Ribosomal RNA Metabolism Research Articles

SBDS Gene Mutation Increases ROS Production and Causes DNA Damage as Well as Oxidation of Mitochondrial Membranes in the Murine Myeloid Cell Line 32Dcl3.

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disease caused by mutation in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SDS has a variety of clinical features, including exocrine pancreatic insufficiency and hematological dysfunction. Neutropenia is the most common symptom in patients with SDS. SDS is also associated with an elevated risk of developing myelodysplastic syndromes and acute myeloid leukemia. The SBDS protein is involved in ribosome biogenesis, ribosomal RNA metabolism, stabilization of mitotic spindles and cellular stress responses, yet the function of SBDS in detail is still incompletely understood. Considering the diverse function of SBDS, the effect of SBDS seems to be different in different cells and tissues. In this study, we established myeloid cell line 32Dcl3 with a common pathogenic SBDS variant on both alleles in intron 2, 258 + 2T > C, and examined the cellular damage that resulted. We found that the protein synthesis was markedly decreased in the mutant cells. Furthermore, reactive oxygen species (ROS) production was increased, and oxidation of the mitochondrial membrane lipids and DNA damage were induced. These findings provide new insights into the cellular and molecular pathology caused by SBDS deficiency in myeloid cells.

SBDS interacts with RNF2 and is degraded through RNF2-dependent ubiquitination

Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder caused by mutation in the Shwachman-Bodian-Diamond syndrome (SBDS) gene that has a variety of clinical features, including exocrine pancreatic insufficiency and hematological dysfunction. The SBDS protein is considered to be involved in ribosome biogenesis, ribosomal RNA metabolism, stabilization of mitotic spindles and cellular stress responses, yet the function of SBDS in detail is still incompletely understood. The multiple functions imply that certain proteins might associate with SBDS and affect its function. In this study, we identified Ring finger protein 2 (RNF2) as a candidate for the SBDS interactor by yeast two-hybrid screening. Moreover, we confirmed the interaction by GST-pull down assay using recombinant proteins and co-immunoprecipitation in HEK293T cells overexpressing RNF2. In addition, it is shown that RNF2 ubiquitinates SBDS and promotes its proteasomal degradation in HEK293T cells. These findings provide new insights into the regulation of SBDS.

Mutational Landscape of Relapsed Core-Binding Factor Acute Myeloid Leukemia (CBF-AML)

Mutational Landscape of Relapsed Core-Binding Factor Acute Myeloid Leukemia (CBF-AML)

Prokaryotic and Mitochondrial Lipids: A Survey of Evolutionary Origins.

Mitochondria and bacteria share a myriadof properties since it is believed that the powerhouses of the eukaryotic cell have evolved from a prokaryotic origin. Ribosomal RNA sequences, DNA architecture and metabolism are strikingly similar in these two entities. Proteins and nucleic acids have been a hallmark for comparison between mitochondria and prokaryotes. In this chapter, similarities (and differences) between mitochondrial and prokaryotic membranes are addressed with a focus on structure-function relationship of different lipid classes. In order to be suitable for the theme of the book, a special emphasis is reserved to the effects of bioactive sphingolipids, mainly ceramide, on mitochondrial membranes and their roles in initiating programmed cell death.

BORIS/CTCFL is an RNA-binding protein that associates with polysomes

BackgroundBORIS (CTCFL), a paralogue of the multifunctional and ubiquitously expressed transcription factor CTCF, is best known for its role in transcriptional regulation. In the nucleus, BORIS is particularly enriched in the nucleolus, a crucial compartment for ribosomal RNA and RNA metabolism. However, little is known about cytoplasmic BORIS, which represents the major pool of BORIS protein.ResultsWe show, firstly, that BORIS has a putative nuclear export signal in the C-terminal domain. Furthermore, BORIS associates with mRNA in both neural stem cells and young neurons. The majority of the BORIS-associated transcripts are different in the two cell types. Finally, by using polysome profiling we show that BORIS is associated with actively translating ribosomes.ConclusionWe have demonstrated the RNA binding properties of cellular BORIS and its association with actively translating ribosomes. We suggest that BORIS is involved in gene expression at both the transcriptional and post-transcriptional levels.

Functional ribosome biogenesis is a prerequisite for p53 destabilization: impact of chemotherapy on nucleolar functions and RNA metabolism

The production and processing of ribosomal RNA is a complex and well-coordinated nucleolar process for ribosome biogenesis. Progress in understanding nucleolar structure and function has lead to the unexpected discovery of the nucleolus as a highly sensitive sensor of cellular stress and an important regulator of the tumor suppressor p53. Inhibition of ribosomal RNA metabolism has been shown to activate a signaling pathway for p53 induction. This review elucidates the potential of classical and recently developed chemotherapeutic drugs to stabilize p53 by inhibiting nucleolar functions.

HSC Aging Epigenome: Widespread Alterations in DNA Methylation and Transcription.

AbstractAbstract 2329Age is the most important risk factor for myelodysplastic syndrome (MDS), a premalignant state that transforms into acute myelogenous leukemia in one third of cases. Indeed with normal aging, hematopoietic stem cell (HSC) regenerative potential diminishes and differentiation skews from lymphopoiesis toward myelopoiesis. The expansion in the HSC pool with aging provides sufficient but abnormal blood production, and animals experience a decline in immune function. Previous studies from our lab established that the DNA methyltransferase 3a (Dnmt3a) enables efficient differentiation by critically regulating epigenetic silencing of HSC genes (Challen et al. 2012) Interestingly, Dnmt3a expression is decreased in old HSCs, leading us to hypothesize that epigenetic changes in old HSCs may partially mimic the changes seen in Dnmt3a mutant HSCs. We propose that revealing the genome-wide DNA methylation and transcriptome signatures will lead to a greater understanding of HSC aging and MDS, which is characterized by frequent epigenetic abnormalities.In this study, we investigated genome-wide DNA methylation and transcripts by whole genome bisulfite sequencing (WGBS) and transcriptome sequencing (mRNA-seq)in young and old HSCs. For WGBS, we generated ∼600M raw reads resulting in ∼ 60 raw Gb of paired-end sequence data and aligned them to either strand of the reference genome (mm9), providing an average 40-fold sequencing depth. Globally, there was a 1.1% difference in the DNA methylation between young and old HSCs. Of these differences, 38% (172,609) of the CpG dinucleotides were hypo-methylated, and 62% (275,557) were hyper-methylated in old HSCs. To understand where the methylation changes predominantly occurred, the genome was subdivided into 77 features. Among these features, SINEs, especially Alu elements, exhibited the highest level of DNA methylation (90.94% in young HSCs, and 91.87% in old HSCs). CpG islands (CGIs) adjacent to the transcription start sites (TSS) exhibited the lowest level of DNA methylation (2.02% in young HSCs, and 2.11% in old HSCs). Interestingly strong hypo-methylation was observed in ribosomal RNA regions (68.04% in young HSCs, 59.04% in old HSCs), and hyper-methylation was observed in LINEL1 repetitive elements (88.62% in young HSCs, 90.12% in old HSCs). Moreover, the examination of differentially methylated promoters identified enrichment of developmentally important transcription factors such as Gata2, Runx1, Gfi1b, Erg, Tal1 Eto2, Cebpa and Pu.1. Additionally, we compare our ∼10,000 differentially methylation regions (DMRs, regions with clustered DNA methylation changes) with a chip-seq data set containing binding of 160 ChIP-seq analyses of hematopoietic transcription factors in different hematopoietic cells. We found significant overlaps between DMRs and transcription factor binding regions. We found DMRs which were hypermethylated showed association with differentiation-promoting Ets factors, in particular Pu.1 from a range of different blood cell types. In contrast, hypomethylated DMRs showed associations with HSC-associated transcription factors such as Scl and Gata2. Further examination of the differentially methylated gene bodies, intragenic and intergenic DMRs identified some previously noted targets for epigenetic silencing or alteration in AML and also novel transcripts including long non-coding RNAs (lincRNA) and upstream regulatory elements (URE). We found significant correlation between RNA-seq expression and DMRs within +1kb upstream of TSS. RNA-sequencing provided complementary and distinct information about HSC aging. We identified differentially expressed genes, novel RNA transcripts, differential promoter, coding sequence, and splice variant usage with age. Gene set enrichment analysis of up- and down- regulated genes, revealed ribosomal protein and RNA metabolism as critical contributors to HSC aging.In conclusion, our study marks a milestone in the mouse HSC epigenome, reporting the first complete methylome and transcriptome of pure HSC using whole-genome bisulfite sequencing and RNA-seq. These provide novel information about the magnitude and specificity of age-related epigenetic changes in a well-defined HSC population. Understanding the roles of DNA methylation and transcription in normal HSC function will allow for greater therapeutic exploitation of HSCs in the clinic. Disclosures:No relevant conflicts of interest to declare.

Structure and Functional Studies of the CS Domain of the Essential H/ACA Ribonucleoparticle Assembly Protein SHQ1

H/ACA ribonucleoprotein particles are essential for ribosomal RNA and telomerase RNA processing and metabolism. Shq1p has been identified as an essential eukaryotic H/ACA small nucleolar (sno) ribonucleoparticle (snoRNP) biogenesis and assembly factor. Shq1p is postulated to be involved in the early biogenesis steps of H/ACA snoRNP complexes, and Shq1p depletion leads to a specific decrease in H/ACA small nucleolar RNA levels and to defects in ribosomal RNA processing. Shq1p contains two predicted domains as follows: an N-terminal CS (named after CHORD-containing proteins and SGT1) or HSP20-like domain, and a C-terminal region of high sequence homology called the Shq1 domain. Here we report the crystal structure and functional studies of the Saccharomyces cerevisiae Shq1p CS domain. The structure consists of a compact anti-parallel beta-sandwich fold that is composed of two beta-sheets containing four and three beta-strands, respectively, and a short alpha-helix. Deletion studies showed that the CS domain is required for the essential functions of Shq1p. Point mutations in residues Phe-6, Gln-10, and Lys-80 destabilize Shq1p in vivo and induce a temperature-sensitive phenotype with depletion of H/ACA small nucleolar RNAs and defects in rRNA processing. Although CS domains are frequently found in co-chaperones of the Hsp90 molecular chaperone, no interaction was detected between the Shq1p CS domain and yeast Hsp90 in vitro. These results show that the CS domain is essential for Shq1p function in H/ACA snoRNP biogenesis in vivo, possibly in an Hsp90-independent manner.

Arabidopsis thaliana mutants reveal a role for CSP41a and CSP41b, two ribosome-associated endonucleases, in chloroplast ribosomal RNA metabolism

A proteomic analysis of Chlamydomonas reinhardtii 70S ribosomes identified two proteins, RAP38 and RAP41, which associate in stoichiometric amounts with intact ribosomes. In this work we show results that suggest the Arabidopsis thaliana homologs, CSP41b and CSP41a, participate in ribosomal RNA metabolism. Csp41a-1 and csp41b-1 single mutants show little phenotype, while the loss of both proteins is lethal. Plants homozygous for the csp41b-1 mutation and heterozygous for the csp41a-1 mutation (csp41b-1/csp41a-1*) fail to accumulate CSP41b and show a marked reduction in the levels of CSP41a. These mutants have reduced chlorophyll content, grow slower and over-accumulate 23S precursor rRNAs compared to their wild-type (WT) siblings, whereas other rRNAs or mRNAs are unaffected. Chloroplast polysome assembly is reduced in csp41b-1/csp41a-1* mutants, which also contain increased amounts of pre-ribosomal particles compared to mature 70S ribosomes. Our results also indicate that CSP41b associates with pre-ribosomal particles in vivo. In vitro, the pattern of 23S precursors and mature rRNAs is altered upon incubation with recombinant CSP41a and CSP41b. Taken together, these results suggest that CSP41a and CSP41b have a role in chloroplast ribosomal RNA metabolism, most likely acting in the final steps of 23S rRNA maturation.

Interaction of the plant glycine-rich RNA-binding protein MA16 with a novel nucleolar DEAD box RNA helicase protein from Zea mays.

The maize RNA-binding MA16 protein is a developmentally and environmentally regulated nucleolar protein that interacts with RNAs through complex association with several proteins. By using yeast two-hybrid screening, we identified a DEAD box RNA helicase protein from Zea mays that interacted with MA16, which we named Z. maysDEAD box RNA helicase 1 (ZmDRH1). The sequence of ZmDRH1 includes the eight RNA helicase motifs and two glycine-rich regions with arginine-glycine-rich (RGG) boxes at the amino (N)- and carboxy (C)-termini of the protein. Both MA16 and ZmDRH1 were located in the nucleus and nucleolus, and analysis of the sequence determinants for their cellular localization revealed that the region containing the RGG motifs in both proteins was necessary for nuclear/nucleolar localization The two domains of MA16, the RNA recognition motif (RRM) and the RGG, were tested for molecular interaction with ZmDRH1. MA16 specifically interacted with ZmDRH1 through the RRM domain. A number of plant proteins and vertebrate p68/p72 RNA helicases showed evolutionary proximity to ZmDRH1. In addition, like p68, ZmDRH1 was able to interact with fibrillarin. Our data suggest that MA16, fibrillarin, and ZmDRH1 may be part of a ribonucleoprotein complex involved in ribosomal RNA (rRNA) metabolism.

The roles of endonucleolytic cleavage and exonucleolytic digestion in the 5'-end processing of S. cerevisiae box C/D snoRNAs.

Small nucleolar RNAs (snoRNAs) play important roles in ribosomal RNA metabolism. In Saccharomyces cerevisiae, box C/D snoRNAs are synthesized from excised introns, polycistronic precursors, or independent transcription units. Previous studies have shown that only a few independently transcribed box C/D snoRNAs are processed at their 5' end. Here we describe 12 additional independently transcribed box C/D snoRNAs that undergo 5'-end processing. 5' Extensions found in the precursors of these snoRNAs contain cleavage sites for Rnt1p, the S. cerevisiae homolog of RNase III, and unprocessed precursors accumulate in vivo in the absence of Rnt1p. Rnt1p cleavage products were identified in vivo when the 5' --> 3' exonucleases Xrn1p and Rat1p are inactivated (xrn1delta rat1-1) and in vitro using model RNA substrates and recombinant Rnt1p. Some of these snoRNAs show increased levels of unprocessed precursors when the rnt1Delta deletion is combined to the xrn1delta rat1-1 mutation, suggesting that these exonucleases participate in the 5' processing or the degradation of the snoRNA precursors. Unprocessed precursors are not significantly destabilized in the absence of the trimethylguanosine capping enzyme Tgs1p, suggesting that a 5' monomethyl cap is sufficient to ensure stabilization of these precursors. These results demonstrate that the majority of independently transcribed box C/D snoRNAs from the yeast genome undergo 5'-end processing and that the Rnt1p endonuclease and the Xrn1p and Rat1p 5' --> 3'exonucleases have partially redundant functions in the 5'-end processing of these snoRNAs.

The Shq1p·Naf1p Complex Is Required for Box H/ACA Small Nucleolar Ribonucleoprotein Particle Biogenesis

Small nucleolar ribonucleoprotein particles (snoRNPs) are essential cofactors in ribosomal RNA metabolism. Although snoRNP composition has been thoroughly characterized, the biogenesis process of these particles is poorly understood. We have identified two proteins from the yeast Saccharomyces cerevisiae, Yil104c/Shq1p and Ynl124w/Naf1p, which are essential and required for the stability of box H/ACA snoRNPs. Depletion of either Shq1p or Naf1p leads to a dramatic and specific decrease in box H/ACA snoRNA levels in vivo. A severe concomitant defect in ribosomal RNA processing is observed, consistent with the depletion of this family of snoRNAs. Shq1p and Naf1p show nuclear localization and interact with Nhp2p and Cbf5p, two core proteins of mature box H/ACA snoRNPs. Shq1p and Naf1p form a complex, but they are not strongly associated with box H/ACA snoRNPs. We propose that Shq1p and Naf1p are involved in the early biogenesis steps of box H/ACA snoRNP assembly.

Ribosomal RNA metabolism in macrophages.

Macrophages are ubiquitous cells critical for host defense (Wiltrout and Varesio 1987; Varesio 1985b). Metchnikoff’s original report in the late nineteenth century first suggested that phagocytes are the body’s prime detectors of foreign invaders; the functions of macrophages have since been delineated, initially at a cellular level and more recently at a molecular level.

Monoclonal antibodies against a nucleolar protein from differentiating chick muscle cells.

Two monoclonal antibodies, NORM and NORA, bind specifically to a 37 000 molecular weight protein in total protein extracts of chick skeletal muscle cell cultures. They have been used to follow changes in the amount and distribution of this protein during terminal differentiation and the associated cessation of cell division. Immunofluorescence microscopy shows that the antigen is found almost exclusively in the nucleolus in interphase cells and is dispersed in the cytoplasm during mitosis. Low doses of actinomycin D cause segregation of nucleoli into DNA fibrils and ribonucleoprotein granules, and the antigen is clearly associated with the latter. High doses of actinomycin D cause shrinkage of the nucleolus and the antigen is dispersed within the nucleoplasm, but not released into the cytoplasm. The amount and distribution of antigen is similar in all cell types (myoblasts, myotubes and fibroblasts) and there are no major changes during muscle differentiation. From known changes in ribosomal RNA metabolism during myogenesis and after actinomycin D treatment, we suggest that the 37 000 Mr protein is involved in some post-transcriptional aspect of ribosome production and that its concentration and distribution may be related to the concentration of ribosomal RNA in the nucleolus.

Analysis of ribosomal RNA metabolism during development of Dictyostelium discoideum

Using double labelling protocols we have compared the developmental metabolism of ribosomal subunits fabricated during vegetative growth of Dictyostelium discoideum with those accumulated during subsequent development. Unlike vegetative growth when ribosomal subunits are accumulated in equal amounts, early development is characterized by the accumulation of approximately twice as much large as small subunit. The unusual paucity of small subunit was not due to selective sequestration by the nucleus as previously thought nor cytoplasmic degradation. Ribosomal subunits, whether synthesized during growth or development, were degraded at equivalent rates by the developing cell indicating the lack of preferential conservation at the degradative level.

Muscle growth and development in chick embryos—thyroidal influence on ribosomal RNA metabolism

Thyroidal influence on muscle ribosomal RNA (rRNA) metabolism was examined during late embryogenesis of the chick, when there is normally considerable growth. RNA polymerase I activity of leg muscle nuclei was 52% of the control value at 20 days of incubation, following 1 week of methimazole (MMI) treatment ( P < 0.01). The enzyme activity for nuclei isolated from muscle of 19-day, MMI-treated embryos at 48 hr after the administraton of 300 ng of thyroxine (T 4) was 31% greater than for embryos receiving MMI alone ( P < 0.01). At 24 hr after the administration of [ 14C]orotic acid, and 23.5 hr after the administration of 300 ng of T 4, the amount of 14C incorporated into the RNA from leg muscles of 18-day, MMI-treated embryos was 29% higher than for MMI-treated embryos receiving Ringer's solution at the time of hormone injection ( P < 0.02); T 4 did not influence incorporation into total RNA at 3, 6, or 12 hr after administration of labeled precursor. The average specific activity of the cytoplasmic ribosomal-polysomal RNA fraction was increased approximately 43% in the T 4-treated embryos at 24 hr after administration of [ 14C]orotic acid, and 23.5 hr after hormone, compared to that of MMI controls ( P < 0.01), and the specific activity of the combined 18-S and 28-S rRNA, isolated from the ribosomal-polysomal RNA fraction, was increased 39% in the T 4-treated embryos ( P < 0.01). Data on the 24-hr, cumulative incorporation of [ 14C]orotic acid into perchloric acid-soluble components of leg muscle indicate that the increased incorporation of labeled precursor into RNA was not due to greater uptake of [ 14C]orotic acid into muscle or a higher specific activity of uridine nucleotide precursors. The present report of thyroidal influence on muscle rRNA metabolism supports earlier observations which indicated that thyroid function is important to muscle growth during late embryogenesis in the chick.

Interferon-mediated effect on ribosomal RNA metabolism

Ribosomal RNA (rRNA) has been shown to be involved in the binding of bacterial messenger RNA (mRNA) and an analogous 18 S rRNA · mRNA complex has been reported in eukaryotic systems. Thus, qualitative changes in host rRNA may be involved in the development of the interferon mediated antiviral state, a process thought to involve the inability of host ribosomes to bind and recognize viral mRNA. Data are reported which suggest that trisomy 21 human fibroblasts respond to human interferon with a marked reduction in cytoplasmic rRNA. [ 3H]Uridine was used to radioactively label the polysomal RNAs for 24 h beginning 12 h after interferon addition. Subsequent sucrose gradient analysis of the phenol or SDS-extracted RNA revealed that the reduction in radioactive rRNA was nearly complete for the 28 S rRNA. In contrast, considerable residual uridine incorporation was found in the 18 S rRNA species. Corollary data suggesting a net increase in mRNA synthesis and a net decrease in protein synthesis are reported.

Discoordination of ribosomal RNA metabolism during metabolic shifts of Spirodela plants

The effects of metabolic shifts on nucleic acid syntheses have been widely studied in prokaryotes, but not in plants because of a paucity of suitable systems. Spirodela (Duckweed) was thus used to ascertain the response of the nucleocytoplasmic (nc) and plastid ribosomal RNA metabolisms to partial and total carbon deprivation. The 0.56 × 10 6 M m plastid rRNA is the one species of RNA most affected by metabolic shifts; unlike other species, its appearance is delayed by deprivation and it appears more rapidly than other species on transfer from dark to light. The data suggest a discoordination between the transcription and processing of plastid ribosomal precursors. Incorporation into all nc and plastid rRNAs was severely reduced and all rRNA precursors accumulated in green plants that were completely deprived of carbon by transferring to the dark, without sucrose. The amounts of nc and plastid precursors transcribed readjusted to the reduced amounts processed to mature RNA only after long periods in the dark with sucrose. This delay involved the formation of new colorless plants. Less plastid RNAs, compared to nc RNAs are found in the dark steady state.

Ribosomal RNA metabolism in synchronized plasmacytoma cells

Ribosome synthesis and metabolism has been studied in a plasmacytoma cell line synchronized by isoleucine deprivation. Ribosomal RNA (rRNA) was characterized by gel electrophoresis. The rate of ribosome synthesis (as measured by the appearance of labelled rRNA in the cytoplasm) varied greatly during the cell cycle. It was low during the G l phase, increased rapidly during the S phase, remained high during part of the G 2 phase, and dropped to a minimum during mitosis. A slowdown in the increasing rate of RNA synthesis was observed during the middle of the S phase. No significant decrease in the total nucleotide pool per cell could be observed during the S phase. The accumulation of RNA (as determined by absorbance measurements) was highest during the S and G 2 phases. Pulse labelling of rRNA and pulse chase experiments demonstrated that newly synthesized ribosomal subunits entered into free polysomes to the highest extent during the S phase. The percentage of membrane-bound polysomes of total polysomes increased during the G 1 phase, as did the percentage of labelled rRNA in the membrane-bound fraction.

Studies on messenger and ribosomal RNA synthesis in plant tissue cultures induced to undergo synchronus cell division.

Messenger and ribosomal RNA metabolism was tudied in a plant tissue culture system: cells from the quiescent tubers of Helianthus tuberosus (Jerusalem artichoke) were induced to divide synchronously and dedeifferentiate by excision and culture of explants in nutrient medium. Large accumulations of ribosomal RNA and protein started early in the 20-h lag-period preceding the first division. In pulse-labelling experiments, two types of polydisperse messenger-like RNAs were detected one with and one without a poly(adenylic acid) sequence. In the first 2 h of culture the two polydisperse RNA fractions were the predominant types of RNA synthesised. Ribosomal RNA synthesis was very low during the first 2 h, but acclerated later. Low concentrations of actinomycin-D strongly inhibited ribosomal RNA synthesis, but had little effect on the synthesis of transfer RNA or either type of polydisperse messenger-like RNA. In explants cultured with low concentrations of actinomycin-D ribosomal RNA accumulation was completely inhibited, but cell division and protein accumulation occurred, though at a reduced rate. It is concluded that the synthesis and accumulation of new ribosomal RNA which normally occur during culture are not required for the induction of cell division or for protein accumulation, i.e. the ribosomal RNA existing in the quiescent tuver cells can support protein accumulation and cell division induced by excision and culture. The quiescent tuber tissue is also shown to contain significant amounts of messenger-like RNA.