K-UBF axis by either RNAi or pharmaceutical approaches leads to reduction of pre-45s rRNA expression, which subsequently suppresses cell proliferation in colon cancer cells by causing cell-cycle arrest. Knockdown of APC activates CaMKII-S6K-UBF cascade and thus enhances pre-45s rRNA expression. Moreover, the high expression level of pre-45s rRNA is associated with poor survival of CRC patients in two independent cohorts. Our study identifies a novel mechanism in CRC pathogenesis mediated by pre-45s rRNA and a prognostic factor of pre-45s rRNA in CRC patients.

Both absolute and relative quantification of urinary mRNA are useful for non-invasive diagnosis of acute kidney allograft rejection.

Urinary mRNA analysis with three-gene set (18S rRNA, CD3ε, and IP-10) has been suggested as a non-invasive biomarker of acute rejection (AR) in kidney transplant recipients using quantitative real-time PCR (qPCR). Application of droplet digital PCR (ddPCR), which has been suggested to provide higher sensitivity, accuracy, and absolute quantification without standard curves, could be a useful method for the quantifying low concentration of urinary mRNA. We investigated the urinary expression of these three genes in Korean patients with kidney transplantation and also evaluated the usefulness of ddPCR. 90 urine samples were collected at time of allograft biopsy in kidney recipients (n = 67) and from patients with stable renal function more than 10 years (n = 23). Absolute quantification with both PCR system showed significant higher mRNA levels of CD3ε and IP-10 in AR patients compared with stable transplants (STA), but there was no difference in 18S rRNA expression across the patient groups. To evaluate discrimination between AR and STA, ROC curve analyses of CTOT-4 formula yielded area under the curve values of 0.72 (95% CI 0.60–0.83) and 0.77 (95% CI 0.66–0.88) for qPCR and ddPCR, respectively. However, 18S normalization of absolute quantification and relative quantification with 18S showed better discrimination of AR from STA than those of the absolute method. Our data indicate that ddPCR system without standard curve would be useful to determine the absolute quantification of urinary mRNA from kidney transplant recipients. However, comparative method also could be useful and convenient in both qPCR and ddPCR analysis.

Methylation of the ribosomal RNA gene promoter is associated with aging and age-related decline.

SummaryThe transcription of ribosomal RNA genes (rDNA) is subject to epigenetic regulation, as it is abrogated by the methylation of CpG dinucleotides within their promoter region. Here, we investigated, through Sequenom platform, the age‐related methylation status of the CpG island falling into the rDNA promoter in 472 blood samples from 20‐ to 105‐year‐old humans and in different tissues (blood, heart, liver, kidney, and testis) of 15 rats 3–96 weeks old. In humans, we did not find a consistently significant correlation between CpG site methylation and chronological age. Furthermore, the methylation levels of one of the analyzed CpG sites were negatively associated with both cognitive performance and survival chance measured in a 9‐year follow‐up study. We consistently confirmed such result in a replication sample. In rats, the analysis of the homologous region in the tissues revealed the existence of increased methylation in old rats. rRNA expression data, in both humans and rats, were consistent with observed methylation patterns, with a lower expression of rRNA in highly methylated samples. As chronological and biological ages in rats of a given strain are likely to be much closer to each other than in humans, these results seem to provide the first evidence that epigenetic modifications of rDNA change over time according to the aging decline. Thus, the methylation profile of rDNA may represent a potential biomarker of aging.

Stimulation of Pol III‐dependent 5S rRNA and U6 snRNA gene expression by AP‐1 transcription factors

RNA polymerase III transcribes structurally diverse group of essential noncoding RNAs including 5S ribosomal RNA (5SrRNA) and U6 snRNA. These noncoding RNAs are involved in RNA processing and ribosome biogenesis, thus, coupling Pol III activity to the rate of protein synthesis, cell growth, and proliferation. Even though a few Pol II-associated transcription factors have been reported to participate in Pol III-dependent transcription, its activation by activator protein 1 (AP-1) factors, c-Fos and c-Jun, has remained unexplored. Here, we show that c-Fos and c-Jun bind to specific sites in the regulatory regions of 5S rRNA (type I) and U6 snRNA (type III) gene promoters and stimulate their transcription. Our chromatin immunoprecipitation studies suggested that endogenous AP-1 factors bind to their cognate promoter elements during the G1/S transition of cell cycle apparently synchronous with Pol III transcriptional activity. Furthermore, the interaction of c-Jun with histone acetyltransferase p300 promoted the recruitment of p300/CBP complex on the promoters and facilitated the occupancy of Pol III transcriptional machinery via histone acetylation and chromatin remodeling. The findings of our study, together, suggest that AP-1 factors are novel regulators of Pol III-driven 5S rRNA and U6 snRNA expression with a potential role in cell proliferation.

Phosphorylation of WHIRLY1 by CIPK14 Shifts Its Localization and Dual Functions in Arabidopsis

Plastid-to-nucleus retrograde signaling is critical for normal growth and development in plants. The dual-function and dual-located ssDNA binding protein WHIRLY1 (WHY1) has been proposed to coordinate the retrograde signaling from plastids to the nucleus. However, the regulatory mechanism governing the functional switch of WHY1 for mediating plastid-to-nucleus retrograde signaling remains unknown. Here, we report that the Calcineurin B-Like-Interacting Protein Kinase14 (CIPK14) interacts with and phosphorylates WHY1 in Arabidopsis. Phosphorylation of WHY1 results in increased accumulation in the nucleus and enhanced binding with the promoter of WRKY53, which encodes a key transcription factor regulating leaf senescence in Arabidopsis. Transgenic plants overexpressing CIPK14 showed an increased nuclear isoform but decreased plastid isoform of WHY1, among which 95% of transgenic lines showed the stay-green phenotype and 5% of lines showed the variegated pale-green phenotype. Interestingly, the phenotypes of both types of transgenic plants could be recovered by overexpression of plastid-form WHY1. In contrast, knockdown of CIPK14 caused early senescence and even seedling-lethal phenotypes along with elevated expression of senescence-related genes such as WRKY53, SAG12, and NDHF but decreased expression of MER11, RAD50, and POR genes, which could be rescued by overexpression of CIPK14 but not by overexpressing plastid-form or nuclear-form WHY1; the stay-green plants overexpressing CIPK14 showed reduced expression of WRKY53, SAG12, NDHF, and large plastid rRNA. Consistently, the accumulation of nuclear-form WHY1 was significantly reduced in the CIPK14 knockdown lines, resulting in a low ratio of nuclear-/plastid-form WHY1. Taken together, our results demonstrate that CIPK14 regulates the phosphorylation and organellar distributions of WHY1 and pinpoint that CIPK14 may function as a cellular switch between leaf senescence and plastid development for coordinating the intercellular signaling in Arabidopsis.

Unraveling The Role of Helicases in Nonfunctional rRNA Decay

rRNAs are essential, long‐lived, and highly abundant, comprising up to 90% of total cellular RNA. However, when structurally intact rRNAs in Saccharomyces cerevisiae are functionally compromised, they are instead rapidly degraded through a quality control pathway termed nonfunctional ribosomal RNA decay (NRD). Currently, two mechanistically distinct types of NRD are known: 18S NRD and 25S NRD. 18S NRD is translation dependent and removes rRNAs with loss of function mutations in the small subunit decoding site. The 18S NRD mechanism involves the translation factor‐like protein Dom34 and its binding partner Hbs1. On the other hand, 25S NRD, which occurs prior to subunit joining, is translation independent and removes nonfunctional rRNAs containing mutations in the large subunit peptidyl transferase center. The 25S NRD mechanism involves polyubiquination of proteins associated with a nonfunctional 25S rRNA through the E3 ligase components Rtt101 and Mms1. While some of the proteins that are involved in either 18S or 25S NRD are known, we have not yet elucidated the complete molecular mechanism of NRD. Since RNA helicases have been implicated in other RNA degradation pathways, we hypothesize that one or more helicases may be important for either or both NRD mechanisms. S. cerevisiae contains 44 different helicases, 14 of which are non‐essential. Thus, we are initially investigating whether any of these 14 helicases are involved in NRD using single gene deletion yeast strains. We transformed BY4741 and the 14 non‐essential helicase knock‐out strains with various rRNA expression plasmids that encode for functional and/or nonfunctional versions of the 18S and 25S rRNAs. These plasmid‐derived rRNAs also contain short, unique sequence tags that allows for specific detection of these rRNAs in the presence of total cellular rRNA. We measured the growth phenotypes of these transformed strains at various temperatures in liquid cultures. At standard temperatures, we see a slow growth phenotype in some of the deletion strains transformed with either nonfunctional 18S or 25S rRNA expression plasmids. Since some helicase deletion strains exhibit either cold‐ or temperature‐sensitivities, we are currently measuring growth phenotypes at these normally restrictive temperatures. In addition, we are measuring the rRNA expression levels in these deletion strains using an RT‐qPCR assay to quantify the level of NRD activity. We hope this work will provide insight into the role of helicases in RNA degradation mechanisms and contribute to our understanding of NRD.Support or Funding InformationThis work is supported by the Howard Hughes Medical Institute.

Treatment with Streptozotocin (STZ) causes hypoglycemia and alters the stability of reference genes for real‐time PCR analysis in the liver of channel catfish

Genetically selected channel catfish for increased growth often develops phenotype similar to human obesity, suggesting that channel catfish may be a model organism to investigate human metabolic disorders. Streptozotocin (STZ) treatment induces hyperglycemia in mammals and some fish species. However, the effects of STZ treatment on blood glucose level as well as other physiological parameters in channel catfish have never been examined in detail. In this study, we tested the hypothesis that treatment with STZ affects body weight, blood glucose level, and hepatic expression of house‐keeping genes. Juvenile channel catfish (28.3 ± 2g) were treated with 0, 3.6, 36, 180, or 360 mg/kg STZ intraperitoneally on day 0 (n=4 fish/treatment). On day 7, blood glucose level was measured and blood, liver, muscle and brain samples were collected. Expression of five common housekeeping genes (beta‐macroglobulin, alpha‐tubulin, 18s ribosomal RNA, beta‐actin, and GAPDH) were measured using real‐time PCR. The weight of fish did not change between days 0 and 7 among all treatments. Blood glucose levels were lower in fish treated with 180 or 360 mg/kg STZ compared to fish treated with 0 mg/kg STZ. Treatment with STZ at higher doses altered expression of 4 housekeeping genes. Expression of beta‐macroglobulin, alpha‐tubulin, and beta‐actin mRNA was higher in fish treated with higher doses of STZ compared to 0 mg/kg STZ. In contrast, expression of GAPDH mRNA tended to be less in 360 mg/kg STZ than 0 mg/kg STZ. Expression of 18s rRNA was not affected by STZ treatment. Rather than causing hyperglycemia, treatment with high doses of STZ decreased blood glucose level in channel catfish, suggesting a difference in glucose metabolism exists between channel catfish and humans.Support or Funding InformationThis study is supported by APS IOSP fellowship program and Kansas Idea Network of Biomedical Research Excellence (P20GM10348).

RdRP-synthesized antisense ribosomal siRNAs silence pre-rRNA via the nuclear RNAi pathway.

Expression of rRNA affects cell growth and proliferation, but mechanisms that modulate rRNA levels are poorly understood. We conducted a genetic screen for factors that negatively regulate generation of endogenous short interfering RNA (endo-siRNA) in Caenorhabditis elegans and identified a suppressor of siRNA (susi-1) and antisense ribosomal siRNAs (risiRNAs). risiRNAs show sequence complementary to 18S and 26S rRNAs and require RNA-dependent RNA polymerases (RdRPs) for their production. They act through the nuclear RNA interference (RNAi) pathway to downregulate pre-rRNA. Stress stimuli, including low temperature and UV irradiation, induced the accumulation of risiRNAs. SUSI-1 is a homolog of the human DIS3L2 exonuclease involved in 3'-5' degradation of oligouridylated RNAs. In susi-1 mutant and in low temperature-treated animals, 3'-tail oligouridylated 26S rRNA accumulated. The injection of oligouridylated rRNA elicited nuclear accumulation of NRDE-3. Our findings identify a new subset of 22G-RNAs that regulate pre-rRNA expression and a mechanism to maintain rRNA homeostasis.