scholarly journals MicroRNAs Cause Accelerated Decay of Short-Tailed Target mRNAs

2019 ◽  
Author(s):  
Timothy J. Eisen ◽  
Stephen W. Eichhorn ◽  
Alexander O. Subtelny ◽  
David P. Bartel
Keyword(s):  
Steady State ◽  
Length Distribution ◽  
Tail Length ◽  
Translational Efficiency ◽  
List Type ◽  
Mrna Levels ◽  
Target Mrnas ◽  
Mrna Targets ◽  
Target Molecules ◽  
Nascent Mrna

SummaryMicroRNAs (miRNAs) specify recruitment of deadenylases to mRNA targets. Despite this recruitment, we find that miRNAs have almost no effect on steady-state poly(A)-tail lengths of their targets in mouse fibroblasts, which motivates acquisition of pre-steady-state measurements of the effects of miRNAs on tail lengths, mRNA levels, and translational efficiencies. Effects on translational efficiency are minimal compared to effects on mRNA levels—even for newly transcribed target mRNAs. Effects on target mRNA levels accumulate as the mRNA population approaches steady state, whereas effects on tail lengths peak for recently transcribed target mRNAs and then subside. Computational modeling of this phenomenon reveals that miRNAs cause not only accelerated deadenylation of their targets but also accelerated decay of short-tailed target molecules. This unanticipated effect of miRNAs largely prevents short-tailed target mRNAs from accumulating despite accelerated target deadenylation. The net result is a nearly imperceptible change to the steady-state tail-length distribution of targeted mRNAs.HighlightsmiRNAs cause accelerated decay of short-tailed target moleculesThis accelerated decay largely prevents accumulation of short-tailed target mRNAsmiRNAs are similarly effective on short-lived and long-lived target mRNAsIn 3T3 cells, miRNA effects on translation are negligible—even for nascent mRNA

scholarly journals MicroRNA and their target mRNAs change expression in whole blood of patients after intracerebral hemorrhage

2019 ◽  
Vol 40 (4) ◽  
pp. 775-786 ◽  
Author(s):  
Xiyuan Cheng ◽  
Bradley P Ander ◽  
Glen C Jickling ◽  
Xinhua Zhan ◽  
Heather Hull ◽  
...  
Keyword(s):  
Intracerebral Hemorrhage ◽  
Whole Blood ◽  
Killer Cell ◽  
Transcriptome Profiling ◽  
Rna Degradation ◽  
Differentially Expressed ◽  
Mrna Levels ◽  
Target Mrnas ◽  
Mrna Targets ◽  
Differentially Expressed Mirnas

Previous studies showed changes in mRNA levels in whole blood of rats and humans, and in miRNA in whole blood of rats following intracerebral hemorrhage (ICH). Thus, this study assessed miRNA and their putative mRNA targets in whole blood of humans following ICH. Whole transcriptome profiling identified altered miRNA and mRNA levels in ICH patients compared to matched controls. Target mRNAs of the differentially expressed miRNAs were identified, and functional analysis of the miRNA-mRNA targets was performed. Twenty-nine miRNAs (22 down, 7 up) and 250 target mRNAs (136 up, 114 down), and 7 small nucleolar RNA changed expression after ICH compared to controls (FDR < 0.05, and fold change ≥ |1.2|). These included Let7i, miR-146a-5p, miR210-5p, miR-93-5p, miR-221, miR-874, miR-17-3p, miR-378a-5p, miR-532-5p, mir-4707, miR-4450, mir-1183, Let-7d-3p, miR-3937, miR-4288, miR-4741, miR-92a-1-3p, miR-4514, mir-4658, mir-3689d-1, miR-4760-3p, and mir-3183. Pathway analysis showed regulated miRNAs/mRNAs were associated with toll-like receptor, natural killer cell, focal adhesion, TGF-β, phagosome, JAK-STAT, cytokine–cytokine receptor, chemokine, apoptosis, vascular smooth muscle, and RNA degradation signaling. Many of these pathways have been implicated in ICH. The differentially expressed miRNA and their putative mRNA targets and associated pathways may provide diagnostic biomarkers as well as point to therapeutic targets for ICH treatments in humans.

Salubrinal Increases Human Fetal Hemoglobin Production Through a Post-Transcriptional Mechanism

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 250-250
Author(s):  
Cynthia K Hahn ◽  
Christopher H. Lowrey
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Steady State ◽  
Stress Response ◽  
Fetal Hemoglobin ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Globin Mrna ◽  
Hbf Induction ◽  
Hemoglobin Production

Abstract Abstract 250 Sickle cell disease and β-thalassemia continue to cause significant morbidity and mortality. Strategies to increase fetal hemoglobin (HbF) levels can ameliorate symptoms and improve the lives of patients with these diseases. While most previous studies have focused on induction of γ-globin gene expression as an approach to induce HbF, there is evidence that HbF may be post-transcriptionally regulated. For example, butyrate was shown to increase the translational efficiency of γ-globin mRNA and 5-azacytidine (5-Aza) induces HbF to a much greater degree than γ-globin mRNA steady state levels. These findings suggest that translational regulation may play an underappreciated yet important role in controlling HbF levels and that investigating the molecular mechanisms involved in this control may provide new therapeutic targets for HbF induction. We hypothesized that the Integrated Stress Response (ISR) pathway is involved in differentially regulating fetal and adult hemoglobin production. The ISR pathway has been shown to modulate globin protein synthesis in response to heme availability and other stresses. In the absence of heme, the heme-regulated inhibitor kinase phosphorylates eIF2α, downregulates general protein synthesis, but enables translation of a limited number of transcripts that are critical for coordinating the stress response. To test our hypothesis, we first evaluated the effects of salubrinal (Sal), a small molecule that activates ISR signaling by selectively inhibiting p-eIF2α dephosphorylation, in K562 cells. 3μM and 6μM Sal increased p-eIF2α and activated ISR signaling as evidenced by increased ATF4 and GADD34 protein levels and increased gene expression of ATF3 and CHOP, two transcriptional targets of ATF4. Once we verified that Sal increased p-eIF2α and ISR signaling, we extended testing to primary human erythroid cells to evaluate its effect on hemoglobin production. We first determined a dose range of Sal that increased p-eIF2α in primary cells without reducing cell viability. Both 3μM and 6μM Sal increased p-eIF2α and only reduced cell number by 15% when applied on days 15 and 18 of differentiation, the period of maximal hemoglobin synthesis. Next, we determined that 3μM and 6μM Sal slightly reduced γ-globin and β-globin steady state mRNA levels but did not change the γ/(γ+β) ratio relative to control. In contrast, Sal significantly induced HbF when evaluated by HPLC at the end of differentiation on day 20. Compared to untreated cells, 3μM Sal increased the percent HbF from 2.7% to 5.0% (1.8 fold) and 6μM Sal resulted in 12.9% HbF (4.7 fold) (n=4, p<0.05). The enhanced %HbF was due to increased HbF but also reduced HbA, providing evidence that HbF and HbA may be differentially or reciprocally regulated at the translational level. Importantly, Sal treatment did not significantly reduce the total hemoglobin content relative to the untreated control and did not alter cellular differentiation when assessed by flow cytometry for CD71 and CD235a. These results suggest that Sal increases HbF by a post-transcriptional mechanism potentially through ISR activation. Sal treatments earlier in the differentiation process (days 9 and 12) before considerable amounts of hemoglobin are synthesized failed to significantly increase HbF, further supporting this conclusion. We then evaluated whether Sal treatment could enhance HbF induction by known activators of γ-globin transcription, such as 5-Aza and hydroxyurea (HU). 200nM 5-Aza alone increased %HbF from 2.7% to 12.4%. When 200nM 5-Aza was combined with 3μM and 6μM Sal, the %HbF increased to 18.0% and 22.8%, respectively. Similarly, 10μM HU alone increased HbF from 2.9% to 4.9%, but co-treatment with 3μM and 6μM Sal increased HbF to 7.7% and 15.0%, respectively. For both HU and 5-Aza, combined treatment with Sal did not alter the γ/(γ+β) ratio from what was seen with HU or 5-Aza alone. Taken together, these results indicate that the novel method of HbF induction by Sal enhances the effect of transcriptional activators of γ-globin. In the future, utilization of transcriptional and translational mechanisms of HbF induction may provide an opportunity for combination therapy to achieve therapeutic HbF levels at reduced doses, thereby reducing toxicity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Conserved Methyltransferase Spb1 Targets mRNAs for Regulated Modification with 2′-O-Methyl Ribose

2018 ◽  
Author(s):  
Kristen M. Bartoli ◽  
Cassandra Schaening ◽  
Thomas M. Carlile ◽  
Wendy V. Gilbert
Keyword(s):  
List Type ◽  
Single Nucleotide ◽  
Target Mrnas ◽  
Mrna Targets ◽  
Non Coding Rna ◽  
Genome Wide ◽  
Non Coding Rnas ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution ◽  
Specific Mrna

SUMMARYNon-coding RNAs contain dozens of chemically distinct modifications, of which only a few have been identified in mRNAs. The recent discovery that certain tRNA modifying enzymes also target mRNAs suggests the potential for many additional mRNA modifications. Here, we show that conserved tRNA 2′-O-methyltransferases Trm3, 7,13 and 44, and rRNA 2′-O-methyltransferase Spb1, interact with specific mRNA sites in yeast by crosslinking immunoprecipitation and sequencing (CLIP-seq). We developed sequencing of methylation at two prime hydroxyls (MeTH-seq) for transcriptome-wide mapping of 2′-O-methyl ribose (Nm) with single-nucleotide resolution, and discover thousands of potential Nm sites in mRNAs. Genetic analysis identified hundreds of mRNA targets for the Spb1 methyltransferase, which can target both mRNA and non-coding RNA for environmentally regulated modification. Our work identifies Nm as a prevalent mRNA modification that is likely to be conserved and provides methods to investigate its distribution and regulation.HIGHLIGHTSMeTH-seq identifies 2′-O-methylribose genome-wide at single-nucleotide resolutionFive conserved methyltransferases interact with yeast mRNASpb1 is a major mRNA 2′-O-methyltransferase, and targets most ribosomal protein mRNAsSPB1 expression is required to maintain normal levels of Spb1 target mRNAs

scholarly journals Genomic structure of murine methylmalonyl-CoA mutase: evidence for genetic and epigenetic mechanisms determining enzyme activity

1993 ◽  
Vol 296 (3) ◽  
pp. 663-670 ◽  
Author(s):  
M F Wilkemeyer ◽  
E R Andrews ◽  
F D Ledley
Keyword(s):  
Enzyme Activity ◽  
Steady State ◽  
Transcription Initiation ◽  
Cultured Cells ◽  
Genomic Structure ◽  
Genetic Regulation ◽  
Regulatory Elements ◽  
Transcription Unit ◽  
Mrna Levels ◽  
Sequence Motifs

Methylmalonyl-CoA mutase (MCM) is a nuclear-encoded mitochondrial matrix enzyme. We have reported characterization of murine MCM and cloning of a murine MCM cDNA and now describe the murine Mut locus, its promoter and evidence for tissue-specific variation in MCM mRNA, enzyme and holo-enzyme levels. The Mut locus spans 30 kb and contains 13 exons constituting a unique transcription unit. A B1 repeat element was found in the 3′ untranslated region (exon 13). The transcription initiation site was identified and upstream sequences were shown to direct expression of a reporter gene in cultured cells. The promoter contains sequence motifs characteristic of: (1) TATA-less housekeeping promoters; (2) enhancer elements purportedly involved in co-ordinating expression of nuclear-encoded mitochondrial proteins; and (3) regulatory elements including CCAAT boxes, cyclic AMP-response elements and potential AP-2-binding sites. Northern blots demonstrate a greater than 10-fold variation in steady-state mRNA levels, which correlate with tissue levels of enzyme activity. However, the ratio of holoenzyme to total enzyme varies among different tissues, and there is no correlation between steady-state mRNA levels and holoenzyme activity. These results suggest that, although there may be regulation of MCM activity at the level of mRNA, the significance of genetic regulation is unclear owning to the presence of epigenetic regulation of holoenzyme formation.

Influence of environmental enrichment on steady-state mRNA levels for EAAC1, AMPA1 and NMDA2A receptor subunits in rat hippocampus

2007 ◽  
Vol 1174 ◽  
pp. 18-27 ◽  
Author(s):  
Josefine Andin ◽  
Martin Hallbeck ◽  
Abdul H. Mohammed ◽  
Jan Marcusson
Keyword(s):  
Steady State ◽  
Environmental Enrichment ◽  
Rat Hippocampus ◽  
Mrna Levels

scholarly journals Determinants of mRNA stability in Dictyostelium discoideum amoebae: differences in poly(A) tail length, ribosome loading, and mRNA size cannot account for the heterogeneity of mRNA decay rates.

1988 ◽  
Vol 8 (5) ◽  
pp. 1957-1969 ◽  
Author(s):  
R A Shapiro ◽  
D Herrick ◽  
R E Manrow ◽  
D Blinder ◽  
A Jacobson
Keyword(s):  
Steady State ◽  
Dictyostelium Discoideum ◽  
Mrna Decay ◽  
Decay Rates ◽  
Time Dependent ◽  
Translational Efficiency ◽  
Cdna Clones ◽  
Dependent Manner ◽  
Significant Difference ◽  
Kinetics Of

As an approach to understanding the structures and mechanisms which determine mRNA decay rates, we have cloned and begun to characterize cDNAs which encode mRNAs representative of the stability extremes in the poly(A)+ RNA population of Dictyostelium discoideum amoebae. The cDNA clones were identified in a screening procedure which was based on the occurrence of poly(A) shortening during mRNA aging. mRNA half-lives were determined by hybridization of poly(A)+ RNA, isolated from cells labeled in a 32PO4 pulse-chase, to dots of excess cloned DNA. Individual mRNAs decayed with unique first-order decay rates ranging from 0.9 to 9.6 h, indicating that the complex decay kinetics of total poly(A)+ RNA in D. discoideum amoebae reflect the sum of the decay rates of individual mRNAs. Using specific probes derived from these cDNA clones, we have compared the sizes, extents of ribosome loading, and poly(A) tail lengths of stable, moderately stable, and unstable mRNAs. We found (i) no correlation between mRNA size and decay rate; (ii) no significant difference in the number of ribosomes per unit length of stable versus unstable mRNAs, and (iii) a general inverse relationship between mRNA decay rates and poly(A) tail lengths. Collectively, these observations indicate that mRNA decay in D. discoideum amoebae cannot be explained in terms of random nucleolytic events. The possibility that specific 3'-structural determinants can confer mRNA instability is suggested by a comparison of the labeling and turnover kinetics of different actin mRNAs. A correlation was observed between the steady-state percentage of a given mRNA found in polysomes and its degree of instability; i.e., unstable mRNAs were more efficiently recruited into polysomes than stable mRNAs. Since stable mRNAs are, on average, "older" than unstable mRNAs, this correlation may reflect a translational role for mRNA modifications that change in a time-dependent manner. Our previous studies have demonstrated both a time-dependent shortening and a possible translational role for the 3' poly(A) tracts of mRNA. We suggest, therefore, that the observed differences in the translational efficiency of stable and unstable mRNAs may, in part, be attributable to differences in steady-state poly(A) tail lengths.

On the steady-state solution of the M/G/2 queue

1979 ◽  
Vol 11 (01) ◽  
pp. 240-255 ◽  
Author(s):  
Per Hokstad
Keyword(s):  
Steady State ◽  
General Solution ◽  
Laplace Transform ◽  
Queue Length ◽  
Joint Distribution ◽  
Length Distribution ◽  
Steady State Solution ◽  
Queue Length Distribution ◽  
The Difference ◽  
Number Of Customers

The asymptotic behaviour of the M/G/2 queue is studied. The difference-differential equations for the joint distribution of the number of customers present and of the remaining holding times for services in progress were obtained in Hokstad (1978a) (for M/G/m). In the present paper it is found that the general solution of these equations involves an arbitrary function. In order to decide which of the possible solutions is the answer to the queueing problem one has to consider the singularities of the Laplace transforms involved. When the service time has a rational Laplace transform, a method of obtaining the queue length distribution is outlined. For a couple of examples the explicit form of the generating function of the queue length is obtained.

Quantitative assessment of ground squirrel mRNA levels in multiple stages of hibernation

2002 ◽  
Vol 10 (2) ◽  
pp. 93-102 ◽  
Author(s):  
L. Elaine Epperson ◽  
Sandra L. Martin
Keyword(s):  
Steady State ◽  
Core Body Temperature ◽  
Ground Squirrels ◽  
Apolipoprotein A1 ◽  
Mrna Levels ◽  
Cdna Subtraction ◽  
Global Issues ◽  
Α2 Macroglobulin ◽  
Steady State Levels ◽  
Molecular Components

Hibernators in torpor dramatically reduce their metabolic, respiratory, and heart rates and core body temperature. These extreme physiological conditions are frequently and rapidly reversed during the winter hibernation season via endogenous mechanisms. This phenotype must derive from regulated expression of the hibernator’s genome; to identify its molecular components, a cDNA subtraction was used to enrich for seasonally upregulated mRNAs in liver of golden-mantled ground squirrels. The relative steady-state levels for seven mRNAs identified by this screen, plus five others, were measured and analyzed for seasonal and stage-specific differences using kinetic RT-PCR. Four mRNAs show seasonal upregulation in which all five winter stages differ significantly from and are higher than summer (α2-macroglobulin, apolipoprotein A1, cathepsin H, and thyroxine-binding globulin). One of these mRNAs, α2-macroglobulin, varies during the winter stages with significantly lower levels at late torpor. None of the 12 mRNAs increased during torpor. The implications for these newly recognized upregulated mRNAs for hibernation as well as more global issues of maintaining steady-state levels of mRNA during torpor are discussed.

scholarly journals Triosephosphate isomerase deficiency: consequences of an inherited mutation at mRNA, protein and metabolic levels

2005 ◽  
Vol 392 (3) ◽  
pp. 675-683 ◽  
Author(s):  
Judit Oláh ◽  
Ferenc Orosz ◽  
László G. Puskás ◽  
László Hackler ◽  
Margit Horányi ◽  
...  
Keyword(s):  
Steady State ◽  
Triosephosphate Isomerase ◽  
Specific Activity ◽  
Energy State ◽  
Dihydroxyacetone Phosphate ◽  
Peptidase Activity ◽  
Mrna Levels ◽  
Regulatory Enzymes ◽  
Computational Data ◽  
Fructose 1,6 Bisphosphate

Triosephosphate isomerase (TPI) deficiency is a unique glycolytic enzymopathy coupled with neurodegeneration. Two Hungarian compound heterozygote brothers inherited the same TPI mutations (F240L and E145Stop), but only the younger one suffers from neurodegeneration. In the present study, we determined the kinetic parameters of key glycolytic enzymes including the mutant TPI for rational modelling of erythrocyte glycolysis. We found that a low TPI activity in the mutant cells (lower than predicted from the protein level and specific activity of the purified recombinant enzyme) is coupled with an increase in the activities of glycolytic kinases. The modelling rendered it possible to establish the steady-state flux of the glycolysis and metabolite concentrations, which was not possible experimentally due to the inactivation of the mutant TPI and other enzymes during the pre-steady state. Our results showed that the flux was 2.5-fold higher and the concentration of DHAP (dihydroxyacetone phosphate) and fructose 1,6-bisphosphate increased 40- and 5-fold respectively in the erythrocytes of the patient compared with the control. Although the rapid equilibration of triosephosphates is not achieved, the energy state of the cells is not ‘sick’ due to the activation of key regulatory enzymes. In lymphocytes of the two brothers, the TPI activity was also lower (20%) than that of controls; however, the remaining activity was high enough to maintain the rapid equilibration of triosephosphates; consequently, no accumulation of DHAP occurs, as judged by our experimental and computational data. Interestingly, we found significant differences in the mRNA levels of the brothers for TPI and some other, apparently unrelated, proteins. One of them is the prolyl oligopeptidase, the activity decrease of which has been reported in well-characterized neurodegenerative diseases. We found that the peptidase activity of the affected brother was reduced by 30% compared with that of his neurologically intact brother.

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