Bases Of mRNA Research Articles

Novel splicing mutations in PATL2 and WEE2 cause oocyte degradation and fertilization failure.

To determine the genetic cause of infertility in two unrelated families of female patients suffering from oocyte degeneration and fertilization failure. Whole exome sequencing and Sanger sequencing were performed to identify the disease-causing genes of infertility in two unrelated female patients. Minigene experiments were conducted to confirm the effect of splice site mutations on mRNA splicing. In two unrelated female infertility patients, a novel compound heterozygous splicing mutation (c.516-1G > T and c.877-1G > A) in PATL2 gene and a novel homozygous splicing mutation (c.1222-1G > A) in WEE2 gene were identified. Minigene splicing assays revealed that the c.516-1G > T mutation in PATL2 resulted in a deletion of 8 bases in mRNA that causes a frameshift (c.516-523delTCCCCCAG, p.P173Q fs*13). The c.877-1G > A mutation led to the skipping of exons 10 and 11 and retention of introns 8-9 in PATL2 mRNA. The c.1222-1G > A mutation resulted in the deletion of exon 9 in WEE2 mRNA, leading to an in-frame deletion of 57 amino acids in the WEE2 protein (p.408-464del). Our study discovered novel splicing mutations in PATL2 and WEE2, further expanding the mutation spectrum of these two genes and providing guidance for genetic counseling and diagnosis of female infertility.

EDITORIAL. El Premio Nobel de Medicina o Fisiología 2023

The 2023 Nobel Prize in Medicine or Physiology, awarded to Katalin Karikó and Drew Weissman, recognizes the fundamental discovery that the modification of bases in mRNA synthesized in vitro prevents inflammatory responses and increases protein production when it is introduced into the cell. Although these discoveries received little attention when they were published between 2005 and 2010, ten years later they made possible the rapid develop-ment of vaccines against COVID-19

The Four mRNA Bases Have Quite Different (Un)folding Free Energies, Applications to RNA Splicing and Translation Initiation with BindOligoNet

Expression of mRNA is often regulated by the binding of a small RNA (miRNA, snoRNA, siRNA). While the pairing contribution to the net free energy is well parameterized and can be computed in O(N) time, the cost of removing pre-existing mRNA secondary structure has not received sufficient attention. Conventional methods for computing the unfolding free energy of a target mRNA are costly, scaling like the cube of the number of target bases O(N3). Here we introduce a model to describe the unfolding costs of the binding site, which features surprisingly big differences in the free energy parameters for the four bases. The model is implemented in our O(N) algorithm, BindOligoNet. Donor splice site prediction is more accurate when using our calculation of spliceosomal U1-snRNA to mRNA net binding free energy. Our base-dependent free energies also correlate with efficient ribosome docking near the start codon.

Progress on genome-scale metabolic models integrated with multi-omics data

The genome-scale metabolic network model (GEM) is a mathematical framework based on gene-protein-reaction associations combined with stoichiometric balance and is capable of facilitating the computation and prediction of multiscale phenotypes by optimizing the objective function of interest. It has been increasingly used as an important tool for understanding cellular metabolism and characterizing cell phenotypes. In cells, metabolism is tightly controlled by intricate regulatory mechanisms at the different system levels and is strictly regulated to ensure the dynamic adaptation of biochemical reaction fluxes for maintaining cell homeostasis to ultimately achieve optimal metabolic fitness. Advances in high-throughput screening and analysis technologies have generated massive amounts of genome sequences, along with transcriptomic, proteomic and metabolomic data, providing quantitative regulatory information to gain insights into cellular metabolism; however, integrating the available omics data into constraint-based metabolic models and quantitatively profiling genotype-phenotype relationships remains an outstanding challenge for computational biology. Here, we describe the recent developments in introducing macromolecular expression into GEMs and generating metabolic expression (ME) models, which increase the complexity and predictive capability of computational frameworks. Various algorithms employ different approaches to combine additional layers of omics data to limit the cone of allowable flux distributions in the metabolic model. In this review, we categorize all methods by five different grouping criteria and evaluate their practical perspectives. The first category of methods utilizes a threshold to distinguish active and inactive states of the corresponding reactions based on the gene expression measurement data. The second uses omics data to build cell- and tissue-specific models of human metabolism by removing unexpressed reactions from the global human metabolic network. The third category of methods involves modifying reaction bounds on the basis of mRNA and protein abundance, in which the width of the “flux cone” is adjusted via the maximum possible flux in the upper bound of the FBA optimization problem dependent on gene and protein expression levels. The imposition of constraints further defines the associated solution space of the model to improve the prediction accuracy. The fourth model incorporates transcriptional regulation networks (TRNs), which describe the phenomenological interactions between different biomolecules in response to genetic and environmental perturbation, into GEMs and avoids the obstacles of information formulation to achieve comprehensive knowledge regarding the metabolic and regulatory events occurring inside the cell. The last category integrates time-series transcriptomics data with flux-based bilevel optimization to comprehend the interplay between metabolism and regulation in time-dependent processes. We compare the advantages and limitations of different categories and explore the application areas of integrated models in analyzing metabolic characteristics, interpreting phenotypic states and the consequences of environmental and genetic perturbations while discovering potential drug targets and screening anti-metabolic drugs for cancer treatment. Finally, we also highlight the future perspectives and challenges for GEM-based reconstruction with omics data integration.

Identifying a Molecular Mechanism That Imparts Species-Specific Toxicity to YoeB Toxins.

The ribosome-dependent E. coli (Ec) mRNase toxin YoeB has been demonstrated to protect cells during thermal stress. Agrobacterium tumefaciens (At), a plant pathogen, also encodes a YoeB toxin. Initial studies indicated that AtYoeB does not impact the growth of Ec, but its expression is toxic to the native host At. The current work examines this species-specific effect. We establish the highly similar structure and function of Ec and AtYoeB toxins, including the ability of the AtYoeB toxin to inhibit Ec ribosomes in vitro. Comparison of YoeB sequences and structures highlights a four-residue helix between β-strands 2 and 3 that interacts with mRNA bases within the ribosome. This helix sequence is varied among YoeB toxins, and this variation correlates with bacterial classes of proteobacteria. When the four amino acid sequence of this helix is transplanted from EcYoeB onto AtYoeB, the resulting chimera gains toxicity to Ec cells and lessens toxicity to At cells. The reverse is also true, such that EcYoeB with the AtYoeB helix sequence is less toxic to Ec and gains toxicity to At cultures. We suggest this helix sequence directs mRNA sequence-specific degradation, which varies among proteobacterial classes, and thus controls growth inhibition and YoeB toxicity.

Oxidative Stress Induced by Loss of Superoxide Dismutases Leads to Activation of No‐Go Decay

Reactive oxygen species (ROS) are known to cause oxidative damage in the cell and have been connected to many neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and cancer. Superoxide dismutases (SODs) are a group of enzymes that act to prevent oxidative damage from ROS by converting O2− to H2O2. In Saccharomyces cerevisiae, there are two SOD proteins that are expressed, each of which is conserved in humans. Sod1p is a cytoplasmic Cu/Zn coordinated enzyme, whereas Sod2p coordinates Mn2+ and localizes to mitochondria. Although there is significant data showing that loss of SOD proteins allows for high levels of intracellular oxidation, it is unknown what effects that would have on the translating pool of mRNA. Current work has revealed that strains lacking Sod1p have a moderate decrease in general translation and an increase in P‐body assembly. Furthermore, this reduction in translation is likely due to the formation of 8‐oxo(G) bases in mRNA, which can cause ribosome stalls leading to the subsequent activation of the No‐Go quality control pathway. No‐Go decay is mediated by the proteins Dom34p and Hbs1p, which act together to remove stalled ribosomes and promote endonucleolytic cleavage of the mRNA at the stall site. The 3′ fragment of mRNA is then localized to P‐bodies to be degraded 5′ to 3′ by Xrn1p, whereas the 5′ fragment is degraded by the cytoplasmic exosome.Sod2p performs a similar role to Sod1p, as it also converts O2− to H2O2, and plays an important role in preventing the formation of strong oxidant peroxynitrate (ONOO−). Given that it localizes to mitochondria, a loss of Sod2p would result in higher levels of mitochondrially derived ROS. To determine the effect of mitochondrially derived ROS on the translating pool of mRNA, both general translation and P‐body assembly will be analyzed in strains lacking Sod2p. If general translation is reduced and P‐body assembly is increased, then it will be ascertained whether No‐Go Decay is being upregulated in the absence of Sod2p. To perform this analysis, double mutant strains lack both Sod2p and No‐Go Decay will be created and further analyzed for general translational control and P‐body assembly.

Determining the Physiological Basis of No‐Go Decay Using sod1Δ Strains in Saccharomycescerevisiae

Chromium VI (Cr(VI)) induces oxidative stress in cells, resulting in altered gene expression, increased apoptosis and cell death. Previous work showed that Cr(VI) exposure results in the formation of 8‐oxo(G) bases in mRNA. The presence of 8‐oxo(G) bases can lead to ribosome stalls during translation and activation of no‐go decay. During no‐go decay, the Dom34p/Hbsp1p complex acts to remove stalled ribosomes and subsequent endonucleolytic cleavage of the damaged mRNA at the stall site. Although oxidative agents and other non‐physiological substrates have been used to activate no‐go decay, very little work exists to elucidate the true physiological role of this pathway.Sod1p (superoxide dismutase 1) acts to scavenge free oxygen species by catalyzing the partitioning of superoxide (O2− ) into O2 and H2O2. Cells that lack functional Sod1p were observed to have increased oxidative stress resulting in increased P‐body assembly. Yet, global translation was only attenuated. To determine whether no‐go decay is being activated in strains lacking Sod1p, double mutant strains were created in which SOD1 was knocked out in conjunction with one of the effectors of no‐go decay. Consistent with no‐go decay being activated in response to oxidation, the hbs1Δsod1Δ strain resulted in a decrease in P‐body assembly, as compared to wild‐type, even in the presence of Cr (VI).Interestingly, Stm1p is thought to potentially to aid in the dissociation and recycling of the ribosomal subunits. In the stm1Δ strain, P‐body assembly was mildly decreased as compared to the wild‐type upon treatment with Cr(VI). This is consistent with Stm1p functioning to recycle ribosomes downstream of no‐go decay. Additionally, a stm1Δ can can suppress sod1Δ growth defects on Cr(VI) containing media.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNA Quality Control Using External Standard RNA.

In this paper, we propose a new evaluation method using external standard RNA for quality control of the extracted RNA. RNA Integrity Number and UV absorption are generally used as a basis for RNA quality control; however, these methods do not always reflect the quality of mRNA. While standard RNA is supposedly designed on the basis of mRNA, it has the potential to be used to evaluate the quality of the mRNA. In this study, we took into consideration the three essential factors, viz., yield of mRNA, inhibition to DNA polymerase, and degradation of mRNA for determining the RNA quality using standard RNA. It would be possible to know yield of mRNA and inhibition of the enzyme reaction by adding standard RNA before RNA extraction and looking at standard RNA loss. Degradation was evaluated by comparing the differences in the 3’ and 5’ regions of the RNA. In our study, it was demonstrated that in the crude extract of Saccharomyces cerevisiae, degradation was comparatively higher at the 3’ end of RNA than at the 5’ end. Hence, the degree of RNA degradation can be evaluated by comparing the ratio of degradation from the 3’ and 5’ end.

Structural basis for mRNA 3′-end processing

Structural Biology The eukaryotic mRNA 3′-end processing machinery interacts with the transcription machinery and adds the polyadenylation tail on the mRNA substrate. Casanal et al. used cryo-electron microscopy, mass spectrometry, and biochemical reconstitutions to show that the mRNA 3′-end

A binary representation of the genetic code

This article introduces a novel binary representation of the canonical genetic code based on both the structural similarities of the nucleotides, as well as the physicochemical properties of the encoded amino acids. Each of the four mRNA bases is assigned a unique 2-bit identifier, so that the 64 triplet codons are each indexed by a 6-bit label. The ordering of the bits reflects the hierarchical organization manifested by the DNA replication/repair and tRNA translation systems. In this system, transition and transversion mutations are naturally expressed as binary operations, and the severities of the different point mutations can be analyzed. Using a principal component analysis, it is shown that the physicochemical properties of amino acids related to protein folding also correlate with certain bit positions of their respective labels. Thus, the likelihood for a point mutation to be conservative, and less likely to cause a change in protein functionality, can be estimated.

Reversible methylation of m6Am in the 5' cap controls mRNA stability.

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N6,2'-O-dimethyladenosine (m6Am), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m6Am we find that m6Am-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m6Am-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m6Am is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m6Am rather than N6-methyladenosine (m6A), and reduces the stability of m6Am mRNAs. Together, these findings show that the methylation status of m6Am in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

The 5' untranslated mRNA region base content can greatly affect translation initiation in the absence of secondary structures in Prevotella bryantii TC1-1.

It has become clear lately that many bacteria and even whole bacterial phyla do not use the classical Shine-Dalgarno sequence mediated pathway of protein translation initiation. The prominent phylum Bacteroidetes is one of them, and this was shown not only using bioinformatic but also functional reporter gene studies in its representative Prevotella bryantii. The latter studies revealed much higher sensitivity toward secondary structures in 5(') untranslated mRNA regions (5(') UTRs) during translation initiation compared to Escherichia coli. It was proposed that in the absence of Shine-Dalgarno sequence interaction the key elements enabling translation initiation are local absence of secondary structures in 5(') UTRs, and the ribosomal protein S1 which binds to mRNA. Here, we evaluate the 5(') UTRs devoid of secondary structures but containing divergent nucleotide compositions in P. bryantii reporter assay. We show that base composition profoundly affects the amount of the reporter synthesized, and further that these amounts were in agreement with S1 protein binding affinity for adenine/uracil bases in mRNA. This is the first, though indirect, clue that S1 is actually involved in translation initiation in Bacteroidetes and adds the second layer of control beside mRNA secondary structure affecting translation initiation in this phylum.

The formation, metabolism and the evolution of blood platelets

Platelets are the smallest, depleted of nucleus blood cells which contain a typical cellular organelles including the mitochondria, so that have active metabolism. Platelets possess the highly organized cytoskeleton, specific secretory granules and unique membrane receptors system responsible for their high reactivity. The key role of blood platelets is to maintain normal hemostasis, but they also play important roles in inflammation, immune processes and the cancer progression. The anucleated, small platelets occur in representatives of all clusters of mammals, so it seems to be an adaptation feature. In other vertebrates similar hemostatic functions are played by large nucleated platelets, which are much more weakly reactive. Small, reactive platelets, appearing in the evolution of mammals, allowed the formation of clots faster and slower blood loss in case of injury, but also increased the risk of thromboembolic and cardiovascular diseases. Daily the human body forms about 1x10¹¹ platelets, which are produced by a process of differentiation, maturation and fragmentation of the cytoplasm of mature megakaryocytes. The emergence of platelets is the final stage of megakaryocyte differentiation and is followed by formation of the direct precursors called proplatelets. The anucleated platelets are regarded as terminally differentiated cells, which are not capable of further cell division. However, despite the absence of a nucleus, in blood platelets the synthesis and transcription of mitochondrial DNA and protein synthesis occurring on the basis of mRNA from megakaryocytes has been confirmed. However, recent studies published in 2012 show that the platelets are capable not only of the process of protein synthesis, but also of generation of new cells, which are functionally and structurally similar to the parent platelets.

Crystal Structures of EF-G–Ribosome Complexes Trapped in Intermediate States of Translocation

Translocation of messenger and transfer RNA (mRNA and tRNA) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome-tRNA-mRNA-EF-G complexes trapped with β,γ-imidoguanosine 5'-triphosphate (GDPNP) or fusidic acid reveal conformational changes occurring during intermediate states of translocation, including large-scale rotation of the 30S subunit head and body. In all complexes, the tRNA acceptor ends occupy the 50S subunit E site, while their anticodon stem loops move with the head of the 30S subunit to positions between the P and E sites, forming chimeric intermediate states. Two universally conserved bases of 16S ribosomal RNA that intercalate between bases of the mRNA may act as "pawls" of a translocational ratchet. These findings provide new insights into the molecular mechanism of ribosomal translocation.

Abstract 4489: Clinical specimen-based investigatory study of biomarkers for gastric cancer

Abstract Objectives: We have identified potential biomarkers for gastric cancer using a cDNA bank on the basis of mRNA extracted from frozen specimens of gastric cancer tissue and adjacent normal gastric mucosa. We report currently available results. Methods: The study group comprised 227 patients in whom 5 or more years had elapsed since gastrectomy. The disease stage was IB in 29 patients, II in 66, III in 103, and IV in 29. Among the 139 patients who postoperatively received fluoropyrimidines, 97 with stage II or III disease were given adjuvant chemotherapy with S-1. A total 104 genes were selected on the basis of the results of comprehensive DNA microarray analysis, extraction from serial analysis of gene expression (SAGE) libraries, and preclinical and other studies of potential targets for molecular-targeted therapy. The relative expression levels of these genes in gastric cancer tissue and adjacent normal mucosa from each patient were measured by a quantitative polymerase chain reaction assay using the cDNA bank described above, and the relations between clinicopathological factors and outcomes were examined. Results: In patients who underwent gastrectomy, high expression of matrix metalloproteinase-9 (MMP9), insulin-like growth factor receptor 1 (IGF-R1), and apolipoprotein E (APOE) genes were associated with significantly worse outcomes. In patients with stage II or III disease who received adjuvant chemotherapy with S-1, high expression of IGF-R1, thymidylate synthase (TS), and regenerating IV (Reg IV) genes were associated with significantly worse outcomes. Conclusions: MMP9, IGFR-1, and APOE gene expression levels were suggested to be useful prognostic factors in patients who underwent gastrectomy for gastric cancer. In patients with stage II or III disease who received adjuvant chemotherapy with S-1, IGF-1R, TS, and Reg IV gene expression levels were suggested to be useful prognostic factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4489. doi:1538-7445.AM2012-4489

MODIFYING MESSENGER RNA

RETURN TO ISSUEPREVNewsNEXTMODIFYING MESSENGER RNACHEMICAL BIOLOGY: Methylated bases in mRNA may have roles in gene regulation and obesitySARAH EVERTSCite this: Chem. Eng. News 2011, 89, 43, 7Publication Date (Print):October 24, 2011Publication History Published online9 March 2012Published inissue 24 October 2011https://doi.org/10.1021/cen-v089n043.p007Copyright © 2011 AMERICAN CHEMICAL SOCIETYArticle Views11Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (292 KB) SUBJECTS:Genetics,Organic reactions Get e-Alerts

Genetic analysis of the ferrochelatase gene in eight Japanese patients from seven families with erythropoietic protoporphyria

A decrease in the activity of ferrochelatase (FECH; EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, results in erythropoietic protoporphyria (EPP; MIM 177000). We analyzed the FECHgene in eight Japanese EPP patients from seven non-consanguineous families and found two distinct genomic DNA abnormalities. In six patients from five families, there was a G-to-A point-mutation at the first position of the intron 9 donor site; it resulted in aberrant splicing and skipping of exon 9 in FECH mRNA. In one patient, we found an A-to-G point-mutation 4 bases from the 3" terminus of intron 4 that led to the in-frame insertion of 3 bases in mRNA. No allelic anomalies, except for 3 single nucleotide polymorphisms were detected in another patient. We analyzed intron polymorphism at IVS3-48, known to be associated with the phenotypic expression of EPP, in these eight patients and 152 healthy Japanese volunteers. All patients were C/C homozygous for IVS3-48. The allelic frequency of IVS3-48C polymorphism in the healthy Japanese volunteers was 67.8% (103/152).

Production and actions of inhibin and activin during folliculogenesis in the rat

Evidence to enhance the premise that inhibin and activin are local regulators of ovarian folliculogenesis is presented in this review. Granulosa cells (GC) have been identified as the source of inhibin/activin in the ovary on the basis of mRNA and protein localisation and the measurement of the inhibin forms in GC conditioned media. Expression of the subunit mRNAs changed with follicular development, being maximal in the ovaries of 8-day-old rats, where secondary follicles predominate. The expression of β subunit mRNAs by GC isolated from diethylstilboestrol (DES)-treated immature rats, was reduced in the absence of any change in α subunit mRNA expression. Dimeric inhibin-A, -B and free α subunit were produced by ovarian cell cultures prepared from 4- to 12-day-old rats. Inhibin-A production by these cultures was responsive to FSH and TGF-β, with preantral follicles of day 8 ovaries exerting effects so profound that the inhibin A/α subunit ratio increased, most likely due to a stimulation of β A subunit production. In contrast, inhibin-B was not stimulated by TGF-β until day 8 and FSH until day 12. Fractionation of GC conditioned media revealed a prominence of free α subunit and inhibin-A, but little inhibin-B, suggesting that inhibin-B production declines with follicular development. Activin receptor types I and II, Smads 1–8 and betaglycan (β-glycan) mRNAs were present in the rat ovary and showed distinct patterns of expression between postnatal days 4 and 12. Oocytes and GC localised activin receptor, Smad and β-glycan proteins, with β-glycan also present in theca cells (TC). These data indicate that activin/TGF-β signalling machinery and factors which influence these pathways, are present in the postnatal rat ovary. Our hypothesis that inhibin and activin play important and changing autocrine/paracrine roles in the growth and differentiation of follicles, including the oocyte, has been supported by these studies.

A geometric model for codon recognition logic

Known types of pairings between mRNA bases and tRNA nucleosiocs are shown to be consistent with the notion of a translation space TS constructed such that certain wobble-pairings cannot be used in the same translation system without engendering confusion between keto-final codon twins like AAU(ASN)/AAG(LYS) and between amino-final codon twins like AAC(ASN)/AAA(LYS). When TS is abstractly formalized using Coxeter's face-first three-dimensional projection of a four-dimensional hypercube, the resulting model suggests a specific configurational logic for codon recognition by cognate tRNAs. Although this logic will in general permit codons and anticodons to form matching configurations whose loci are six lines parallel to the axis of a cylinder, confusion of keto-final and amino-final codon twins may result from wobble-pairings whose loci are the two of these lines off the surface of the cylinder.

Immunological Methods for Determining Phylogenetic Relationships

The concept of organic evolution implies descent of all living and extinct forms from one or more common ancestors in the distant past of earth history. During recent decades, traditional phylogenetic deductions from the anatomy of living and fossil organisms have been supplemented by a vast and growing new body of molecular evidence. Each living creature contains a genome consisting of linear sequences of DNA bases, millions or billions of them, which determine all the developmental, anatomical and physiological characteristics of that species. This DNA is transcribed into messenger RNA (mRNA) which uses the machinery of the cell to make proteins. The sequence of mRNA bases determines the sequence of amino acids of the resultant protein (Lowenstein, 1986).