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The presence of N.sup.1 methyl groups on adenine bases was thought to be widespread in messenger RNAs. It now seems that these modifications are much less prevalent, and occur on mRNAs that structurally mimic transfer RNA. See Letter (https://www.nature.com/articles/nature24456) The basis of m1A modification N.sup.1-methyladenosine (m.sup.1A) modification has been detected on mRNA, but validation of the internal mRNA sites at which it occurs and the functional consequences of it have not been well defined. Schraga Schwartz and colleagues now address these limitations using a method that enables single-nucleotide resolution of such sites in the transcriptome. They show that the level of modification is much lower than reported previously and varies during development and by tissue type. The authors identify a structural motif associated with the modification and define the enzymatic machinery responsible for the methylation. They find that m.sup.1A modification is associated with translational repression, consistent with its tight regulation., Author(s): Anya V. Grozhik [sup.1] , Samie R. Jaffrey [sup.1] Author Affiliations: (1) Department of Pharmacology, Weill Cornell Medicine, Cornell University, New York, USA The fate of messenger RNAs is [...]

Understanding how RNA structure influences its function has been hampered by a lack of approaches that can accurately quantify RNA structure in vivo; here, RNA structure is revealed on a global scale and with nucleotide-level resolution, showing that there is less structure within cells than expected from in vitro and in silico analyses. Probing the in vivo RNA structurome Being single-stranded, RNA can adopt a diversity of secondary structures via inter- and intramolecular base-pairing. Three studies published in this issue of Nature provide an in-depth view of the variety, dynamics and functional influence of RNA structures in vivo. Sarah Assmann and colleagues map the in vivo RNA structure of over 10,000 transcripts in the model plant Arabidopsis thaliana. Their struc-seq (structure-seqence) approach incorporates in vivo chemical (DMS) probing and next-generation sequencing to provide single-nucleotide resolution on a genome-wide scale. Distinct patterns of structure are found to be correlated with coding regions, splice sites and polyadenylation sites. Comparison of these results with those obtained by earlier technologies reveals that, although predictions for some classes of genes were fairly accurate, others, such as those involved in stress response, were poorly predicted and may reflect changes that made them more adapted to that condition. Jonathan Weissman and colleagues have also developed a DMS-seq method to globally monitor RNA structure to single-nucleotide precision in yeast and mammalian cells. Comparing their findings with in vitro data, the authors conclude that there is less structure within cells than expected. Even thermostable RNA structures can be denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Howard Chang and colleagues asked a different question: how does RNA secondary structure change on a transcriptome-wide level in related individuals? By calculating the RNA secondary structures of two parents and their child, they find that about 15% of transcribed single-nucleotide variants affect local secondary structure. These 'RiboSNitches' are depleted in certain locations, suggesting that a particular RNA structure at that site is important. This study illustrates that there is much to be learned about how changes in RNA structure, particularly as imparted by genetic variation, can alter gene expression. RNA has a dual role as an informational molecule and a direct effector of biological tasks. The latter function is enabled by RNA's ability to adopt complex secondary and tertiary folds and thus has motivated extensive computational.sup.1,2 and experimental.sup.3,4,5,6,7,8 efforts for determining RNA structures. Existing approaches for evaluating RNA structure have been largely limited to in vitro systems, yet the thermodynamic forces which drive RNA folding in vitro may not be sufficient to predict stable RNA structures in vivo.sup.5. Indeed, the presence of RNA-binding proteins and ATP-dependent helicases can influence which structures are present inside cells. Here we present an approach for globally monitoring RNA structure in native conditions in vivo with single-nucleotide precision. This method is based on in vivo modification with dimethyl sulphate (DMS), which reacts with unpaired adenine and cytosine residues.sup.9, followed by deep sequencing to monitor modifications. Our data from yeast and mammalian cells are in excellent agreement with known messenger RNA structures and with the high-resolution crystal structure of the Saccharomyces cerevisiae ribosome.sup.10. Comparison between in vivo and in vitro data reveals that in rapidly dividing cells there are vastly fewer structured mRNA regions in vivo than in vitro. Even thermostable RNA structures are often denatured in cells, highlighting the importance of cellular processes in regulating RNA structure. Indeed, analysis of mRNA structure under ATP-depleted conditions in yeast shows that energy-dependent processes strongly contribute to the predominantly unfolded state of mRNAs inside cells. Our studies broadly enable the functional analysis of physiological RNA structures and reveal that, in contrast to the Anfinsen view of protein folding whereby the structure formed is the most thermodynamically favourable, thermodynamics have an incomplete role in determining mRNA structure in vivo., Author(s): Silvi Rouskin [sup.1] , Meghan Zubradt [sup.1] , Stefan Washietl [sup.2] [sup.3] [sup.4] , Manolis Kellis [sup.2] [sup.3] [sup.4] , Jonathan S. Weissman [sup.1] Author Affiliations: (1) Department of [...]

A recent investigation concluded that codon bias did not affect expression of green fluorescent protein (GFP) variants in Escherichia coli, while stability of an mRNA secondary structure near the 5? end played a dominant role. We demonstrate that combining the two variables using regression trees or support vector regression yields a biologically plausible model with better support in the GFP data set and in other experimental data: codon usage is relevant for protein levels if the 5' mRNA structures are not strong. Natural E. coli genes had weaker 5' mRNA structures than the examined set of GFP variants and did not exhibit a correlation between the folding free energy of 5' mRNA structures and protein expression.

Type V myosin (MyoV)-dependent transport of cargo is an essential process in eukaryotes. Studies on yeast and vertebrate MyoV showed that their globular tails mediate binding to the cargo complexes. In Saccharomyces cerevisiae, the MyoV motor Myo4p interacts with She3p to localize asymmetric synthesis of HO 1 (ASH1) mRNA into the bud of dividing cells. A recent study showed that localization of GFP-MS2-tethered ASH1 particles does not require the Myo4p globular tail, challenging the supposed role of this domain. We assessed ASH1 mRNA and Myo4p distribution more directly and found that their localization is impaired in cells expressing globular tail-lacking Myo4p. In vitro studies further show that the globular tail together with a more N-terminal linker region is required for efficient She3p binding. We also determined the x-ray structure of the Myo4p globular tail and identify a conserved surface patch important for She3p binding. The structure shows pronounced similarities to membrane-tethering complexes and indicates that Myo4p may not undergo auto-inhibition of its motor domain. doi/ 10.1083/jcb.201002076

The DExD/H-box RNA-dependent ATPase Dbp5 plays an essential role in the nuclear export of mRNA. Dbp5 localizes to the nuclear pore complex, where its ATPase activity is stimulated by Gle1 and its coactivator inositol hexakisphosphate. Here, we present the crystal structure of the C-terminal domain of Dbp5, refined to 1.8 A. The structure reveals a RecA-like fold that contains two defining characteristics not present in other structurally characterized DExD/H-box proteins: a C-terminal s-helix and a loop connecting [beta]35 and [alpha]4, both of which are composed of conserved and unique elements in the Dbp5 primary sequence. Using structure-guided mutagenesis, we have identified several charged surface residues that, when mutated, weaken the binding of Gle1 and inhibit the ability of Gle 1 to stimulate Dbp5's ATPase activity. In vivo analysis of the same mutations reveals that those mutants displaying the weakest ATPase stimulation in vitro are also unable to support yeast growth. Analysis of the correlation between the in vitro and in vivo data indicates that a threshold level of Dbp5 ATPase activity is required for cellular mRNA export that is not met by the unstimulated enzyme, suggesting a possible mechanism by which DbpS's activity can be modulated to regulate mRNA export. crystal structure | DExD/H-box | nuclear pore complex

RNase R is a 3'-5' highly processive exoribonuclease that can digest RNAs with extensive secondary structure. We analyzed the global elect of eliminating RNase R on the Pseudomonas putida transcriptome and the expression of the rnr gene under diverse conditions. The absence of RNase R led to increased levels of many mRNAs, indicating that it plays an important role in mRNA turnover.

Many viruses avoid immune surveillance during latent infection through reduction in the synthesis of virally encoded proteins. Although antigen presentation critically depends on the level of viral protein synthesis, the precise mechanism used to regulate the generation of antigenic peptide precursors remains elusive. Here, we demonstrate that a purine overloaded virally encoded mRNA lacking secondary structure significantly impacts the efficiency of protein translation and prevents endogenous antigen presentation. Reducing this purine bias through the generation of constructs expressing codon-modified sequences, while maintaining the encoded protein sequence, increased the stem-loop structure of the corresponding mRNA and dramatically enhanced self-synthesis of the viral protein. As a consequence, a higher number of HLA-peptide complexes were detected on the surface of cells expressing this viral protein. Furthermore, these cells were more efficiently recognized by virus-specific T cells compared with those expressing the same antigen expressed by a purine-biased mRNA. These findings delineate a mechanism by which viruses regulate self-synthesis of proteins and offer an effective strategy to evade [CD8.sup.+] T cell-mediated immune regulation. antigen processing | EBV-encoded nuclear antigen 1 | immune evasion | protein synthesis

The initiation of translation in eukaryotes generally involves the recognition of a 'cap' structure at the 5' end of the mRNA. However, for some viral and cellular mRNAs, a cap-independent mechanism occurs through an mRNA structure known as the internal ribosome entry site (IRES). Here, I postulate that the first eukaryotic mRNAs were translated in a cap-independent, IRES-driven manner that was then superseded in evolution by the cap-dependent mechanism, rather than vice versa. This hypothesis is supported by the following observations: (i) IRES-dependent, but not cap-dependent, translation can take place in the absence of not only a cap, but also many initiation factors; (ii) eukaryotic initiation factor 4E (elF4E) and elF4G, molecules absolutely required for cap-dependent translation, are among the most recently evolved translation factors; and (iii) functional similarities suggest the evolution of IRESs from spliceosomal introns. Thus, the contemporary cellular IRESs might be relics of the past.

Degradation of the cspA mRNA in vivo is very rapid at temperatures greater than 30[degrees]C and is moderately dependent on RNase E. Investigations in vitro show that degradosomes prepared from normal or cold-shocked cultures cleave the cspA mRNA preferentially at a single site in vitro between two stem-loops ~24 residues 3' to the termination codon and ~31 residues from the 3' end. The site of cleavage is independent of the temperature and largely independent of the phosphorylation status of the 5' end of cspA mRNA. A 5' stem-loop, potential occlusion of the initiation and termination codons, temperature-dependent translational efficiency, and the position of the RNase E cleavage site can explain the differential stability of the cspA mRNA.

A study was conducted to explore the structural properties, mechanistic details, and physiological significance of tmRNA and its principal protein partners. The evidence indicates that in addition to its quality-control function the tmRNA system might also play a key regulatory role in certain physiological pathways.

There is clinical evidence linking asthma with the trace element, zinc (Zn). Using a mouse model of allergic inflammation, we have previously shown that labile Zn decreases in inflamed airway epithelium (Truong-Tran AQ, Ruffin RE, Foster PS, Koskinen AM, Coyle P, Philcox JC, Rofe AM, Zalewski PD. Am J Respir Cell Mol Biol 27: 286-296, 2002). Moreover, mild nutritional Zn deficiency worsens lung function. Recently, a number of proteins belonging to the Solute Carrier Family 39 (ZIP) and Solute Cartier Family 30 (ZnT) have been identified that bind Zn and regulate Zn homeostasis. Mice were sensitized, and subsequently aerochallenged, with ovalbumin to induce acute and chronic airway inflammation. Mice received 0, 54, or 100 [micro]g of Zn intraperitoneally. Tissues were analyzed for Zn content and histopathology. Inflammatory cells were counted in bronchoalveolar lavage fluid. Cytokine and Zn transporter mRNA levels were determined by eDNA gene array and/or real-time PCR. Zn supplementation decreased bronchoalveolar lavage fluid eosinophils by 40 and 80%, and lymphocytes by 55 and 66%, in the acute and chronic models, respectively. Alterations in Zn transporter expression were observed during acute inflammation, including increases in ZIP1 and ZIP14 and decreases in ZIP4 and ZnT4. Zn supplementation normalized ZIP1 and ZIP14, but it did not affect mRNA levels of cytokines or their receptors. Our results indicate that inflammation-induced alterations in Zn transporter gene expression are directed toward increasing Zn uptake. Increases in Zn uptake may be needed to counteract the local loss of Zn in the airway and to meet an increased demand for Zn-dependent proteins. The reduction of inflammatory cells by Zn in the airways provides support for Zn supplementation trials in human asthmatic individuals. asthma; airway; lung

An AUF1 knockout mouse is developed to understand the function of the AU-rich element (ARE)-binding protein AUF1. The results have provided the first in vivo evidence implicating AUF1 in regulation of the inflammatory response and they have provided a molecular understanding of AUF1 action in selective targeted degradation of inflammatory cytokine mRNAs.

The nucleolus is the largest subnuclear structure and is plurifunctional in nature. Here, we demonstrate that nucleolar localization of a key herpesvirus regulatory protein is essential for its role in virus mRNA nuclear export. The herpesvirus saimiri ORF57 protein is a nucleocytoplasmic shuttle protein that is conserved in all herpesviruses and orchestrates the nuclear export of viral intronless mRNAs. We demonstrate that expression of the ORF57 protein induces nucleolar redistribution of human TREX (transcription/ export) proteins that are involved in mRNA nuclear export. Moreover, we describe a previously unidentified nucleolar localization signal within ORF57 that is composed of two distinct nuclear localization signals. Intriguingly, point mutations that ablate ORF57 nucleolar localization lead to a failure of ORF57-mediated viral mRNA nuclear export. Furthermore, nucleolar retargeting of the ORF57 mutant was achieved by the incorporation of the HIV-1 Rev nucleolar localization signal, and analysis demonstrated that this modification was sufficient to restore viral mRNA nuclear export. This finding represents a unique and fundamental role for the nucleolus in nuclear export of viral mRNA. mRNA export | nucleolus | virus

MicroRNAs (miRNAs) attenuate gene expression by means of translational inhibition and mRNA degradation. They are abundant, highly conserved, and predicted to regulate a large number of transcripts. Several hundred miRNA classes are known, and many are associated with cell proliferation and differentiation. Many exhibit tissue-specific expression, which aids in evaluating their functions, and it has been assumed that their high level of sequence conservation implies a high level of expression conservation. A limited amount of data supports this, although discrepancies do exist. By comparing the expression of [approximately equal to] 100 miRNAs in medaka and chicken with existing data for zebrafish and mouse, we conclude that the timing and location of miRNA expression is not strictly conserved. In some instances, differences in expression are associated with changes in miRNA copy number, genomic context, or both between species. Variation in miRNA expression is more pronounced the greater the differences in physiology, and it is enticing to speculate that changes in miRNA expression may play a role in shaping the physiological differences produced during animal development. chick | evolution | medaka | miRNA | zebrafish

A comparative sequence-structure analysis of hfq genes in bacterial genomes is employed to identify conserved residues that may be involved in binding RNA. A covariance of residue properties at neighboring positions 12 and 39 and conserved surfaces residues with high propensities at binding sites of RNA- binding proteins suggested several sites for Hfq-RNA interactions.

The structural and biochemical studies related to the tmRNA system which performs translational surveillance and ribosome rescue and also some additional roles such as stress management and regulation of transcriptional circuits is presented.

Byline: S. E. Dmitriev (1), I. M. Terenin (1), M. P. Rubtsova (1), I. N. Shatsky (1) Keywords: translation initiation; [beta]-globin mRNA; mRNA secondary structure; 5'-untranslated region; initiation factor eIF2; reconstruction of the 48S initiation complex; mammals Abstract: Nucleotide sequence changes increasing the number of paired bases without producing stable secondary structure in the 5'-untranslated region (5'-UTR) of the [beta]-globin mRNA had a slight effect on its translation in rabbit reticulocyte lysate at low mRNA concentration and dramatically decreased translation efficiency at a high concentration. The removal of paired regions restored translation. Addition of purified eIF2 to the lysate resulted in equal translation efficiencies of templates differing in structure of 5'-UTR. A similar effect was observed for p50, a major mRNP protein. Other mRNA-binding initiation factors, eIF4F and eIF4B, had no effect on the dependence of translation efficiency on mRNA concentration. Analysis of the assembly of the 48S initiation complex from its purified components showed that less eIF2 is required for translation initiation on the [beta]-globin mRNA than on its derivative containing minor secondary structure elements in 5'-UTR. According to a model proposed, eIF2 not only delivers Met-tRNA, but it also stabilizes the interaction of the 40S ribosome subunit with 5'-UTR, which is of particular importance for translation initiation on templates with structured 5'-UTR. Author Affiliation: (1) Belozerskii Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119899, Russia Article History: Registration Date: 12/10/2004

Byline: I. B. Khvorostov (1), M. K. Ivanov (1), G. M. Dymshits (1) Keywords: mitochondrial genome; recombination; transcription; expression regulation Abstract: The multistep regulation of mitochondrial gene expression in higher plants is considered. Data are summarized that concern the structure and function of the transcription system, posttranscriptional changes in the mRNA structure, and other levels of expression regulation. Author Affiliation: (1) Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences, Novosibirsk, 630090, Russia Article History: Registration Date: 09/10/2004

Byline: L. P. Ovchinnikov (1), M. A. Skabkin (1), P. V. Ruzanov (1), V. M. Evdokimova (1) Keywords: mRNA; mRNP; p50; PABP; structural organization; translation; regulation; life time; actin; localization Abstract: The properties of two universal major proteins of cytoplasmic mRNP, p50 and the poly(A)-binding protein (PABP), are summarized. Their roles in formation of polyribosomal and free inactive mRNP are considered, with the focus on the authors' studies of p50. The parts these mRNP proteins play in translation regulation, stability, and localization of mRNA are described, and the possible mechanisms of their function are discussed. Author Affiliation: (1) Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia Article History: Registration Date: 08/10/2004

A cell-free translation mixture and a 27 MHz quartz-crystal microbalance (QCM) was used to develop a system that allowed real-time monitoring of protein synthesis and understand the reactions of protein translation. The developed approach allowed assessing the effects of different antibiotics and mRNA sequences on the different steps of protein translation and the rate-limiting steps in protein synthesis.

The small size and domain mobility has made the PBCV-1 mRNA capping enzyme an ideally suited model system for exploring domain mobility in context of substrate binding. The results have shown that binding efficiency is a function of conformation but that isomerization between efficient and inefficient binding conformations have not affected the substrate association rate.

Hairpin loops from c-src and GABAA [gamma]2 pre-mRNAs and from the 3' terminus of hepatitis C virus (HCV) were compared to a single-stranded polypyrimidine tract from GABAA [gamma]2 pre-mRNA to understand the modular binding function of structured or unstructured polypyrimidine tract binding protein 1 (PTB1) RNA. The structured or single-stranded binding of short and long polypyrimidine tracts provided insights into the versatile role of PTB as an adaptor protein that facilitates formation of RNA-protein regulatory complexes.

A study was conducted to probe the crystal structures and CstF-50 binding characteristics of BARD1 C-terminal ankyrin and tandem BRCT repeat domains which modulate mRNA processing and RNAP II stability in response to DNA damage. The results revealed that BARD1 structure and plasticity imparted by the ANK-BRCT liner facilitate the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

The kinetic and thermodynamic characterization of the reaction catalyzed by an RNA guanylyltransferase capping enzyme involved in the synthesis of the 5' end of eukaryotic mRNAs, from Paramecium bursaria Chorella virus 1 is described. The findings provide insight into the highest energy required by the RNA substrates to promote the catalytic activities of the enzyme.

A comparative analysis of bacterial mRNA sequences of Bacillus stearothermophilus were carried out to define the features recognized by binding site of an unusual ribosomal protein complex, L10[(L12).sub.4]. The mRNA and ribosome targets were found to use similar RNA features, held in different structural contexts, to recognize L10[(L12).sub.4], while the ribosome is found to ensure the saturation of its L10[(L12).sub.4] binding site by means of an additional protein-protein interaction.

A combination of the hydrogen-deuterium exchange method along with electrospray mass spectrometry is applied to monitor structural rearrangement of eukaryotic initiation factor 4E (eIF4E) upon binding of the mRNA 5' cap analogues. The results suggest that the additional double methylation at the [N.sup.2]-amino group of the cap, which causes sterical effects upon binding to mammalian eIF4E, influences the overall solution dynamics of the protein.

Results show that mRNA, the eukaryotic initiation factor 4G (elF4G), and elF4E exist as a stable mRNP, which mediates ribosomal loading during translation initiation in protein synthesis. The solution structure of the complex between yeast elf4E/cap and elF4G is presented.