Cells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates that are enriched in enzymes important for mRNA degradation and have been identified as sites of ...both storage and decay. How these opposing outcomes can be achieved in condensates remains unresolved. mRNA decapping immediately precedes degradation, and the Dcp1/Dcp2 decapping complex is enriched in P-bodies. Here, we show that Dcp1/Dcp2 activity is modulated in condensates and depends on the interactions promoting phase separation. We find that Dcp1/Dcp2 phase separation stabilizes an inactive conformation in Dcp2 to inhibit decapping. The activator Edc3 causes a conformational change in Dcp2 and rewires the protein-protein interactions to stimulate decapping in condensates. Disruption of the inactive conformation dysregulates decapping in condensates. Our results indicate that the regulation of enzymatic activity in condensates relies on a coupling across length scales ranging from microns to ångstroms. We propose that this regulatory mechanism may control the functional state of P-bodies and related phase-separated compartments.
7-Methylguanosine 5' cap on mRNA is necessary for efficient protein expression in vitro and in vivo. Recent studies revealed structural diversity of endogenous mRNA caps, which carry different ...5'-terminal nucleotides and additional methylations (2'-O-methylation and m6A). Currently available 5'-capping methods do not address this diversity. We report trinucleotide 5' cap analogs (m7GpppN(m)pG), which are utilized by RNA polymerase T7 to initiate transcription from templates carrying Φ6.5 promoter and enable production of mRNAs differing in the identity of the first transcribed nucleotide (N = A, m6A, G, C, U) and its methylation status (±2'-O-methylation). HPLC-purified mRNAs carrying these 5' caps were used to study protein expression in three mammalian cell lines (3T3-L1, HeLa and JAWS II). The highest expression was observed for mRNAs carrying 5'-terminal A/Am and m6Am, whereas the lowest was observed for G and Gm. The mRNAs carrying 2'-O-methyl at the first transcribed nucleotide (cap 1) had significantly higher expression than unmethylated counterparts (cap 0) only in JAWS II dendritic cells. Further experiments indicated that the mRNA expression characteristic does not correlate with affinity for translation initiation factor 4E or in vitro susceptibility to decapping, but instead depends on mRNA purity and the immune state of the cells.
COVID-19 vaccinations are about to begin in various countries or are already ongoing. This is an unprecedented operation that is also met with a loud response from anti-vaccine communities-currently ...using all available channels to manipulate public opinion. At the same time, the strategy to educate on vaccinations, explain their mechanism of action, and build trust in science is subdued in different world parts. Such actions should go much beyond campaigns promoting the COVID-19 vaccines solely on the information provided by the health institutions and national authorities. In this paper, actions provided by independent expert groups needed to counteract the anti-vaccine propaganda and provide scientific-based information to the general public are offered. These actions encompass organizing groups continuously communicating science on COVID-19 vaccines to the general public; tracking and tackling emerging and circulating fake news; and equipping celebrities and politicians with scientific information to ensure the quality of messages they communicate, as well as public letters, and statements of support for vaccination by healthcare workers, recognized scientists, VIPs, and scientific societies; and no tolerance to false and manipulated claims on vaccination spread via traditional and social media as well as by health professionals, scientists, and academics. These activities should be promptly implemented worldwide, regardless of the current status and availability of the COVID-19 vaccine in a particular region. If we are about to control the pandemic for the sake of public benefit, it is high time to collectively speak out as academic and medical societies with support from decision-makers. Otherwise, the battle will be lost to those who stand against scientific evidence while offering no feasible solution to the problem.
Chemical modifications enable preparation of mRNAs with augmented stability and translational activity. In this study, we explored how chemical modifications of 5',3'-phosphodiester bonds in the mRNA ...body and poly(A) tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogs modified at the α-phosphate (containing either O-to-S or O-to-BH
substitutions) and three different RNA polymerases-SP6, T7, and poly(A) polymerase. To verify the efficiency of incorporation of ATP analogs in the presence of ATP, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantitative assessment of modification frequency based on exhaustive degradation of the transcripts to 5'-mononucleotides. The method also estimated the average poly(A) tail lengths, thereby providing a versatile tool for establishing a structure-biological property relationship for mRNA. We found that mRNAs containing phosphorothioate groups within the poly(A) tail were substantially less susceptible to degradation by 3'-deadenylase than unmodified mRNA and were efficiently expressed in cultured cells, which makes them useful research tools and potential candidates for future development of mRNA-based therapeutics.
Synthetic nucleotide and nucleic acid analogues are useful research tools and modern therapeutics. Hence, methods for the rapid and unambiguous identification of mononucleotides derived from organic ...syntheses or biological materials are of broad interest. Here, we analysed over 150 mononucleotides (mostly nucleoside 5'-mono-, 5'-di-, and 5'-triphosphates) and their structurally related nucleobase-, phosphate-, and ribose-modified analogues by electrospray tandem mass spectrometry (ESI/MS/MS), identifying characteristic fragmentation ions that may be helpful in structure determination. While positive-ion mode yielded fragments derived mainly from nucleobases, negative-ion mode provided insight into the structures of phosphoryl and phosphoribosyl moieties, enabling the determination of structural features such as the number of phosphate groups and the presence of ribose or phosphate substitutions. Based on these data, we proposed fragmentation pathways that were confirmed by experiments with
O-isotopologues. We demonstrated the utility of ESI(-)/MS/MS in the analysis of structurally related compounds by analysing isomeric and isobaric nucleotides and applying ESI(-)/MS/MS to rapid identification of nucleotide synthesis products. We formulated general rules regarding nucleotide structure-fragmentation pattern relationships and indicating characteristic fragmentation ions for the interpretation of ESI(-)/MS/MS spectra of nucleotides and their analogues. The ESI(-)/MS/MS spectra of all nucleotides are available in an on-line database, msTide, at www.msTide-db.com.
Fluorescence anisotropy (FA) is a powerful technique for the discovery of protein inhibitors in a high-throughput manner. In this study, we sought to develop new universal FA-based assays for the ...evaluation of compounds targeting mRNA 5' cap-binding proteins of therapeutic interest, including eukaryotic translation initiation factor 4E and scavenger decapping enzyme. For this purpose, a library of 19 carboxyfluorescein probes based on 7-methylguanine nucleotides was evaluated as FA probes for these proteins. Optimal probe:protein systems were further investigated in competitive binding experiments and adapted for high-throughput screening. Using a small in-house library of compounds, we verified and confirmed the accuracy of the developed FA assay to study cap-binding protein binders. The applications of the most promising probes were then extended to include evaluation of allosteric inhibitors as well as RNA ligands. From this analysis, we confirmed the utility of the method to study small molecule ligands and evaluate differently 5' capped RNAs.
Gold nanoparticles (AuNPs) decorated with biologically relevant molecules have variety of applications in optical sensing of bioanalytes. Coating AuNPs with small nucleotides produces particles with ...high stability in water, but functionality-compatible strategies are needed to uncover the full potential of this type of conjugates. Here, we demonstrate that lipoic acid-modified dinucleotides can be used to modify AuNPs surfaces in a controllable manner to produce conjugates that are stable in aqueous buffers and biological mixtures and capable of interacting with nucleotide-binding proteins. Using this strategy we obtained AuNPs decorated with 7-methylguanosine mRNA 5' cap analogs and showed that they bind cap-specific protein, eIF4E. AuNPs decorated with non-functional dinucleotides also interacted with eIF4E, albeit with lower affinity, suggesting that eIF4E binding to cap-decorated AuNPs is partially mediated by unspecific ionic interactions. This issue was overcome by applying lipoic-acid-Tris conjugate as a charge-neutral diluting molecule. Tris-Lipo-diluted cap-AuNPs conjugates interacted with eIF4E in fully specific manner, enabling design of functional tools. To demonstrate the potential of these conjugates in protein sensing, we designed a two-component eIF4E sensing system consisting of cap-AuNP and 4E-BP1-AuNP conjugates, wherein 4E-BP1 is a short peptide derived from 4E-BP protein that specifically binds eIF4E at a site different to that of the 5' cap. This system facilitated controlled aggregation, in which eIF4E plays the role of the agent that crosslinks two types of AuNP, thereby inducing a naked-eye visible absorbance redshift. The reported AuNPs-nucleotide conjugation method based on lipoic acid affinity for gold, can be harnessed to obtain other types of nucleotide-functionalized AuNPs, thereby paving the way to studying other nucleotide-binding proteins.
The conserved decapping enzyme Dcp2 recognizes and removes the 5' eukaryotic cap from mRNA transcripts in a critical step of many cellular RNA decay pathways. Dcp2 is a dynamic enzyme that functions ...in concert with the essential activator Dcp1 and a diverse set of coactivators to selectively and efficiently decap target mRNAs in the cell. Here we present a 2.84 Å crystal structure of K. lactis Dcp1-Dcp2 in complex with coactivators Edc1 and Edc3, and with substrate analog bound to the Dcp2 active site. Our structure shows how Dcp2 recognizes cap substrate in the catalytically active conformation of the enzyme, and how coactivator Edc1 forms a three-way interface that bridges the domains of Dcp2 to consolidate the active conformation. Kinetic data reveal Dcp2 has selectivity for the first transcribed nucleotide during the catalytic step. The heterotetrameric Edc1-Dcp1-Dcp2-Edc3 structure shows how coactivators Edc1 and Edc3 can act simultaneously to activate decapping catalysis.
The RNA-binding proteins Roquin-1 and Roquin-2 redundantly control gene expression and cell-fate decisions. Here, we show that Roquin not only interacts with stem-loop structures, but also with a ...linear sequence element present in about half of its targets. Comprehensive analysis of a minimal response element of the Nfkbid 3'-UTR shows that six stem-loop structures cooperate to exert robust and profound post-transcriptional regulation. Only binding of multiple Roquin proteins to several stem-loops exerts full repression, which redundantly involved deadenylation and decapping, but also translational inhibition. Globally, most Roquin targets are regulated by mRNA decay, whereas a small subset, including the Nfat5 mRNA, with more binding sites in their 3'-UTRs, are also subject to translational inhibition. These findings provide insights into how the robustness and magnitude of Roquin-mediated regulation is encoded in complex cis-elements.
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