Differential modes of processing and decay for the major N-dependent RNA transcript of coliphage lambda.
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Bacteriophage lambda↗
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Decay of individual Escherichia coli trp messenger RNA molecules is sequentially ordered.
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Base Sequence↗
Spatial mapping of RNA turnover kinetics in the mouse brain.
Gene regulation requires coordinated control of RNA synthesis and degradation, yet measuring RNA turnover across intact tissues remains challenging. Here we present spatial NT-seq, a method that combines transgenesis-free metabolic RNA labeling with in situ chemical recoding on spatial transcriptomics platforms to co-map newly synthesized and pre-existing RNAs. Applying spatial NT-seq to the mouse brain reveals pronounced regional heterogeneity in RNA turnover and identifies the dentate gyrus as a spatial hotspot marked by coordinated upregulation of basal RNA synthesis and decay. Moreover, spatial NT-seq uncovers rapid, brain region-specific transcriptional and post-transcriptional responses to electroconvulsive stimulation, a clinically relevant treatment for refractory depression. Finally, we leverage computational modeling to identify sequence features and post-transcriptional regulators that shape transcriptome-wide mRNA stability across spatial and cellular contexts in the mouse brain. Together, this integrated 'in vivo timescope' framework provides a spatially resolved view of RNA turnover kinetics and reveals the regulatory architecture of RNA stability in vivo.
Journal Article↗
Evidence for endonucleolytic cleavage at the 5'-proximal segment of the trp messenger RNA in Escherichia coli.
The 5'-proximal trp leader RNA segment (about 5S) decays at 2 to 3 times slower rates than the distal trp mRNA sequence. This has been demonstrated by employing the deletion mutants which lack a large portion of the structural genes but retain the promoter-proximal region of the trp operon. Relative stability of the leader RNA is not merely due to the presence of an untranslatable region in the segment; the internal untranslatable segment of trp mRNA downstream from the nonsense alteration site of a double mutant trpAD28.trpE9758 decays as fast as the normal trp mRNA sequence. These results suggest that the trp mRNA is endonucleolytically cleaved to yield the small 5'-proximal leader RNA segment before the distal mRNA decays and that the leader RNA sequence is not subject to usual mode of mRNA decay in the 5' to 3' direction.
DNA, Viral↗
Stability of messenger RNA for nitrate reductase in Neurospora crassa.
A method has been developed to study the synthesis and decay of the messenger RNA for nitrate reductase in Neurospora crassa. Glutamine prevents the synthesis of the mRNA which appears to have a half-life of approximately 8.5 min.
Enzyme Repression↗
Studies on ribosomal protein biosynthesis in an RNA polymerase temperature sensitive E. coli mutant.
In E. coli strain XH56 the synthesis of all RNA species is blocked upon shifting the culture to the non-permissive temperature. The decay of specific messenger RNA species coding for individual ribosomal (r) proteins was followed by measuring the rate of r-protein synthesis by pulse labelling at various times after the shift. The half-lives of the average 30S r-protein and 50S r-protein mRNA species are identical (1.75 min) and shorter than those of the average messenger coding for total cell proteins (2.75 min). Most individual r-protein messengers have a half-life in the same range (1.50-2.00). Only a few r-protein messengers have significantly longer half-lives: S1 (2.80 min), S17 (3.29 min), L29 (2.30 min), L31 (2.30 min), L32 (2.33 min) and L16 (2.60 min). The results indicate that the degradation of most individual r-protein mRNA species is not specifically controlled. After a few min at the non-permissive temperature, all protein synthesis is blocked. The restart of r-protein synthesis was followed after shifting the culture back to the permissive temperature. The recovery of cell growth is very slow. During this period preferential r-protein synthesis was observed. Moreover differential rates of bisynthesis of r-proteins was obtained, it may be indicative of specific regulatory process(es).
DNA-Directed RNA Polymerases↗
A change in the stability of globin mRNA during the induction of murine erythroleukemia cells.
The stability of globin mRNA in murine erythroleukemia cells (Friend cells) before and during DMSO-induced differentiation was investigated. Cells were exposed to 3H-uridine for 2 hr and then transferred to medium without the radioactive precursor. The loss of radioactivity in total RNA, poly(A)-containing RNA and globin mRNA was followed. The globin mRNA was isolated using a highly specific globin cDNA column. In uninduced cells and cells early in differentiation, the globin mRNA decays with a half-life of less than 50 hr. After 4 days of induction, the globin mRNA decays with a half-life of 17 hr, demonstrating a change in stability during the induction process. Although the stability of globin mRNA changes during induction, this is not true for total poly(A)-containing RNA. At all times of induction, the poly(A)-containing RNA decays as two populations, one with a half-life of 6 hr and the other with a half-life of 36 hr. The half-life of the rRNA also remains unchanged during differentiation.
Cell Differentiation↗
Individual messenger RNA half lives in Saccharomyces cerevisiae.
We have measured the decay half-life of functional messenger RNA (mRNA) for some thirty different proteins in the yeast Saccharomyces cerevisiae. Production of newly synthesized mRNA was halted by raising the temperature of a culture of a temperature-sensitive mutant, ts 136. Aliquots of this culture were pulsed-labelled with [35S]-methionine at various times after the temperature shift and the radioactive proteins separated on the two-dimensional gel electrophoresis system of O'Farrell. We find a range in the decay half lives of individual mRNA species which varies from 3.5 min to greater than 70 min. We find three general classes of decay curves, (a) simple exponential (first order); some of these showed a shoulder before onset of exponential decay; (b) bi-component or multi-component concave upward; (c) initial stimulation of rate of mRNA synthesis, followed by virtually undetectable decay.
Electrophoresis↗
Steroid induction of mouse mammary tumor virus: effect upon synthesis and degradation of viral RNA.
Steroid hormones have been demonstrated to induce in tissue culture the production of mouse mammary tumor viral (MMTV) RNA, proteins, and particles 10-fold compared with constitutive levels. However, previous data of increased viral RNA levels did not distinguish between an increased rate of viral-specific RNA synthesis and a slower rate of viral RNA degradation. According to the recently developed assay of Coffin et al. (1974) for measuring rates of viral RNA synthesis, short-term labeling experiments of a mouse mammary tumor cell line indicate that the glucocorticoid hormone dexamethasone stimulates a 3-fold increase in the synthesis of MMTV-specific RNA within 10 min after the addition of hormone and that stimulation of RNA synthesis reaches 5- to 10-fold within 30 to 60 min, while the synthesis of Moloney leukemia virus-specific RNA in the same cell is unaffected by steroids. The decay rates of pulse-labeled and accumulated MMTV RNA in the presence or absence of dexamethasone show this RNA to have a half-life of greater than 8 h. In addition, hormone-stimulated MMTV RNA appears to have an increased rate of decay compared to basal MMTV RNA, thus ruling out an increased stability of MMTV RNA in the presence of steroid hormones as the basis for increased RNA levels. Thus, the magnitude, rapidity, and specificity of hormone action on MMTV RNA synthesis indicate a primary effect upon transcription.
Dactinomycin↗
Minimal Perturbation Analysis of mRNA Degradation Rates with Tet-Off and RT-qPCR.
Messenger RNA stability is an important variable in gene expression and its dynamics. High stability ensures a constant level of synthesized protein, whereas mRNA instability can be critical for regulatory processes in which protein production needs to be stopped, such as development, inflammation, or adaptation to stress. Accurate measurements of RNA degradation rates are important for understanding how RNA features and RNA binding proteins affect the posttranscriptional life of an mRNA. As an alternative to global transcriptional inhibition methods, the use of a Tet-off repressible promoter has the advantage that cells are minimally perturbed by the addition of doxycyclin during the assay. We illustrate the use of a reporter mRNA expressed from a plasmid in Saccharomyces cerevisiae cells, but similar methods can be applied to other regulated promoters, on plasmids or by genome editing, and in other organisms. RNA levels are measured by reverse transcription followed by quantitative PCR. An exponential decay law is then used to estimate how well the measurements follow this expected trend for the simplest possible mechanism of RNA degradation, where the decay is proportional to the amount of RNA present at any given time.
RNA Stability↗
Viral mimicry escape as a necessary feature of malignant transformation.
Malignant transformation is driven by disruption of pathways regulating proliferation and cell fate, but these same disruptions can create a collateral vulnerability: loss of transcriptional and epigenetic control over transposable elements and other normally silenced genomic regions. Consequently, emerging cancer cells can accumulate transposable element-derived and other endogenous immunogenic nucleic acids capable of triggering antiviral responses, a process termed viral mimicry. Increasing evidence indicates that viral mimicry can eliminate precancerous cells and shape tumour evolution, positioning it as an intrinsic tumour-suppressive mechanism. Here we highlight how cancer-associated changes in DNA methylation, histone modifications, splicing and RNA processing can lead to the presence of immunogenic nucleic acids that can activate viral mimicry pathways. We outline how cancer cells suppress viral mimicry, including compensatory epigenetic repression, RNA editing, nucleic acid decay and dampening of interferon signalling to enable cancer cell growth. Finally, we highlight the evidence suggesting that escaping viral mimicry is a fundamental process for cancer initiation and progression, and suggest that viral mimicry escape is necessary for cancer transformation and a therapeutic target in combination with immunotherapies. By framing viral mimicry escape as a necessary part of cancer transformation, this Review provides a unifying conceptual model for its translational exploitation.
Journal Article↗
Stability of bacterial messenger RNA in mesophiles and thermophiles.
The decay of [3H]uridine-labeled mRNA was measured in the mesophile, Bacillus licheniformis (grown at 37 degrees C and 46 degrees C), and in the thermophile, Bacillus stearothermophilus (grown at 46 degrees C and 55 degrees C). For each organism, the half-life of the mRNA decreased as the growth temperature was increased. The stability index (half-life of mRNA/doubling time of cells), however, was remarkably constant for each organism regardless of the growth temperature. It is concluded that these results support the concept that kinetic considerations play a significant role in the explanation of thermophily.
Bacillus↗
Interaction of E. coli RNA polymerase with promotors of coliphage T5: the rates of complex formation and decay and their correlation with in vitro and in vivo transcriptional activity.
The genome of virulent coliphage T5 contains about 30 sites which form stable complexes with E. coli RNA polymerase. Some of these sites bind RNA polymerase with high rates, others form extremely stable complexes as compared with promotors of other E. coli systems. The transcriptional activity of these promotors in vivo and in vitro reflects the rate of complex formation with RNA polymerase rather than the stability of the enzyme/promotor complex. The fastest, i.e. the most active promotors are found in the "early" region of gene expression followed by promotors of the "preearly" class. The few binding sites for the E. coli holoenzyme within the "late" region react more slowly with the enzyme.
Binding Sites↗
The capacity of polyadenylated RNA from myogenic cells treated with actinomycin D to direct protein synthesis in a cell-free system.
Cytoplasmic polyadenylated RNA of myogenic cells was shown to decay with biphasic kinetics, suggesting the existence of two main populations of mRNA with respect to stability. In the present study, the stability of mRNA extracted from actinomycin-D-treated cultures of a myogenic cell line was tested by its capacity to direct protein synthesis in the wheat germ cell-free system. The products were analyzed by dodecylsulphate/polyacrylamide gel electrophoresis. All major radioactive bands found in gels used for analyzing the products of the cell-free system directed by polyadenylated RNA extracted from untreated cultures were also found in similar gels containing products of RNA extracted after many hours of application of actinomycin D. The capacity to code for specific protein bands decays with a half-life ranging between 11 and 40 h. No fast-decaying translatable mRNA could be detected by this method. Instead, it was found that during the first 4--6 h following application of actinomycin D, the capacity of RNA to stimulate incorporation of amino acids into total acid-insoluble material increased by 20--30%. The synthesis of specific products increased by up to 100%. The possibility that the fast-decaying polyadenylated RNA or part of it is nontranslatable RNA is discussed.
Cell Differentiation↗
Size and turnover of polyadenylic acid-containing ribonucleic acids in a fragile mutant of Saccharomyces cerevisiae.
Ribonucleic acid-containing polyadenylic acid [poly(A)+-RNA] was studied in lysates from an osmotic-sensitive mutant of Saccharomyces cerevisiae characterized by low nuclease activity. The poly(A)+-RNA fraction, analyzed by electrophoresis in polyacrylamide-formamide gels, constitutes a heterogeneous population of molecules, with molecular weights ranging from 0.2 X 10(6) to 3 X 10(6) and having an average of 1.2 X 10(6). The turnover rate of poly(A)+-RNA was determined by the decay of radioactivity after a cold uracil chase, and the observed half-life of 21 min corresponds to about 10% of the cell doubling time. Poly(A)+-RNA was analyzed by gel electrophoresis under denaturing and non-denaturing conditions. A correlation was established between the apparent secondary structure and the turnover rate of poly(A)+-RNA species.
Electrophoresis, Polyacrylamide Gel↗
Stability of polysome-associated polyadenylated RNA from soybean suspension culture cells.
The half-life of polysome-associated, poly(A)-RNA in exponentially growing soybean (Glycine max) suspension culture cells was determined with pulse-chase experiments. Based on a best fit from a computer analysis of the data, two decay components for poly(A)-RNA were found. One component had a half-life of approximately 0.6 h, while the other had a half-life of about 30 h, similar to the doubling time of the cultures. At the beginning of the chase period, the short-lived component represented approximately 90% of the total poly(A)-RNA in the polysomes. This percentage decreased with time so that, under steady-state conditions, the long-lived component probably represented the majority of poly(A)-RNA.
Cells, Cultured↗
Induction of endogenous guinea pig retrovirus by 5-bromodeoxyuridine: amplification of virus-specific RNA.
Treatment of normal guinea pig embryo cells with 5-bromodeoxyuridine (BUdR) activates endogenous guinea pig retrovirus. In this report the effect of BUdR treatment upon the level of endogenous retroviral RNA in normal guinea pig embryo cells was determined by using hybridization of viral complementary DNA (cDNA) to cellular RNA. We found that 0.0075% (120 copies per cell) of total RNA of untreated cells was virus-specific, whereas 0.32% (5,120 copies per cell) of total cellular RNA obtained from cells 48 h after BUdR treatment was virus-specific. Thus, BUdR causes an approximately 40-fold amplification of virus-specific RNA after 48 h of treatment. Several lines of evidence favor the hypothesis that the amplification of virus-specific RNA observed after BUdR treatment involves enhancement of transcription rather than an alteration of post-transcriptional processing. At different times after BUdR treatment, similar increases in virus-specific RNA concentration occur in both nucleus and cytoplasm. After 48 h of BUdR treatment, nuclear virus-specific RNA increased 99-fold, from 29 copies per cell to 2,880 copies per cell, whereas cytoplasmic virus-specific RNA increased 47-fold from 85 copies per cell to 4,000 copies per cell. Decay rates of virus-specific RNA in the presence of actinomycin D were similar in the presence or absence of BUdR, indicating that BUdR does not stabilize virus-specific RNA. In BUdR-treated cells the t1/2 of virus-specific RNA was 170 min either in the continued presence of BUdR or after the removal of BUdR, and 150 min in untreated cells. The size distribution of nuclear virus-specific RNA sequences, after denaturation with dimethyl sulfoxide, was similar in untreated and BUdR-treated cells, suggesting similar nuclear processing of viral RNA in both untreated and BUdR-treated cells. The accumulation of nuclear precursors to 38S virus-specific RNA was not observed at steady-state levels in untreated or BUdR-treated cells. Similar species of virus-specific RNA (14S 24S, 38S, and 70S) were present in the total cellular RNA of untreated and BUdR-treated cells. Additionally, virus-specific RNA was present in purified polyribosomes of untreated cells. Finally, direct analysis of the amount of radiolabeled virus-specific RNA in nuclear RNA pulse-labeled for 30 min with [3H]uridine was performed by the method of Coffin et al. (J. Mol. Biol. 86:373-396, 1977) for quantitative determination of pulse-labeled virus-specific RNA. It was found that labeled virus-specific RNA comprised 0.0035 to 0.004% of the total pulse-labeled nuclear RNA of cells treated for 48 h with BUdR. This 50-fold increase in radiolabeled virus-specific RNA may full- account for the 40-fold increase in steady-state levels of virus-specific RNA observed after 48 h of BUdR treatment.
Animals↗
hnRNPC facilitates coronavirus replication by directly binding the frameshift-stimulatory element of viral genomic RNA.
Translation of key viral replicative proteins in coronaviruses requires a programmed -1 ribosomal frameshifting (-1 PRF) event controlled by the viral frameshift-stimulatory element (FSE). Although previous studies have analyzed host factor dependencies of coronaviruses, how host cellular factors alter -1 PRF efficiency and affect viral replication remains poorly understood. Here, using RNA pull-down combined with LC-MS/MS analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNPC) as a major interacting protein of FSE RNA. Coronavirus infection triggers hnRNPC mRNA decay, alters hnRNPC protein levels, and induces its cytoplasmic relocalization, where it appears to bind directly to FSE RNA through residues Asn7 and Asn83. This binding is associated with increased -1 PRF efficiency and may facilitate coronavirus replication. Deletion mapping analysis shows that hnRNPC preferentially binds U-rich regions of the FSE RNA. Finally, we demonstrated that the small molecule Elbasvir directly binds hnRNPC, disrupting the interaction between hnRNPC and FSE RNA and inhibiting coronavirus replication by decreasing -1 PRF efficiency. Collectively, our study identifies hnRNPC as a key host cofactor for coronaviruses and provides a novel target for broad-spectrum antiviral drug development.
RNA, Viral↗