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Results for “Ribosomal RNA depletion”

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At least 19 recordsLinked to original sources
PubMed2025

Transcriptome-Wide Analysis of the 5' Cap Status of RNA Using 5' Monophosphate-Dependent Exonuclease Digestion and RNA Sequencing.

Eukaryotic mRNAs carry an N7-methylguanosine (m7G) cap structure at their 5' extremity, which protects them from the degradation by 5'-3' exoribonucleases and plays a pivotal role in mRNA metabolism, promoting splicing, nuclear export, and translation. Decapping, the enzymatic process that removes this structure, is a key event during cytoplasmic mRNA 5'-3' decay, leading to the degradation of the transcript body by Xrn1. In this chapter, we describe a procedure to assess the cap status of RNA at the transcriptome level. It is based on a treatment of total RNA extracts with a 5' monophosphate-dependent exonuclease, which like Xrn1 specifically degrades decapped RNAs harboring 5' monophosphate extremities, but not RNAs with intact m7G cap. The digested RNAs are then analyzed by RNA sequencing.

Exoribonucleases↗
PubMed2025

Dual RNA isolation from blood: an optimized protocol for host and bacterial RNA purification for dual RNA-sequencing analysis in whole blood sepsis samples.

Dual RNA-sequencing (dual RNA-seq) holds significant promise for deciphering bacterial virulence mechanisms during systemic infections. However, its application in sepsis research is hindered by technical challenges, including a low bacterial burden in blood and limited sample volumes and RNA yield from vulnerable populations, such as neonates. We developed an optimized protocol [dual RNA isolation from blood (DRIB)] for simultaneous stabilization, isolation and purification of high-quality host leukocyte and bacterial RNA from low-volume whole blood samples (0.5 ml). This protocol is compatible with clinical sample collection workflows and high-throughput RNA sequencing. The feasibility of DRIB for dual RNA-seq was validated using a pilot cohort of clinical adult sepsis samples, enabling the investigation of host-bacterial gene expression during sepsis. The DRIB protocol yielded 2.10-6.91 µg of total RNA per clinical sample in our pilot cohort. Dual-species ribosomal RNA (rRNA) depletion and RNA-seq generated 16.6-24.8 million filtered reads per sample, with 63±7% of reads uniquely mapped to host or bacterial sequences. Host genes accounted for 51-68% (8.4-10.9 million) reads, while 0.5-6.7% (79,496-789,808 reads) mapped to bacterial genomes. Bioinformatic analysis revealed that both shared and individual transcriptional patterns were identified in host and bacterial responses, including pathways related to immune metabolism and metal-ion binding. Our optimized DRIB protocol and RNA-seq pipeline effectively captured both host and bacterial RNA transcription in clinical sepsis samples. Expanding this approach to larger cohorts and varying disease timepoints will provide crucial new insights into host-bacterial gene co-expression dynamics in sepsis progression and outcomes.

Humans↗
PubMed2025

RNA Sequencing Protocols for Short-Read Sequencing.

RNA sequencing (RNA-seq) methodologies allow the discovery of novel variants and transcripts. These comprise three general steps: (1) capture of RNA species of interest, (2) conversion of RNA to complementary DNA (cDNA), and (3) modification of cDNA to fit the sequencing platform. Here we describe four different library preparation protocols for short-read sequencing: cDNA synthesis with poly(A) selection, library preparation with ribosomal depletion, and cDNA synthesis with SMART® (Switching Mechanism at 5' end of RNA Template) technology for low and Pico inputs.

Gene Library↗
PubMed1979

Distribution of newly formed ribosomal proteins in HeLa cell fractions.

The distribution of newly formed ribosomal proteins between cytoplasmic, nucleoplasmic, and nucleolar fractions of HeLa cells was determined. All but a few of the newly formed ribosomal proteins were concentrated 10- to 50-fold in the nucleolus and two- to fivefold in the nucleoplasm. Nevertheless, substantial amounts were found in the cytoplasm. Pretreatment of cells with actinomycin D to deplete the nucleolar pool of ribosomal precursor RNA had no effect on the concentration of newly formed ribosomal proteins in the nucleus, but did lead to an increased amount in the nucleoplasm at the expense of the nucleolus.

Cell Nucleolus↗
PubMed1979

Synthesis and degradation of lac mRNA in E. coli depleted of 30S ribosomal subunits.

Escherichia coli was depleted of active ribosomes by a thermal shock at 47 degrees C which quantitatively destroyed the 30S ribosomal subunits. During recovery, RNA is synthesized while protein synthesis resumes only after about 90 minutes. It is shown that lac mRNA is synthesized in the complete absence of ribosomal activity and hence RNA synthesis is not coupled to protein synthesis. Transcription time and average transcript length were slightly less than in untreated cells. lac mRNA was degraded much more slowly in bacteria depleted of ribosomes. In E. coli W both functional half life (T 1/2 = 28 min vs. 2.25 in untreated cells) and chemical stability. The analysis of rna and pnp mutants showed that polynucleotide phosphorylase is involved in lac mRNA degradation in heat treated cells but that RNase I is not. The functional T 1/2 was increased in pnp mutants and was 95 min during the recovery period. The rate of chemical decay is so slow that the half-life cannot be accurately determined.

Bacterial Proteins↗
PubMed1977

Requirement of chain initiation factor 3 and ribosomal protein S1 in translation of synthetic and natural messenger RNA.

Amino acid incorporation directed by poly(A), poly(U) or R17 RNA has been examined in S1-depleted protein synthesizing systems. We observe that the translation of either synthetic or natural messenger RNA is strictly dependent on the presence of chain initiation factor 3 and ribosomal protein S1. With poly(A) or poly(U) both IF-3 and S1 stimulate amino acid incorporation at least 25-fold, and with R17 RNA the stimulation is approximately 15-fold. More than one copy of S1 per ribosome decreases amino acid incorporation directed by poly(U) or R17 RNA. Initiation complex formation with R17 RNA is also stimulated optimally by the addition of one copy of S1 per ribosome. The function of IF-3 and S1 in protein synthesis is considered.

Bacterial Proteins↗
PubMed1979

Different nucleosome structures on transcribing and nontranscribing ribosomal gene sequences.

Monomeric DNA lengths from Physarum nuclear chromatin occur in two subunit forms which differ from each other and from higher oligomers of nucleosomes in content of transcribed ribosomal DNA sequences. Labeled DNA restriction fragments from ribosomal RNA coding regions reanneal most rapidly with DNA from a monomeric subunit fraction. A particles, isolated from growing plasmodia and containing 144 base pairs of DNA in an extended conformation. Higher oligomers of nucleosomes are depleted in sequences from transcribing gene regions but are enriched in sequences from the nontranscribed central spacer of the ribosomal DNA palindrome. Nucleosome configuration on two 26S gene intervening sequences resembles that on adjacent coding regions.

Cell Nucleolus↗
PubMed1977

mRNA-dependent in vitro synthesis of ribosomal proteins L12 and L10 and elongation factor Tu.

RNA extracted from growing Escherichia coli can direct the in vitro synthesis of ribosomal proteins L12 and L10 and elongation factor Tu when an E. coli system is used. The synthesized L12 can be bound to L12-depleted ribosomes and the synthesized elongation factor Tu can form complexes with both elongation factor Ts and GDP. Guanosine 5'-diphosphate 3'-diphosphate has no effect on the synthesis of these proteins from an RNA template but inhibits their synthesis when a DNA template is used.

Cell-Free System↗
PubMed2025

RNA Pol I activity is required for meiotic chromatin organization and the H3K4me3 gradient essential for oogenesis, independent of ribosome synthesis.

Oogenesis requires extensive and dynamic chromatin remodeling that primes gene promoters for later transcriptional activation during embryonic development. Here, we uncover a pivotal, non-canonical role for RNA Polymerase I (Pol I) in driving these chromatin state transitions during Caenorhabditis elegans oogenesis. Using the auxin-inducible degron system to selectively deplete either Pol I catalytic subunits or ribosome assembly factors, we disentangle the consequences of impaired nucleolar integrity from reductions in ribosome biogenesis. Strikingly, although disrupting ribosome assembly caused minimal effects on oocyte production, loss of Pol I activity led to widespread changes in chromatin accessibility, a dampening of the distal-proximal H3K4me3 gradient required for oogenesis, reduced synapsis, and elevated ATM/ATR phosphorylation, resulting in fewer but significantly larger oocytes. Despite their promoters becoming more accessible, oogenesis genes did not show large changes in steady-state mRNA, consistent with transcriptional repression prior to fertilization. Instead, Pol I depletion prematurely remodeled oogenic chromatin, through a misdirection of H3K4me3 deposition towards promoters normally primed for zygotic genome activation. These findings reveal an epigenetic gating function for nucleolar integrity in oocyte maturation: Pol I preserves three-dimensional chromatin organization and maintains proper spatiotemporal regulation of histone modifications, independent of ribosome production. Given the evolutionary conservation of nucleolar dynamics and histone modifications during gametogenesis, our work suggests that nucleolar stress, whether from environmental factors, aging, or genetic disorders, could broadly compromise fertility by disrupting oogenic chromatin priming.

Journal Article↗
PubMed1975

Effect of brain, liver and muscle cytosols on in vitro incorporation of labelled amino acids into the ribosomes of these organs during protein depletion.

The functional state of the rat liver, brain and muscle proteosynthetic apparatus during developing protein depletion was studied, with special reference to the effect of brain cytosols on the synthetic capacity of liver and muscle ribosomes. The results indicate that both stimulant and inhibitory agents (so far unidentified) may be present in brain cytosols during protein depletion.

Adaptation, Physiological↗
PubMed1979

Relief of polarity in E. coli depleted of 30S ribosomal subunits.

Escherichia coli was depleted of ribosomes by a thermal shock at 47 degrees C which quantitatively destroyed the 30S ribosomal subunits. During recovery in minimal medium at 30 degrees C RNA is synthesized while protein synthesis resumes only after about 90 min. It is shown that lac mRNA is synthesized in the complete absence of ribosomal activity and hence RNA synthesis is not coupled to protein synthesis. Lac mRNA from a series of lac nonsense mutants was examined in both heated and untreated cells. It was found that the polar effect of nonsense mutation is relieved in the absence of ribosomes and that this relief is due to the synthesis of larger mRNA molecules. Since Rho remained active in thermally treated cells, premature termination at secondary signals within the lac operon must also depend on the presence of active ribosomes.

Escherichia coli↗
PubMed2025

Altered translation elongation contributes to key hallmarks of aging in the killifish brain.

Aging is a major risk factor for neurodegeneration and is characterized by diverse cellular and molecular hallmarks. To understand the origin of these hallmarks, we studied the effects of aging on the transcriptome, translatome, and proteome in the brain of short-lived killifish. We identified a cascade of events in which aberrant translation pausing led to altered abundance of proteins independently of transcriptional regulation. In particular, aging caused increased ribosome stalling and widespread depletion of proteins enriched in basic amino acids. These findings uncover a potential vulnerable point in the aging brain's biology-the biogenesis of basic DNA and RNA binding proteins. This vulnerability may represent a unifying principle that connects various aging hallmarks, encompassing genome integrity, proteostasis, and the biosynthesis of macromolecules.

Animals↗
PubMed2026

rRNA expansion segments mediate ribosome dimerization as a conserved stress response.

Inhibition of messenger RNA translation is a common feature in proteostatic stress cellular responses. Puromycin, a widely used compound for studying translation, disrupts protein synthesis by mimicking the 3' end of aminoacyl-transfer RNAs. Despite its extensive use as a research tool to probe the connection between translation activity and various physiological and pathological states, the cellular response associated with puromycin-induced translation stress remains incompletely understood. Here, we used electron tomography and topology analysis to define the effects of puromycin on the translation machinery in situ. We show that puromycin-treated neuronal cells exhibit an accumulation of eIF5A-bound ribosomes in a translationally inactive "idle" state, and thereby defining a broader role of eIF5A in ribosome homeostasis. Additionally, the idle ribosomes formed dimeric complexes mediated by ribosomal RNA expansion segments, suggesting an evolved mechanism involving these regions in translational hibernating and protecting idle ribosomes. We further show that the hibernating disome formation is not unique to puromycin administration but represents a conserved mechanism as a response to different cellular stressors including endoplasmic reticulum stress and amino acid depletion. Collectively, our findings illuminate distinct states of mammalian ribosome hibernation and dimerization, providing new insights into the relationship of cellular stress and the dynamic regulation of ribosomal activity.

Ribosomes↗
PubMed1976

Poliovirus proteins associated with the replication complex in infected cells.

Viral polypeptides associated with the membrane-free replication complex of poliovirus RNA were multiple in nature. The structural protein precursors [VP0, VP1, VP3] predominated, and because they were found in a cytoplasmic component with the same S value and density as the replication complex are likely to be attached to it in vivo. They were not present in the form of empty capsids. The electrophoretic polypeptide pattern of the membrane-bound replication complex was similar but showed a predominance of NCVPX or VP1, unless the cells were slightly depleted in amino acids when the non-structural polypeptide NCVP2 became important. Cystine was the only amino acid capable of reversing this depletion effect on its own.

Amino Acids↗
PubMed1978

Regulation of macromolecular synthesis during hyphal germ tube emergence from Mucor racemosus sporangiospores.

Protein and RNA syntheses were examined during hyphal germ tube emergence from sporangiospores of a dimorphic phycomycete, Mucor racemosus. Both classes of macromolecules were synthesized immediately upon introduction of the dormant sporangiospores into nutrient medium. The specific rates of synthesis of both protein and RNA accelerated during initial germ tube emergence and reached a maximum when the emergence of new germ tubes ended. The specific rates of synthesis later decreased during further hyphal elongation. The distribution of ribosomes between active polysomes and monosomes and inactive subunits was determined by sucrose density gradient centrifugation, and the rate of amino acid addition to nascent polypeptide chains was calculated throughout the developmental sequence. The results showed that both the percentage of ribosomes active in protein synthesis and the velocity of ribosome movement along the mRNA were continuously adjusted throughout hyphal germ tube development. The free intracellular amino acid pools were measured throughout development. Alanine, glutamate, and aspartate were present at very high concentrations in the dormant spores but were rapidly depleted during hyphal germ tube emergence. The results of these studies are discussed in relation to hyphal germ tube development from yeast cells of Mucor and dormant spores of other fungal species.

Amino Acids↗
PubMed1978

Effect of starvation on initiation of protein synthesis in skeletal muscle and heart.

Psoas muscle of rats starved for 2 or 4 days contained increased levels of ribosomal subunits and exhibited reduced rates of protein synthesis in vitro, demonstrating a starvation-induced inhibition of peptide-chain initiation. The activity of an eIF-2-like initiation factor, assayed in postribosomal supernatants, decreased in psoas during starvation, parallel to a 25% reduction in the RNA level. Reduced eIF-2 activity did not result from nucleotide depletion or increased deacylation of initiator tRNA, nor was it abolished by extensive dialysis. Perfusion of psoas muscle in the presence of insulin reversed the starvation-induced block in peptide-chain initiation, but did not alter the activity of eIF-2 or level of RNA. Furthermore, heart muscle did not manifest a starvation-induced block in peptide-chain initiation even though the activity of eIF-2 and the level of RNA decreased as a result of food deprivation. Thus loss of eIF 2 activity in psoas and heart did not parallel changes in peptide-chain initiation but was associated with a reduction in tissue RNA. These results indicate that the level of eIF-2 is not rate-limiting for peptide-chain initiation under the conditions tested in this study.

Amino Acids↗
PubMed1978

The effects of tiamulin, a semisynthetic pleuromutilin derivative, on bacterial polypeptide chain initiation.

Tiamulin, a water-soluble and highly effective semisynthetic derivative of pleuromutilin leads to the formation of physiologically inactive polypeptide chain initiation complexes which readily decompose and do not enter the phase of peptide chain elongation. Once elongation has begun it continues even in the presence of tiamulin as has been shown by measuring the formation of N-acetylphenylalanine-poly(phenylalanine). The formation of abortive initiation complexes was observed regardless of whether AcPhe-tRNA of fMet-tRNA was used as an initiator or whether artificial messengers or a natural messenger, like R17 bacteriophage RNA, was used. When this drug was acting on whole cells, it led to the disappearance of polysomes. The only structures which could be detected were of the monosome size. Therefore, polysomes seem to elongate the polypeptide chains in whole cells in the presence of this antibiotic, but since effective reinitiation is blocked, the polysome pool of the cell soon becomes depleted.

Anti-Bacterial Agents↗
PubMed1977

Impairment of reovirus mRNA methylation in extracts of interferon-treated Ehrilich ascites tumor cells: further characteristics of the phenomenon.

We reported earlier that the methylation of unmethylated reovirus mRNA (reo mRNAU) by the cellular methylating enzymes is impaired in extracts of uninfected, interferon-treated Ehrilich ascites tumor cells (S30INT). We find now that after the methylation of reo mRNAU has stopped in S30INT, the RNA can be reisolated and further methylated in an extract of control cells (S30C). Thus the impairment of methylation in S30INT cannot be due to cleavage or irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is due to the depletion of S-adenosylme thionine (the methyl donor), the accumulation of S-adenosylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of reo mRNAU. Freshly added reo mRNAU can be methylated in S30INT in which the methylation of previously added reo mRNAU has stopped. This indicates that the impairment is not due to the depletion of S-adenosylmethionine (the methyl donor), the accumulation of S-adenoxylhomocysteine (an inhibitor of methylation), or the irreversible inactivation of the methylating enzymes. It may be due, however, to the unavailability of reo mRNAU for methylation. The extent of the impairment of reo mRNAU methylation in S30INT decreases with an increasing concentration of reo mRNAU but is not affected by added poly (U), ribosomal RNA, or encephalomyocarditis virus RNA (an mRNA that is probably not capped or methylated at its 5' end). The methylation of reo mRNAU is also impaired in an extract from cells that have not been treated with interferon but with the interferon inducer poly(I) - poly(C). The inhibitor is apparently a macromolecule that is inactivated during incubation. It decreases the methylation at the 7 position of the 5' terminal guanylate residue. In vitro, the rate of reo mRNA synthesis by reovirus cores in the presence of S30INT is the same as in the presence of S30C. However, the methylation of the de novo synthesized reo mRNA by the core-associated methylating enzyme(s) in vitro is inhibited by S30INT but not by S30C. The relevance of these phenomena to the inhibition of reovirus replication in interferon-treated cells remains to be established.

Animals↗