Molecular Determinants and Specificity of mRNA with Alternatively-Spliced UPF1 Isoforms,Influenced by an Insertion in the ‘Regulatory Loop’

The nonsense-mediated mRNA decay (NMD) pathway rapidly detects and degrades mRNA containing premature termination codons (PTCs). UP-frameshift 1 (UPF1), the master regulator of the NMD process, has two alternatively-spliced isoforms; one carries 353-GNEDLVIIWLR-363 insertion in the ‘regulatory loop (involved in mRNA binding)’. Such insertion can induce catalytic and/or ATPase activity, as determined experimentally; however, the kinetics and molecular level information are not fully understood. Herein, applying all-atom molecular dynamics, we probe the binding specificity of UPF1 with different GC- and AU-rich mRNA motifs and the influence of insertion to the viable control over UPF1 catalytic activity. Our results indicate two distinct conformations between 1B and RecA2 domains of UPF1: ‘open (isoform_2; without insertion)’ and ‘closed (isoform_1; with insertion)’. These structural movements correspond to an important stacking pattern in mRNA motifs, i.e., absence of stack formation in mRNA, with UPF1 isoform_2 results in the ‘open conformation’. Particularly, for UPF1 isoform_1, the increased distance between 1B and RecA2 domains has resulted in reducing the mRNA–UPF1 interactions. Lower fluctuating GC-rich mRNA motifs have better binding with UPF1, compared with AU-rich sequences. Except CCUGGGG, all other GC-rich motifs formed a 4-stack pattern with UPF1. High occupancy R363, D364, T627, and G862 residues were common binding GC-rich motifs, as were R363, N535, and T627 for the AU-rich motifs. The GC-rich motifs behave distinctly when bound to either of the isoforms; lower stability was observed with UPF1 isoform_2. The cancer-associated UPF1 variants (P533L/T and A839T) resulted in decreased protein–mRNA binding efficiency. Lack of mRNA stacking poses in the UPF1_P533T system significantly decreased UPF1-mRNA binding efficiency and increased distance between 1B-RecA2. These novel findings can serve to further inform NMD-associated mechanistic and kinetic studies.     

Imaging target mRNA and siRNA-mediated gene silencing in vivo with ribozyme-based reporters

Noninvasive imaging of specific mRNAs in living subjects promises numerous biological and medical applications. Common strategies use fluorescently or radioactively labelled antisense probes to detect target mRNAs through a hybridization mechanism, but have met with limited success in living animals. Here we present a novel molecular imaging approach based on the group I intron of Tetrahymena thermophila for imaging mRNA molecules in vivo. Engineered trans-splicing ribozyme reporters contain three domains, each of which is designed for targeting, splicing, and reporting. They can transduce the target mRNA into a reporter mRNA, leading to the production of reporter enzymes that can be noninvasively imaged in vivo. We have demonstrated this ribozyme-mediated RNA imaging method for imaging a mutant p53 mRNA both in single cells and noninvasively in living mice. After optimization, the ribozyme reporter increases contrast for the transiently expressed target by 180-fold, and by ten-fold for the stably expressed target. siRNA-mediated specific gene silencing of p53 expression has been successfully imaged in real time in vivo. This new ribozyme-based RNA reporter system should open up new avenues for in vivo RNA imaging and direct imaging of siRNA inhibition.     

G-Quadruplex Regulation of VEGFA mRNA Translation by RBM4

In eukaryotes, mRNAs translation is mainly mediated in a cap-dependent or cap-independent manner. The latter is primarily initiated at the internal ribosome entry site (IRES) in the 5′-UTR of mRNAs. It has been reported that the G-quadruplex structure (G4) in the IRES elements could regulate the IRES activity. We previously confirmed RBM4 (also known as LARK) as a G4-binding protein in human. In this study, to investigate whether RBM4 is involved in the regulation of the IRES activity by binding with the G4 structure within the IRES element, the IRES-A element in the 5′-UTR of vascular endothelial growth factor A (VEGFA) was constructed into a dicistronic reporter vector, psiCHECK2, and the effect of RBM4 on the IRES activity was tested in 293T cells. The results showed that the IRES insertion significantly increased the FLuc expression activity, indicating that this G4-containing IRES was active in 293T cells. When the G4 structure in the IRES was disrupted by base mutation, the IRES activity was significantly decreased. The IRES activity was notably increased when the cells were treated with G4 stabilizer PDS. EMSA results showed that RBM4 specifically bound the G4 structure in the IRES element. The knockdown of RBM4 substantially reduced the IRES activity, whereas over-expressing RBM4 increased the IRES activity. Taking all results together, we demonstrated that RBM4 promoted the mRNA translation of VEGFA gene by binding to the G4 structure in the IRES.     

Platelet-activating factor stimulates expression of IL-1 beta mRNA in THP-1 cells

A human monocytic cell line (THP-1) was used to study the effects of PAF (platelet-activating factor) on the expression of IL-1 beta mRNA. THP-1 cells were incubated with 10 pM PAF in the presence or absence of 0.1 μg/mL endotoxin for 4 hr, after which cytoplasmic RNA was extracted and subjected to Northern hybridizations. PAF, alone and in combination with endotoxin, caused an increase in mRNA levels for IL-1 beta. The magnitude of the effects of PAF on IL-1 beta mRNA levels matched closely the effects seen at the level of protein synthesis, suggesting that the effects of PAF on IL-1 beta release may result largely from its effects on IL-1 beta mRNA levels.     

ATP-Independent Initiation during Cap-Independent Translation of m6A-Modified mRNA

The methylation of adenosine in the N⁶ position (m⁶A) is a widely used modification of eukaryotic mRNAs. Its importance for the regulation of mRNA translation was put forward recently, essentially due to the ability of methylated mRNA to be translated in conditions of inhibited cap-dependent translation initiation, e.g., under stress. However, the peculiarities of translation initiation on m⁶A-modified mRNAs are not fully known. In this study, we used toeprinting and translation in a cell-free system to confirm that m⁶A-modified mRNAs can be translated in conditions of suppressed cap-dependent translation. We show for the first time that m⁶A-modified mRNAs display not only decreased elongation, but also a lower efficiency of translation initiation. Additionally, we report relative resistance of m⁶A-mRNA translation initiation in the absence of ATP and inhibited eIF4A activity. Our novel findings indicate that the scanning of m⁶A-modified leader sequences is performed by a noncanonical mechanism.     

Megalocytivirus Induces Complicated Fish Immune Response at Multiple RNA Levels Involving mRNA,miRNA, and circRNA

Megalocytivirus is an important viral pathogen to many farmed fishes, including Japanese flounder (Paralichthys olivaceus). In this study, we examined megalocytivirus-induced RNA responses in the spleen of flounder by high-throughput sequencing and integrative analysis of various RNA-seq data. A total of 1327 microRNAs (miRNAs), including 368 novel miRNAs, were identified, among which, 171 (named DEmiRs) exhibited significantly differential expressions during viral infection in a time-dependent manner. For these DEmiRs, 805 differentially expressed target mRNAs (DETmRs) were predicted, whose expressions not only significantly changed after megalocytivirus infection but were also negatively correlated with their paired DEmiRs. Integrative analysis of immune-related DETmRs and their target DEmiRs identified 12 hub DEmiRs, which, together with their corresponding DETmRs, formed an interaction network containing 84 pairs of DEmiR and DETmR. In addition to DETmRs, 19 DEmiRs were also found to regulate six key immune genes (mRNAs) differentially expressed during megalocytivirus infection, and together they formed a network consisting of 21 interactive miRNA-messenger RNA (mRNA) pairs. Further analysis identified 9434 circular RNAs (circRNAs), 169 of which (named DEcircRs) showed time-specific and significantly altered expressions during megalocytivirus infection. Integrated analysis of the DETmR-DEmiR and DEcircR-DEmiR interactions led to the identification of a group of competing endogenous RNAs (ceRNAs) constituted by interacting triplets of circRNA, miRNA, and mRNA involved in antiviral immunity. Together these results indicate that complicated regulatory networks of different types of non-coding RNAs and coding RNAs are involved in megalocytivirus infection.     

Random Copolymers of Lysine and Isoleucine for Efficient mRNA Delivery

Messenger RNA (mRNA) is currently of great interest as a new category of therapeutic agent, which could be used for prevention or treatment of various diseases. For this mRNA requires effective delivery systems that will protect it from degradation, as well as allow cellular uptake and mRNA release. Random poly(lysine-co-isoleucine) polypeptides were synthesized and investigated as possible carriers for mRNA delivery. The polypeptides obtained under lysine:isoleucine monomer ratio equal to 80/20 were shown to give polyplexes with smaller size, positive ζ-potential and more than 90% encapsulation efficacy. The phase inversion method was proposed as best way for encapsulation of mRNA into polyplexes, which are based on obtained amphiphilic copolymers. These copolymers showed efficacy in protection of bound mRNA towards ribonuclease and lower toxicity as compared to lysine homopolymer. The poly(lysine-co-isoleucine) polypeptides showed greater than poly(ethyleneimine) efficacy as vectors for transfection of cells with green fluorescent protein and firefly luciferase encoding mRNAs. This allows us to consider obtained copolymers as promising candidates for mRNA delivery applications.     

Psoriasis-Associated Inflammatory Conditions Induce IL-23 mRNA Expression in Normal Human Epidermal Keratinocytes

Psoriasis is a multifactorial, chronic inflammatory skin disease, the development of which is affected by both genetic and environmental factors. Cytosolic nucleic acid fragments, recognized as pathogen- and danger-associated molecular patterns, are highly abundant in psoriatic skin. It is known that psoriatic skin exhibits increased levels of IL-23 compared to healthy skin. However, the relationship between free nucleic acid levels and IL-23 expression has not been clarified yet. To examine a molecular mechanism by which nucleic acids potentially modulate IL-23 levels, an in vitro system was developed to investigate the IL-23 mRNA expression of normal human epidermal keratinocytes under psoriasis-like circumstances. This system was established using synthetic nucleic acid analogues (poly(dA:dT) and poly(I:C)). Signaling pathways, receptor involvement and the effect of PRINS, a long non-coding RNA previously identified and characterized by our research group, were analyzed to better understand the regulation of IL-23 in keratinocytes. Our results indicate that free nucleic acids regulate epithelial IL-23 mRNA expression through the TLR3 receptor and specific signaling pathways, thereby, contributing to the development of an inflammatory milieu favorable for the appearance of psoriatic symptoms. A moderate negative correlation was confirmed between the nucleic-acid-induced IL-23 mRNA level and the rate of its decrease upon PRINS overexpression.     

Metal ion effects on the cis/trans isomerization equilibrium of proline in short-chain peptides: a solution NMR study

The effect of copper(II) ions on the probabilities of existence of the four detectable conformers of the tetrapeptide Tyr-Pro-Phe-Pro (beta-casomorphin 4) in [2H6]DMSO was investigated by 1H NMR spectroscopy. Integration of the Phe-NH signals provided the relative populations in the free state as tt/tc/ct/cc=28:34:29:9 at 293 K (c=cis, t=trans). Copper(II) was shown to bind to all four isomers, yielding complexes with two different structures, depending on the conformation of Pro(2). The interpretation of paramagnetic relaxation rates of Pro(2)-Halpha signals provided the corresponding isomeric probabilities in the metal-bound state as 13:36:20:31. The observed stabilization of the conformation with the lowest probability of existence (cc) may be relevant for the biological role of copper and other metal ions.     

Giant vesicles as microreactors for enzymatic mRNA synthesis

Giant vesicles have attracted much attention as possible microreactors for the conduction of enzymatic reactions in an artificial, cell-sized compartment. In this context, we demonstrated in the first part of the present work that giant vesicles formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in an alternating electric field can be made more permeable to Ca(2+) ions or nucleotide triphosphates by addition of ethanol. This methodology is then applied in a second step whereby these giant vesicles are used as microreactors in which mRNA synthesis can occur. The macromolecules (the DNA template and the enzyme T7 RNA polymerase) are microinjected into a selected giant vesicle, while the substrate molecules (nucleotide triphosphates) are added from the external medium. The fact that mRNA synthesis can be detected is a further step towards our aim: the design of a microreactor that can be seen as a model for a protocell.     

Chemoenzymatic Preparation of Functional Click-Labeled Messenger RNA

Synthetic mRNAs are promising candidates for a new class of transformative drugs that provide genetic information for patients’ cells to develop their own cure. One key advancement to develop so-called druggable mRNAs was the preparation of chemically modified mRNAs, by replacing standard bases with modified bases, such as uridine with pseudouridine, which can ameliorate the immunogenic profile and translation efficiency of the mRNA. Thus the introduction of modified nucleobases was the foundation for the clinical use of such mRNAs. Herein we describe modular and simple methods to chemoenzymatically modify mRNA. Alkyne- and/or azide-modified nucleotides are enzymatically incorporated into mRNA and subsequently conjugated to fluorescent dyes using click chemistry. This allows visualization of the labeled mRNA inside cells. mRNA coding for the enhanced green fluorescent protein (eGFP) was chosen as a model system and the successful expression of eGFP demonstrated that our modified mRNA is accepted by the translation machinery.