Bone Marrow Cells from Patients with Shwachman-Diamond Syndrome Abnormally Express Ribosomal Protein and Ribosomal Biogenesis-Related Genes.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3303-3303
Author(s):  
Piya Rujkijyanont ◽  
Joseph Beyene ◽  
Yigal Dror
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Rna Processing ◽  
Real Time Pcr ◽  
Ribosomal Proteins ◽  
Ribosomal Biogenesis ◽  
Rrna Processing ◽  
Rrna Transcription ◽  
Shwachman Diamond Syndrome ◽  
Marrow Cells

Abstract Background and rational: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by varying degrees of cytopenia and high propensity for myelodysplastic syndrome and acute leukemia. SBDS, the gene associated with SDS, has recently been identified and is postulated to play a role in ribosomal biogenesis and RNA processing, but its functions are still unknown. Defects in ribosomal biogenesis can be characterized by abnormal synthesis of rRNA synthesis or ribosomal proteins or both. Determining the mRNA expression pattern of the various RP genes in SBDS deficient cells will help deciphering the role of SBDS in ribosomal biogenesis. Objectives: To determine whether the primary SDS marrow cells which carry homozygous SBDS mutations abnormally express genes which code for ribosomal proteins (RP) or for proteins that are involved in its transcription. Methods: Total RNA from marrow cells from 9 SDS patients who had hypocellular marrow with normal differential and no malignant transformation and 7 healthy age-matched donors of bone marrows for transplantation was extracted. RNA was labeled and hybridized to Affymetrix HG_U133_Plus2.0 GeneChip. Data were pre-processed using robust multichip analysis (RMA) and differentially expressed genes were identified with permutation-based methods. False discovery rate (FDR)-adjusted p-values were used to rank genes and cluster analysis grouped genes and samples. T-statistic values were used to screen for differentially expressed RP-related genes. Real-time PCR was performed to confirm differential expression of genes found by oligonucleotide microarray. Results: Of the 38,500 genes on the HG_133_Plus2.0 we analyzed 375 known ribosomal protein and RNA processing-related genes. Interestingly, there were differences in the expression pattern of the RP genes, suggesting differential regulation of these genes in Sbds-deficient cells. Interestingly, despite uniform decrease in RP gene expression in reduced cell growth conditions, only 27 of the 85 RP genes were downregulated. Downregulation of representative 2 genes was confirmed by real-time PCR. Further, one of the RP genes, RPL27L was upregulated. This gene, which is a target of p53, has a non-ribosomal function and lead to accelerated apoptosis. It is noteworthy that several genes involved in mRNA transcription such as GABPA and YY1were downregulated without dysregulation of genes involved in mRNA degradation, suggesting that the downregulation of the RP gene expression is at the transcription level. In addition to dysregulation of the RP mRNA we also found dysregulation of genes involved in rRNA transcription (e.g. MKI67IP) and pre rRNA processing (e.g. FBL). Conclusions: SBDS-deficiency results in dysregulation of selective group of RP genes as well as genes related to rRNA processing and rRNA transcription. Future studies should focus on the mechanism of the abnormal expression as well as its biological consequences.

Leukemia-Related Gene Expression of Bone Marrow Cells from Patients with Shwachman-Diamond Syndrome at the Pre-Leukemic Phase.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3433-3433
Author(s):  
Piya Rujkijyanont ◽  
Joseph Beyene ◽  
Kuiru Wei ◽  
Yigal Dror
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Real Time ◽  
Differential Expression ◽  
Real Time Pcr ◽  
Myeloid Leukemia ◽  
P Values ◽  
Shwachman Diamond Syndrome ◽  
Acute Myeloid ◽  
Marrow Cells

Abstract Background: Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure disorder characterized by varying degrees of cytopenia and a high propensity for myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) in up to 36% of the patients by the age of 30 years. Although the gene associated with SDS, SBDS, has recently been identified, its function, the link with MDS/AML and the mechanism for the development of MDS/AML in SDS is unclear and the molecular events occurring during transformation haven’t been yet identified. It is likely that several events occur many years before overt transformation occurs, and might be identifiable by comprehensive analysis. Objectives: To use oligonucleotide microarray to identify leukemogenic gene expression before overt transformation, which can explain a propensity for MDS/AML. Methods: Total RNA from marrow cells from 9 SDS patients and 7 healthy age-matched donors of bone marrows for transplantation was extracted, labeled and hybridized to Affymetrix HG_U133_Plus2.0 GeneChip. Data were pre-processed using robust multichip analysis (RMA) and differentially expressed genes were identified with permutation-based methods. False discovery rate (FDR)-adjusted p-values were used to rank genes and cluster analysis grouped genes and samples. Real-time PCR was performed to confirm differential expression of genes found by microarray. Results: Of the 38,500 genes on the HG_133_Plus2.0 we analyzed 52 known leukemia-related genes. We identified several genes with small FDR-adjusted p-values. Clustering of arrays resulted in two clusters that clearly separated patients from controls. Interestingly among the leukemia-related genes, the most differentially expressed gene (T=4.2) was ARHGEF12, a member of the Rho GEF family. Rho GEFs are oncogenes; many of them can transform NIH 3T3 cells into a malignant phenotype by altering expression and activation of Rho GTPases. ARHGEF12 is mapped at 11q23, telomeric to MLL, and is a novel MLL fusion partner in acute myeloid leukemia. Real time PCR after normalization against beta-actin confirmed statistically higher expression of the ARHGEF12 (p=0.03) in SDS marrow cells. In addition to ARHGEF12, we have found striking expression changes in several other genes, related to MDS/AML including TAL1, whose differential expression was also confirmed by real-time PCR. Conclusions: SDS marrow cells exhibit abnormal gene expression pattern, which might results in continuous stimulation favoring evolution or progression of malignant clones. Additional molecular and cytogenetic events are likely necessary for the malignant process to be irreversible and complete. Although analysis of whole marrow cells may not enable the detection of genes with lower differential expression between SDS and normal, it may still assist identifying molecular pathways involved in leukemogenesis. This is critically important when studying marrow failure disorders as obtaining sufficient amount of RNA from purified cell population is largely impossible.

Gene Expression Changes in Bone Marrow Cells from Diamond-Blackfan Anemia Patients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 720-720 ◽  
Author(s):  
Hanna T. Gazda ◽  
Despina Sanoudou ◽  
Alvin T. Kho ◽  
Jan M. Zaucha ◽  
Colin A. Sieff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Ribosomal Protein ◽  
Cell Population ◽  
Bone Marrow Cells ◽  
Principal Component ◽  
Fold Change ◽  
Ribosomal Protein Genes ◽  
Diamond Blackfan Anemia ◽  
Marrow Cells

Abstract Diamond-Blackfan anemia is usually characterized by anemia, absence or insufficiency of erythroid precursors in bone marrow, growth retardation and diverse congenital anomalies that are present in approximately half of patients, indicating that DBA is a broad disorder of development. Mutations of RPS19 are found in approximately 25% of DBA patients. There is good evidence for a second DBA gene, located on chromosome 8, and further genetic heterogeneity of the disease is likely. The aim of this study is to determine the most disturbed molecular pathways in DBA patients, based on gene expression changes in bone marrow cells. Knowing these pathways will possibly enable us to decipher the pathogenic mechanisms of DBA and find other genes involved in the disease. Bone marrow cells from 6 normal individuals and 3 DBA patients with RPS19 mutations, currently in remission, were FACS separated into 3 populations: primitive (P), erythroid (E) and myeloid (M) containing CD34+CD71-CD45RA-, CD34+CD71hiCD45RA- and CD34+CD71lowCD45RA+ cells, respectively. The purity of each sorted population was >97%. As a control for cell sorting accuracy, methylcellulose assay demonstrated that the P populations were highly enriched in primitive BFU-E and CFU-GEMM colonies, the E populations gave rise to BFU-E and CFU-E colonies in more than 90% of the CFCs, while more than 99% colonies from M populations were CFU-G, CFU-M and CFU-GM. RNA targets from these three FACS sorted cellular subsets was hybridized to Affymetrix HG-U133A chips (>22,000 probe sets). The data from all 27 samples were analyzed by hierarchical clustering and Principal Component Analysis, and each cell population was also studied separately. All pairwise comparisons among 27 datasets showed correlations with r=0.86–0.99. Hierarchical clustering identified three major specimen clusters, perfectly overlapping with the three different cell populations under study. Principal Component 1 and 2 separated the three studied subgroups P, E, and M. In each cell population analysis, 3 patient samples were compared to 6 control samples using 1)Significance Analysis of Microarrays with fold change 2 or greater and false discovery rate 1%, 2)Geometric Fold Change analysis and 3)Filter on Fold Change GeneSpring application (arithmetic analysis). All fold change analyses revealed the most significantly changed transcripts in patients vs. control individuals in E (45 upregulated and 184 downregulated) and P populations. The most changed genes in E subgroup were apoptosis related genes, namely TNFRSF10B and TNFRSF6 (CD95/Fas), upregulated in patients 10 and 3 fold, respectively. Other most changed genes were cancer related and genes involved in developmental processes and nucleic acid binding. Additionally, several ribosomal protein genes, namely RPL10L, RPL28, RPL36, RPL13, RPL27a and RPL37a were significantly underexpressed in P and E populations of DBA patients. All three analyses showed that RPL10L, RPL28 and RPL36 are underexpressed in the M population. This finding indicates that ribosomal protein genes are closely co-regulated and that RPS19 protein abnormalities result in downregulation of the additional ribosomal protein genes in both erythroid and nonerythroid cells in DBA patients.

scholarly journals Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

2000 ◽  
Vol 11 (11) ◽  
pp. 3777-3789 ◽  
Author(s):  
Tracy Stage-Zimmermann ◽  
Ute Schmidt ◽  
Pamela A. Silver
Keyword(s):  
Ribosomal Protein ◽  
Nuclear Export ◽  
Ribosomal Proteins ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Factors Affecting ◽  
Processing Factors

In Saccharomyces cerevisiae, the 60S ribosomal subunit assembles in the nucleolus and then is exported to the cytoplasm, where it joins the 40S subunit for translation. Export of the 60S subunit from the nucleus is known to be an energy-dependent and factor-mediated process, but very little is known about the specifics of its transport. To begin to address this problem, an assay was developed to follow the localization of the 60S ribosomal subunit inS. cerevisiae. Ribosomal protein L11b (Rpl11b), one of the ∼45 ribosomal proteins of the 60S subunit, was tagged at its carboxyl terminus with the green fluorescent protein (GFP) to enable visualization of the 60S subunit in living cells. A panel of mutant yeast strains was screened for their accumulation of Rpl11b–GFP in the nucleus as an indicator of their involvement in ribosome synthesis and/or transport. This panel included conditional alleles of several rRNA-processing factors, nucleoporins, general transport factors, and karyopherins. As predicted, conditional alleles of rRNA-processing factors that affect 60S ribosomal subunit assembly accumulated Rpl11b–GFP in the nucleus. In addition, several of the nucleoporin mutants as well as a few of the karyopherin and transport factor mutants also mislocalized Rpl11b–GFP. In particular, deletion of the previously uncharacterized karyopherin KAP120 caused accumulation of Rpl11b–GFP in the nucleus, whereas ribosomal protein import was not impaired. Together, these data further define the requirements for ribosomal subunit export and suggest a biological function for KAP120.

scholarly journals Autoregulation of yeast ribosomal proteins discovered by efficient search for feedback regulation

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Basab Roy ◽  
David Granas ◽  
Fredrick Bragg ◽  
Jonathan A. Y. Cher ◽  
Michael A. White ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Binding Sites ◽  
Ribosomal Proteins ◽  
Feedback Regulation ◽  
Ribosomal Protein Genes ◽  
Efficient Search ◽  
Fold Reduction

AbstractPost-transcriptional autoregulation of gene expression is common in bacteria but many fewer examples are known in eukaryotes. We used the yeast collection of genes fused to GFP as a rapid screen for examples of feedback regulation in ribosomal proteins by overexpressing a non-regulatable version of a gene and observing the effects on the expression of the GFP-fused version. We tested 95 ribosomal protein genes and found a wide continuum of effects, with 30% showing at least a 3-fold reduction in expression. Two genes, RPS22B and RPL1B, showed over a 10-fold repression. In both cases the cis-regulatory segment resides in the 5’ UTR of the gene as shown by placing that segment of the mRNA upstream of GFP alone and demonstrating it is sufficient to cause repression of GFP when the protein is over-expressed. Further analyses showed that the intron in the 5’ UTR of RPS22B is required for regulation, presumably because the protein inhibits splicing that is necessary for translation. The 5’ UTR of RPL1B contains a sequence and structure motif that is conserved in the binding sites of Rpl1 orthologs from bacteria to mammals, and mutations within the motif eliminate repression.

scholarly journals A novel stress response pathway regulates rRNA biogenesis

2020 ◽  
Author(s):  
Witold Szaflarski ◽  
Mateusz Sowiński ◽  
Marta Leśniczak ◽  
Sandeep Ojha ◽  
Anaïs Aulas ◽  
...  
Keyword(s):  
Stress Response ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Protein Translation ◽  
Rrna Processing ◽  
Stress Response Pathway ◽  
Cellular Translation ◽  
Response To Stress ◽  
Ribosomal Protein Synthesis

ABSTRACTProduction of ribosomes is an energy-intensive process owing to the intricacy of these massive macromolecular machines. Each human ribosome contains 80 ribosomal proteins and four non-coding RNAs. Accurate assembly requires precise regulation of protein and RNA subunits. In response to stress, the integrated stress response (ISR) rapidly inhibits global translation. How rRNA is coordinately regulated with the rapid inhibition of ribosomal protein synthesis is not known. Here we show that stress specifically inhibits the first step of rRNA processing. Unprocessed rRNA is stored within the nucleolus, and, when stress resolves, it re-enters the ribosome biogenesis pathway. Retention of unprocessed rRNA within the nucleolus aids in the maintenance of this organelle. This response is independent of the ISR or inhibition of cellular translation but represents an independent stress-response pathway that we term Ribosome Biogenesis Stress Response (RiBiSR). Failure to coordinately regulate ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils a novel stress response pathway that aims at conserving energy, preserving the nucleolus, and prevents further stress by regulation of rRNA processing.

scholarly journals Ribosomal proteins: mutant phenotypes by the numbers and associated gene expression changes

Open Biology ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 200114
Author(s):  
Michael Polymenis
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Homo Sapiens ◽  
Ribosome Profiling ◽  
Ribosomal Protein Genes ◽  
Future Studies ◽  
Protein Mutants ◽  
Structural Parts ◽  
Mutant Phenotypes

Ribosomal proteins are highly conserved, many universally so among organisms. All ribosomal proteins are structural parts of the same molecular machine, the ribosome. However, when ribosomal proteins are mutated individually, they often lead to distinct and intriguing phenotypes, including specific human pathologies. This review is an attempt to collect and analyse all the reported phenotypes of each ribosomal protein mutant in several eukaryotes ( Saccharomyces cerevisiae , Caenorhabditis elegans , Drosophila melanogaster , Danio rerio , Mus musculus , Homo sapiens ). These phenotypes were processed with unbiased computational approaches to reveal associations between different phenotypes and the contributions of individual ribosomal protein genes. An overview of gene expression changes in ribosomal protein mutants, with emphasis on ribosome profiling studies, is also presented. The available data point to patterns that may account for most of the observed phenotypes. The information presented here may also inform future studies about the molecular basis of the phenotypes that arise from mutations in ribosomal proteins.

scholarly journals Translational initiation factor expression and ribosomal protein gene expression are repressed coordinately but by different mechanisms in murine lymphosarcoma cells treated with glucocorticoids.

1989 ◽  
Vol 9 (9) ◽  
pp. 3679-3684 ◽  
Author(s):  
S Huang ◽  
J W Hershey
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Protein Gene ◽  
Initiation Factor ◽  
Mrna Levels ◽  
Translational Initiation ◽  
Ribosomal Protein Synthesis ◽  
Translational Initiation Factor

P1798 murine lymphosarcoma cells cease to proliferate upon exposure to 10(-7) M dexamethasone and exhibit a dramatic inhibition of rRNA and ribosomal protein synthesis (O. Meyuhas, E. Thompson, Jr., and R. P. Perry, Mol. Cell Biol. 7:2691-2699, 1987). These workers demonstrated that ribosomal protein synthesis is regulated primarily at the level of translation, since dexamethasone did not alter mRNA levels but shifted the mRNAs from active polysomes into inactive messenger ribonucleoproteins. We have examined the effects of dexamethasone on the biosynthesis of initiation factor proteins in the same cell line. The relative protein synthesis rates of eIF-4A and eIF-2 alpha were inhibited by about 70% by the hormone, a reduction comparable to that for ribosomal proteins. The mRNA levels of eIF-4A, eIF-4D, and eIF-2 alpha also were reduced by 60 to 70%, indicating that synthesis rates are proportional to mRNA concentrations. Analysis of polysome profiles showed that the average number of ribosomes per initiation factor polysome was only slightly reduced by dexamethasone, and little or no mRNA was present in messenger ribonucleoproteins. The results indicate that initiation factor gene expression is coordinately regulated with ribosomal protein synthesis but is controlled primarily by modulating mRNA levels rather than mRNA efficiency.

scholarly journals Knockdown of the Ribosomal Protein eL29 in Mammalian Cells Leads to Significant Changes in Gene Expression at the Transcription Level

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1228 ◽  
Author(s):  
Alexander V. Gopanenko ◽  
Alena V. Kolobova ◽  
Maria I. Meschaninova ◽  
Alya G. Venyaminova ◽  
Alexey E. Tupikin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ribosomal Protein ◽  
Mammalian Cells ◽  
Ribosomal Proteins ◽  
Protein A ◽  
Hek293 Cells ◽  
Transcription Level ◽  
Noticeable Effect ◽  
Cellular Processes ◽  
The One

An imbalance in the synthesis of ribosomal proteins can lead to the disruption of various cellular processes. For mammalian cells, it has been shown that the level of the eukaryote-specific ribosomal protein eL29, also known as the one interacting with heparin/heparan sulfate, substantially affects their growth. Moreover, in animals lacking this protein, a number of anatomical abnormalities have been observed. Here, we applied next-generation RNA sequencing to HEK293 cells transfected with siRNAs specific for the mRNA of eL29 to determine what changes occur in the transcriptome profile with a decrease in the level of the target protein. We showed that an approximately 2.5-fold decrease in the content of eL29 leads to statistically significant changes in the expression of more than a thousand genes at the transcription level, without a noticeable effect on cell viability, rRNA level, and global translation. The set of eL29-dependent genes included both up-regulated and down-regulated ones, among which there are those previously identified as targets for proteins implicated in oncogenesis. Thus, our findings demonstrate that an insufficiency of eL29 in mammalian cells causes a significant reorganization of gene expression, thereby highlighting the relationship between the cellular balance of eL29 and the activities of certain genes.

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