scholarly journals The Inherited MDS Gene DDX41 Is Required for Ribosome Biogenesis and Cell Viability

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 773-773
Author(s):  
Timothy M Chlon ◽  
Emily Stepanchick ◽  
Kwangmin Choi ◽  
Yi Zheng ◽  
Kathleen Hueneman ◽  
...  
Keyword(s):  
Cell Viability ◽  
Ribosomal Subunit ◽  
Protein Translation ◽  
Mass Spectrometry Analysis ◽  
Mouse Bone Marrow ◽  
Recipient Mouse ◽  
Rrna Processing ◽  
Small Ribosomal Subunit ◽  
Hematopoietic Stem ◽  
Congenital Diseases

Myelodysplastic syndromes (MDS) are hematopoietic stem cell (HSC) disorders in which myeloid cell differentiation is impaired, causing blood lineage cytopenias and potentially leading to acute myeloid leukemia (AML) through malignant transformation. MDS occurs in adults with a median age of 71 years, and is associated with multiple cytogenetic and genetic abnormalities in the diseased HSC. Younger patients can also have MDS as a result of underlying congenital diseases or secondary effects from cancer therapy. It has recently been discovered that some families with high rates of MDS incidence bear heterozygous inherited mutations in DDX41, a member of the DEAD box RNA helicase family of genes. These patients typically have normal hematopoietic indices into adulthood and present with MDS at a median age of 61 years, slightly younger than the general population. Inherited DDX41 mutations are always heterozygous and are typically frame-shift mutations, indicating they are likely loss of function. Approximately half of MDS patients with inherited DDX41 mutations acquire a second-hit, often R525H, in the healthy DDX41 allele in their disease clones. This mutation is also observed in 1-2% of de novo AML patients, suggesting it causes gain of function or dominant negative activity. Multiple functions have been ascribed to DDX41, such as functioning as an innate immune sensor and as an RNA splicing regulator, but its role in the pathogenesis of MDS remains unknown. We set out to model DDX41 mutations by generating conditional DDX41 knockout and R525H-knock-in mice. Combining these alleles and crossing to Rosa-Cre-ERT expressing mice allowed for tamoxifen-inducible acquisition of knockout (KO), heterozygous (HET), heterozygous knock-in (KI/+) and knock-in alone (KI/-) HSPC. The KO and KI/- HSPC were incapable of engrafting into recipient mice and underwent rapid cell cycle arrest and apoptosis, indicating that Ddx41 is required for HSPC cell viability and that the R525H mutation causes loss of the required function. In contrast, the HET and KI/+ HSPC survived and proliferated normally in culture and successfully engrafted irradiated recipient mouse bone marrow. HET and KI/+ transplanted mice had increased numbers of LSK cells, and subset of mice developed a myeloid malignancy, resembling the human disease. To determine the function of DDX41 that is critical for hematopoiesis, we performed a tandem-pulldown followed by mass spectrometry analysis to identify relevant DDX41 interacting proteins in human AML cells. We found that DDX41 interacts with multiple proteins in the small ribosomal subunit, including RPS3 and RPS14. Consistent with disruption of the assembly and function of the small ribosomal subunit, KO and KI/-HSPC exhibited rapid and robust impairment of global protein translation. Polysome profiling indicated an increase in monosomes and a decrease in polysomes in KO cells, consistent with an inability of ribosomes to initiate translation and move along the mRNA. To determine the role of the translation defect in the cell growth deficiency of DDX41-deficient cells, we treated WT, HET, and KO HSPC with the translation inhibitor puromycin and determined that KO cells were relatively more sensitive to translation inhibition, indicating that DDX41-deficient cells are specifically sensitive to further reduction in protein translation. This data supports the conclusion that the cell lethality caused by DDX41 loss is related to ribosome dysfunction. Mechanistically, we find that DDX41-deficient cells have stalled ribosomal RNA (rRNA) processing, characterized by increased unprocessed rRNA and decreased processed rRNA intermediates. In conclusion, we identify a novel function of DDX41 in regulating rRNA processing and ribosome formation that is essential for the survival and proliferation of HSPC. The loss of DDX41 may contribute to MDS as a result of impaired ribosome function, as has been previously reported in patients bearing mutations in other ribosome regulators. Disclosures Starczynowski: Kurome Therapeutics: Consultancy.

MiR-29a is Essential in Leukemic Transformation and Maintaining Hematopoietic Stem Cell Self-Renewal

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4792-4792
Author(s):  
Wenhuo Hu ◽  
James Dooley ◽  
Luisa Cimmino ◽  
Adrian Liston ◽  
Christopher Y. Park
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Protein Translation ◽  
Epigenetic Mark ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Leukemic Transformation ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract MicroRNAs are small non-coding RNAs that interfere with gene expression by degrading messenger RNAs (mRNAs) or blocking protein translation. Expression profiling studies has identified miRNAs that regulate normal and malignant hematopoietic stem cell function. Our previous studies showed that ectopic expression of miR-29a in mouse bone marrow cells induced a myeloproliferative disorder that progressed to acute myeloid leukemia (AML). Over-expression of miR-29b in AML cell lines has been reported to induce apoptosis by negatively regulating Dnmt3a. We recently found that miR-29a positively regulates hematopoietic stem cell (HSC) self-renewal and proliferation using a knockout mouse model of miR-29ab. miR-29a null mice contained significantly lower HSC numbers and miR-29a null HSCs exhibited markedly decreased reconstitution ability in both competitive and non-competitive transplantation assays. To investigate the mechanism of miR-29a action, we performed transcriptomal profiling of miR-29a null HSCs and found that miR-29a null HSCs exhibit a gene expression pattern more similar to wild-type committed progenitors than wild-type HSCs. We identified Dnmt3a as one dysregulated miR-29a target as showing increased expression in miR-29a null HSCs, and haplodeficiency of Dnmt3a partly restores miR-29a deficient HSC function. In order to test the requirement for miR-29a in myeloid leukemogenesis, we transduced miR-29a deficient Lin-c-Kit+Sca-1+ (LSK) cells with the oncogenic MLL-AF9 fusion gene, and found that the development of AML from these cells was markedly delayed. We found that Meis1, Ccna2, Hoxa5 and Hoxa9 transcripts were significantly downregulated in miR-29a null LSK cells compared to WT LSK cells, but they were similarly induced in MLL-AF9 transformed c-Kit+Mac-1+ cells. To investigate whether the epigenetic dysregulation resulting from miR-29a deletion may underlie this transformation-resistant phenotype, we examined the distribution of the active epigenetic mark, H3K79me2, in c-Kit+Mac-1+ miR-29a null cells using a ChIP-Seq assay. After analyzing H3K39me2 peaks using model-based analysis of ChIP-Seq, we identified 4281 and 3649 genes associated with this active epigenetic mark using a duplicated ChIP-Seq analysis, with an overlap of 3164 genes (66.39%). Using public available ChIP-Seq data, we compared our results with the genes associated with the H3K79me2 mark in normal immature LSK cells (9282 genes), granulocyte-macrophage progenitors (GMPs, 8556 genes), and MLL-AF9 transformed GMP cells (L-GMP, 8578 genes), and found 4234, 4111, 4046, and 4766 genes were also identified have an active H3K79me2 mark in MLL-AF9 transformed miR-29a null cells. These data indicate that miR-29a loss inactivates a large group of genes activated by the MLL-AF9 oncogene. We also found that 379 genes were associated with H3K79me2 peaks in both normal LSK and MLL-AF9 transformed miR-29a null c-Kit+Mac-1+ cells, but were absent of this epigenetic marker in L-GMP, suggesting that these genes confer self-renewal and proliferation capacities to normal HSCs. In addition, suppression of these genes are important in leukemic transformation by MLL-AF9, and finally the reactivation of these genes in miR-29a null cells compromises the leukemogenesis ability of MLL-AF9. Interestingly, out of these 379 genes, we were able to identify 18 genes that were potential miR-29a targets including Akt3, Map4k4, Dnmt3a, et al. This suggests the direct and indirect effects from miR-29a in regulating its target gene networks at transcriptional and post-transcriptional levels. Our studies found miR-29a is essential in maintaining HSC function and loss of miR-29a abrogate the leukemogenesis capacity of MLL-AF9. Disclosures No relevant conflicts of interest to declare.

RBM15-Mediated RNA Splicing Fine-Tunes Epigenetic Program through Interaction with SF3B1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4110-4110
Author(s):  
Tran Ngoc-Tung ◽  
Hairui Su ◽  
Yuheng Lu ◽  
Christina S Leslie ◽  
Xinyang Zhao
Keyword(s):  
Transcription Factors ◽  
Alternative Splicing ◽  
Binding Sites ◽  
Rna Splicing ◽  
Rna Binding ◽  
Protein Translation ◽  
Mass Spectrometry Analysis ◽  
Next Generation Sequencing Data ◽  
Hematopoietic Stem ◽  
Associated Proteins

Abstract RBM15, an RNA binding protein belongs to the SPEN family, which is evolutionally conserved from plant to mammals. The relevance of RBM15 to blood diseases came to spot light when RBM15 was discovered to be involved in chromosome translocation t(1;22) in acute megakaryoblastic leukemia. RBM15 is indispensible for the self-renewal of hematopoietic stem cells in response to stress and for megakaryocyte terminal differentiation. RBM15 knockdown results in the unchecked generation of megakaryocyte progenitors in mouse models. At the molecular level, RBM15 have been shown to be a transcriptional repressor in NOCTH signaling pathway by recruiting HDAC complexes like another family member SHARP. RBM15 interacts with transcriptional coactivator SET1/ASH2. RBM15 controls the alternative splicing of thrombopoietin receptor (MPL). However, the molecular mechanisms on how RBM15 regulates alternative RNA splicing is less known. We identified the RBM15-associated proteins with mass spectrometry analysis. Pathway analysis found that RBM15 is mainly associated with proteins involved in RNA splicing, which is also validated by immune-fluorescent microscopy with anti-RBM15 antibody. Among the RBM15-associated proteins are SF3B1 and U2AF, which are heavily mutated in myeloid dysplasia syndrome and chronic lymphocytic leukemia. The interaction between RBM15 and SF3B1 is further validated by co-immunoprecipitation assays. Furthermore, we found that RBM15-SF3B1 interaction is regulated by protein arginine methylation of RBM15. RBM15 is responsible for recruiting SF3B1 complex to the pre-mRNA intronic regions with weak U2snRNP binding sites. We did pair-end, 100bp read RNA-seq of RNA isolated from RBM15 knockdown leukemia cells, and used RBM15 antibody to immunoprecipiate sonicated RNAs to identify intronic regions bound by RBM15. Combining these next generation sequencing data, we found about 50% of the RBM15 binding sites are in the intronic regions and 45% of the RBM15 binding sites are in the 3'UTR regions. RBM15 regulates alternative splicing of a few known transcription factors involved in hematopoiesis such as GATA1, Fli1, GFIb, and RUNX1 as well as chromatin binding proteins such as BRD4, HDAC7 etc.. Therefore, RBM15-SF3B1 interaction fine-tunes the optimal dose of transcription factors needed for the generation of blood lineages in response to environmental stress. How the mutant SF3B1 proteins disrupt its interaction with RBM15 will be discussed. In addition to RBM15-mediated RNA splicing, we also found that RBM15 per se is regulated by its antisense counterpart (AS-RBM15). AS-RBM15 is a long non-coding RNA whose transcription is activated by RUNX1. AS-RBM15 promotes megakaryocyte differentiation by activating RBM15 protein translation. Therefore, RBM15 and AS-RBM15 are at the center of a RNA regulatory network, which is linked to epigenetic programs regulated by RUNX1 and GATA1. Given that both RUNX1 and SF3B1 are often mutated in myeloid dysplasia syndrome, understanding RBM15-mediated gene regulation offers much needed knowledge for understanding MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Local Role for the Small Ribosomal Subunit Primary Binder rpS5 in Final 18S rRNA Processing in Yeast

PLoS ONE ◽  
2010 ◽  
Vol 5 (4) ◽  
pp. e10194 ◽  
Author(s):  
Andreas Neueder ◽  
Steffen Jakob ◽  
Gisela Pöll ◽  
Jan Linnemann ◽  
Rainer Deutzmann ◽  
...  
Keyword(s):  
18S Rrna ◽  
Ribosomal Subunit ◽  
Rrna Processing ◽  
Small Ribosomal Subunit

Unexpected and Sustained Symmetry in the Repopulation Programs Displayed by Clonally Amplified Hematopoietic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1651-1651
Author(s):  
Brad Dykstra ◽  
David Kent ◽  
Melisa Hamilton ◽  
Merete Kristiansen ◽  
Kristin Lyons ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Adult Life ◽  
Mouse Bone Marrow ◽  
Recipient Mouse ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Over Time

Abstract Heterogeneity in progeny output by individual pluripotent hematopoietic cells is a well documented but poorly understood paradigm. Importantly, the extent to which this functional heterogeneity is pre-determined by intrinsic mechanisms that specify distinct programs, as opposed to conditions that result in a series of stochastic events, is still debated. The prospective isolation of phenotypically defined subpopulations with more restricted behaviors has lent recent support to the concept of predetermined hierarchies with preset, but alternative pathways of lineage restriction and differentiated cell output. Here we have used highly purified starting populations to compare the long-term cell output dynamics of individual multipotent repopulating cells in sublethally irradiated W41/W41 mice transplanted with single Ly-5 congenic CD45midlin−Rho−SP adult mouse bone marrow cells (158 mice) or their clonal progeny generated after 4 days in vitro in 300 ng/ml SF, 20 ng/ml IL-11 and 1 ng/ml Flt3-L (194 mice). WBC samples collected 4, 8, 12, 16, and 24 weeks post-transplant were analyzed for donor contributions to the myeloid (GM) and lymphoid (B and T) lineages. In 49 of the 158 mice (31%) and 44 of the 194 mice (23%), the cells produced in vivo contributed ≥1% of all the WBCs present at ≥16 weeks. The overall and lineage-specific contributions to the WBCs in each recipient mouse varied widely both over time post-transplant and between mice. However, examination of the ratio of the donor contributions to the myeloid and lymphoid lineages (GM:B+T) in each mouse at 16 weeks post-transplant allowed 4 patterns to be readily identified: α and β with GM:B+T ratios of ≥2 and 0.25–2, respectively; γ, with a GM:B+T ratio of <0.25 including a ≥1% contribution to both lymphoid and myeloid lineages at 16 weeks; and δ, also with a GM:B+T ratio of <0.25, but with contribution only to the lymphoid lineages at this time. Secondary transplants performed after 24 weeks showed long-term repopulation (≥16 weeks) of most recipients of type α and β progeny (10/11 and 11/12, respectively) but none of the recipients of type γ and δ progeny were repopulated (0/6 and 0/17, respectively). Interestingly, the variation over time in both the overall and lineage-specific contributions was remarkably similar in pairs of secondary recipients injected with cells from the same primary donor. In addition, the lineage contribution ratios seen in the secondary recipients tended to recapitulate that of the primary donors (i.e., α or β), and these trends remained obvious when tertiary transplants were performed. Preservation of stem cell programming was also evident from sequential analyses of multiple mice injected with aliquots of the same clones generated in vitro after 10 days from single CD45midlin−Rho−SP cells. Very similar patterns of total and lineage-specific contributions were again observed amongst the different recipients of cells from the same clones. Collectively, these findings indicate that by early adult life hematopoietic stem cells have acquired intrinsically fixed patterns of lineage specification that can be stably transmitted through many self-renewal generations.

scholarly journals Pediatric MDS and bone marrow failure-associated germline mutations in SAMD9 and SAMD9L impair multiple pathways in primary hematopoietic cells

Leukemia ◽  
2021 ◽  
Author(s):  
Melvin E. Thomas ◽  
Sherif Abdelhamed ◽  
Ryan Hiltenbrand ◽  
Jason R. Schwartz ◽  
Sadie Miki Sakurada ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Failure ◽  
Transcriptional Profiling ◽  
Hematopoietic Cells ◽  
Protein Translation ◽  
Germline Mutations ◽  
Hematopoietic Stem ◽  
Monosomy 7 ◽  
Cellular Environment ◽  
Primary Mouse

AbstractPediatric myelodysplastic syndromes (MDS) are a heterogeneous disease group associated with impaired hematopoiesis, bone marrow hypocellularity, and frequently have deletions involving chromosome 7 (monosomy 7). We and others recently identified heterozygous germline mutations in SAMD9 and SAMD9L in children with monosomy 7 and MDS. We previously demonstrated an antiproliferative effect of these gene products in non-hematopoietic cells, which was exacerbated by their patient-associated mutations. Here, we used a lentiviral overexpression approach to assess the functional impact and underlying cellular processes of wild-type and mutant SAMD9 or SAMD9L in primary mouse or human hematopoietic stem and progenitor cells (HSPC). Using a combination of protein interactome analyses, transcriptional profiling, and functional validation, we show that SAMD9 and SAMD9L are multifunctional proteins that cause profound alterations in cell cycle, cell proliferation, and protein translation in HSPCs. Importantly, our molecular and functional studies also demonstrated that expression of these genes and their mutations leads to a cellular environment that promotes DNA damage repair defects and ultimately apoptosis in hematopoietic cells. This study provides novel functional insights into SAMD9 and SAMD9L and how their mutations can potentially alter hematopoietic function and lead to bone marrow hypocellularity, a hallmark of pediatric MDS.

scholarly journals Re-Discovery of Giardiavirus: Genomic and Functional Analysis of Viruses from Giardia duodenalis Isolates

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 654
Author(s):  
Gianluca Marucci ◽  
Ilaria Zullino ◽  
Lucia Bertuccini ◽  
Serena Camerini ◽  
Serena Cecchetti ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Diarrheal Disease ◽  
Current Knowledge ◽  
Biological Significance ◽  
Giardia Duodenalis ◽  
Protozoan Parasite ◽  
Protein Translation ◽  
Mass Spectrometry Analysis ◽  
Animal Origin ◽  
Spectrometry Analysis

Giardiasis, caused by the protozoan parasite Giardia duodenalis, is an intestinal diarrheal disease affecting almost one billion people worldwide. A small endosymbiotic dsRNA viruses, G. lamblia virus (GLV), genus Giardiavirus, family Totiviridae, might inhabit human and animal isolates of G. duodenalis. Three GLV genomes have been sequenced so far, and only one was intensively studied; moreover, a positive correlation between GLV and parasite virulence is yet to be proved. To understand the biological significance of GLV infection in Giardia, the characterization of several GLV strains from naturally infected G. duodenalis isolates is necessary. Here we report high-throughput sequencing of four GLVs strains, from Giardia isolates of human and animal origin. We also report on a new, unclassified viral sequence (designed GdRV-2), unrelated to Giardiavirus, encoding and expressing for a single large protein with an RdRp domain homologous to Totiviridae and Botybirnaviridae. The result of our sequencing and proteomic analyses challenge the current knowledge on GLV and strongly suggest that viral capsid protein translation unusually starts with a proline and that translation of the RNA-dependent RNA polymerase (RdRp) occurs via a +1/−2 ribosomal frameshift mechanism. Nucleotide polymorphism, confirmed by mass-spectrometry analysis, was also observed among and between GLV strains. Phylogenetic analysis indicated the occurrence of at least two GLV subtypes which display different phenotypes and transmissibility in experimental infections of a GLV naïve Giardia isolate.

scholarly journals Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

2000 ◽  
Vol 20 (15) ◽  
pp. 5516-5528 ◽  
Author(s):  
Žaklina Strezoska ◽  
Dimitri G. Pestov ◽  
Lester F. Lau
Keyword(s):  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Weight Fraction ◽  
Rnase A ◽  
Mutant Form ◽  
Rrna Processing ◽  
Ribonucleoprotein Complexes ◽  
Nuclear Extracts ◽  
Mouse Tissues ◽  
Mouse Cells

ABSTRACT We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1Δ, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1Δ leads to cell growth arrest in the G1phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1Δ expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1Δ in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.

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