Biology subjects

Kowalska, J.

Publications and source records attributed to Kowalska, J..

16 recordsLinked to original sources

bioRxiv2026

Sequence-encoded autoinhibition couples mRNA decapping activity to phase separation

Removal of the 5' mG cap by the Dcp1/Dcp2 complex commits mRNAs to degradation, yet the mechanisms regulating decapping remain incompletely understood. Here, we identify residue-level determinants within the extended C-terminus of fission yeast Dcp2 that repress activity. Mutations in conserved inhibitory motifs relieve autoinhibition, enhance RNA binding, and bypass the requirement for the activator Edc3. Strikingly, this activation persists within phase-separated condensates, demonstrating that conformational relief in solution is propagated to the dense phase. We further show that long-range interactions between the intrinsically disordered region of Dcp2 and the catalytic core restrict RNA engagement, providing a mechanistic basis for negative regulation. Together, these findings establish that sequence-encoded elements within the Dcp2 C-terminus control catalytic activity and functional output within biomolecular condensates. More broadly, our results reveal that competing interactions encoded within intrinsically disordered regions of proteins are balanced to allosterically tune enzyme activity, providing a general mechanism by which proteins modulate distinct enzymatic functions within biological condensates.

biochemistry↗

bioRxiv2026

FTO separation-of-function mutations alter m6A versus m6Am demethylation selectivity on RNA

The RNA demethylase FTO erases N6-methyladenosine (m6A) and cap-associated N6,2'-O-dimethyladenosine (m6Am) modifications. However, the molecular basis of its substrate selectivity and the biological effects of m6A versus m6Am demethylation in cells remain poorly understood. Here we report two engineered FTO separation-of-function mutants to selectively demethylate either m6A or m6Am modifications on RNA. While investigating the propensity of FTO active site residues to undergo self-hydroxylation, we found that mutations of FTO residue L203 resulted in impaired m6A demethylation but retained wild-type levels of m6Am demethylation, and that FTO L203A could function as a selective m6Am demethylase. Conversely, building on our recent work that identified conserved aromatic residues on FTO involved in mRNA 5' cap recognition, we found that the FTO H232A/W278A double mutant efficiently demethylates m6A modifications while exhibiting substantially impaired m6Am demethylation, making it a selective m6A demethylase. Together, these complementary FTO variants represent the first set of engineered mutations that shift FTO demethylation selectivity between m6A and m6Am substrates. These tools enable selective enzymatic removal of m6A or m6Am modifications in vitro for sequencing applications, and may facilitate understanding of FTO-mediated m6A versus m6Am demethylation in cellular and disease model systems.

biochemistry↗

bioRxiv2026

The trypanosome mRNA decapping enzyme ALPH1 prefers caps without m7G methylation and produces diphosphate-RNA

5'ends of eukaryotic mRNAs are protected by the m7G cap, connected to the mRNA via a three-phosphate-bridge. In mRNA decay, the pyrophosphate bond between the and {beta} phosphate is cleaved by the nudix hydrolase DCP2. Uniquely among eukaryotes, Kinetoplastida lack DCP2 and instead employ the ApaH-like phosphatase ALPH1 for mRNA decapping. ALPH1 consists of an unstructured N-terminus, a catalytic domain and a structured C-terminus that mediates ALPH1 dimerisation. Here, we have analysed Trypanosoma brucei ALPH1 in greater detail. We find that the enzyme has broad substrate specificity and accepts different cap types and even cap analogues. Strikingly, cap-analogues and RNAs without the m7G-methyl group are turned over significantly faster than m7G methylated substrates. Moreover, all methylated and non-methylated cap analogues tested, with at least one additional nucleotide 3' to the NpppN moiety are cleaved at the {beta}-{gamma} pyrophosphate bond, producing the equivalent to a 5 diphosphate-RNA. While the presence of the ALPH1 C-terminal domain is essential for cell viability and increases enzyme activity in vitro, substrate preferences are determined solely by the catalytic domain. Altogether, these ALPH1 enzymatic properties exhibit intriguing differences to the canonical eukaryotic decapping enzyme DCP2, which we critically discuss and which potentially have biotechnological applications.

molecular biology↗

bioRxiv2025

PolyA tail segmentation improves the stability of the template DNA and increases the translatability of in vitro transcribed mRNA

PolyA tail regulates mRNA localization, stability, and translation. PolyA length affects the durability and translational activity of both endogenous and exogenously delivered mRNAs. However, long polyA stretches can undergo recombination during amplification in bacterial plasmids, impairing the production of in vitro transcribed (IVT) mRNA with long polyAs. PolyA tail segmentation with heteronucleotide spacers has recently emerged as a solution. Here, we developed segmented polyA patterns that stabilize the sequence during DNA amplification and enhance mRNA translation. We designed 15 novel genetically modified polyA variants, differing in the length, placement, and frequency of spacers, and the overall length (from [~]120 to [~]200 nucleotides). We evaluated their stability in DNA plasmids and homogeneity, translational activity, and durability in cell culture of the resulting mRNAs, comparing them to A90 tail and other known solutions, including those from existing mRNA vaccines. Selected sequences were validated in vivo. Surprisingly, we found that even frequent heteronucleotide insertions produce functional polyA tails. The most notable enhancements in protein production were observed for a segmented tail exceeding 200 nt in length [A30(CA15)11; up to 6-fold compared to mRNA with A90 tails]. Our findings extend the scope of possible polyA modification strategies, offering new possibilities for advancing mRNA therapeutics.

molecular biology↗

bioRxiv2025

RNA demethylase FTO uses conserved aromatic residues to recognize the mRNA 5' cap and promote efficient m6Am demethylation

The RNA demethylase FTO acts as a methyl eraser to remove either internal N6-methyladenosine (m6A) or 5' end N6-2'-O-dimethyladenosine (m6Am) modifications on mRNA. FTO has an intrinsic preference and significantly faster demethylation rates in vitro for m6Am modifications located at the 5' mRNA cap structure, but the structural basis for FTOs ability to discriminate m6A versus m6Am modifications has remained unknown. Here we utilize molecular dynamics simulations of FTO-RNA cap complexes to identify conserved aromatic residues on the surface of FTO involved in 5 cap recognition. Subsequent mutagenesis and enzymology experiments validate the specificity of these residues in engaging the 5' cap structure to promote m6Am demethylation. We also identify a nonpolar surface on FTO that interacts with the 2'-O-methyl group of m6Am to impact demethylation kinetics. This work provides the first structure-level insights into how FTO selectively catalyzes m6Am versus m6A demethylation on mRNA and advances our understanding of how FTO activity is regulated by diverse mechanisms to help control the epitranscriptome.

biochemistry↗

bioRxiv2025

Differential control of RNA demethylase activity and selectivity by cofactor ascorbate

Across all domains of life, Fe(II)- and 2-oxoglutarate(2-OG)-dependent dioxygenase (FODD) superfamily enzymes carry out pivotal oxidation reactions that underlie key biological processes ranging from hormone biosynthesis to oxygen sensing to DNA repair and RNA modification. This study combines enzymology and structural biology to elucidate a new mechanism of FODD regulation whereby cofactor ascorbate (vitamin C) concentrations tune both the activity and substrate selectivity of FODD enzymes involved in RNA demethylation, and for the first time reveals the structural basis for ascorbates interaction with the FODD superfamily active site. Because ascorbate concentrations vary by over 100-fold across different cell types and disease states, our mechanistic work demonstrates how ascorbate levels likely play a critical, but underappreciated role in regulating RNA modification across the epitranscriptome and, more broadly, in regulating diverse biological oxidation reactions across the cell and human diseases.

biochemistry↗

bioRxiv2024

Chemical circularization of in vitro transcribed RNA opens new avenues for circular mRNA design

Circularization is at the frontier of therapeutic messenger RNA (mRNA) enhancements. Currently available enzymatic and ribozymatic methods for generating circular RNAs (circRNAs) face several challenges related to sequence limitations, purification, and sub-optimal biological activity. The chemical circularization of synthetic RNA fragments potentially overcomes these limitations but is applicable only to extremely short sequences. Here, we report a novel approach for accessing circular RNAs based on the chemical circularization of in vitro transcribed RNA. We efficiently accessed chemically circularized RNAs (chem-circRNAs) by making in vitro transcribed precursor RNAs modified at the 5' end with an ethylenediamine moiety, which undergoes an intramolecular reaction with the periodate-oxidized RNA 3' end under reductive amination conditions. We demonstrate that this method is modification-compatible and applicable to various sequences. Additionally, we report methods for the effective separation of chem-circRNAs from their linear precursors. Using this approach, we prepared multiple chemically-obtained circular RNAs (chem-circRNAs; 35-1500 nt long) with circularization efficiencies reaching up to 60%. We show that protein-coding chem-circRNAs are translationally active in living cells and exhibit increased durability, similar to enzymatically circularized mRNAs. We also demonstrate that this approach enables unprecedented access to chemically modified circRNAs, such as circ-mRNAs incorporating a functional endocyclic N7-methylguanosine cap or modified with N1-methylpseudouridine within the RNA body. Notably, circRNAs containing an endocyclic cap structure engage in the most efficient, cap-dependent mechanism of translation. Our approach makes chemically-modified circularized full-length protein-coding RNAs easily accessible, thereby opening new avenues for the design, modification, and functionalization of circular mRNAs.

biochemistry↗

bioRxiv2024

An MST-based assay reveals new binding preferences of IFIT1 for canonically and non-canonically capped RNAs

IFIT proteins (interferon-induced proteins with tetratricopeptide repeats) are key components of the innate immune response that bind to viral and cellular RNA targets to inhibit viral translation and replication. The RNA target recognition is guided by molecular patterns, particularly at the RNA 5 ends. IFIT1 preferably binds RNAs modified with the 7-methylguanosine (m7G) cap-0 structure, while RNAs with cap-1 structure are recognized with lower affinity. Less is known about the propensity of IFIT1 to recognize non-canonical RNA 5 ends, including hypermethylated and non-canonical RNA caps. Deciphering the structure-function relationship for IFIT1-RNA interaction may improve understanding of cellular selection of IFIT targets and guide the design of exogenously delivered therapeutic RNAs, but requires high-throughput and robust analytical methods. Here, we report a biophysical assay for quick, direct, in-solution affinity assessment of differently capped RNAs with IFIT1. The procedure, which relies on measuring microscale thermophoresis (MST) of fluorescently labelled protein as a function of increasing ligand concentration, is applicable to various RNA lengths and sequences without the need for labelling or affinity tagging. Using the assay, we examined thirteen canonically and non-canonically 5-capped RNAs, revealing new binding preferences of IFIT1. The 5 terminal m6A mark in the m7G cap had a protective function against IFIT1, which was additive with the effect observed for the 2-O position (m6Am cap-1). In contrast, an increased affinity for IFIT1 was observed for several non-canonical caps, including trimethylguanosine (TMG), unmethylated (G), and flavin-adenine dinucleotide (FAD) caps. The results suggest new potential cellular targets of IFIT1 and may contribute to broadening the knowledge on the mechanisms of the innate immune response as well as the more effective design of chemically modified mRNAs.

biochemistry↗

bioRxiv2023

Trinucleotide mRNA cap analog N6-benzylated at the site of posttranscriptional m6Am mark facilitates mRNA purification and confers superior translational properties in vitro and in vivo

Eukaryotic mRNAs undergo co-transcriptional 5-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am - a common epitranscriptomic mark unique to the mRNA 5-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analog of the m6Am-cap - m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as an RP-HPLC purification handle, allowing separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic setting as anti-cancer vaccines. The biochemical characterization suggests several phenomena underlying the biological properties of AvantCap: (i) increased competitiveness of the mRNA 5-end for eIF4E protein by reducing its propensity for unspecific interactions, (ii) direct involvement of eIF3 in alternative translation initiation, (iii) subtle differences in mRNA impurity profiles, or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of the m6Am in mRNA.

biochemistry↗

bioRxiv2023

Extracellular diadenosine tetraphosphate (Ap4A) is recognized by the plasma membrane purinoreceptor P2K1/DORN1 and closes stomata in Arabidopsis thaliana

O_LIDinucleoside polyphosphates (NpnNs) are considered novel signalling molecules involved in the induction of plant defence mechanisms. However, the NpnNs signal recognition and transduction are still enigmatic. Here we report, for the first time, that diadenosine tetraphosphate (Ap4A) is recognized by the Arabidopsis thaliana purinoreceptor P2K1/DORN1 (Does Not Respond to Nucleotides 1) and causes stomatal closure. C_LIO_LIExtracellular Ap4A- and dicytidine tetraphosphate (Cp4C)-induced stomatal closure was observed using a microscope. Reactive oxygen species (ROS) accumulation was determined by staining with nitroblue tetrazolium (NBT) and 3,3'-diaminobenzidine tetrahydrochloride (DAB). Transcriptional changes were determined by quantitative real-time PCR. Wild-type Col-0 and the dorn1-3 A. thaliana knockout mutant were used. C_LIO_LIExamination of the leaf epidermis dorn1-3 mutant provided evidence that P2K1/DORN1 recognizes extracellular Ap4A but not Cp4C. ROS are involved in signal transduction caused by Ap4A and Cp4C, leading to stomatal closure. Ap4A induced and Cp4C suppressed the transcriptional response in wild-type plants. Moreover, in dorn1-3 leaves, the effect of Ap4A on gene expression was impaired. C_LIO_LIOur research demonstrated, for the first time, that P2K1/DORN1 is a plant purinoreceptor for Ap4A. This interaction leads to changes in the transcription of signalling hubs in signal transduction pathways. C_LI

plant biology↗

bioRxiv2022

SARS-CoV-2 mRNA vaccine is re-adenylated in vivo, enhancing antigen production and immune response

Though mRNA vaccines against COVID-19 have revolutionized vaccinology and have been administered in billions of doses, we know incredibly little about how mRNA vaccines are metabolized in vivo. Here we implemented enhanced nanopore Direct RNA sequencing (eDRS), to enable the analysis of single Modernas mRNA-1273 molecules, giving in vivo information about the sequence and poly(A) tails. We show that mRNA-1273, with all uridines replaced by N1-methylpseudouridine (m{Psi}), is terminated by a long poly(A) tail (~100 nucleotides) followed by an m{Psi}Cm{Psi}AG sequence. In model cell lines, mRNA-1273 is swiftly degraded in a process initiated by the removal of m{Psi}Cm{Psi}AG, followed by CCR4-NOT-mediated deadenylation. In contrast, intramuscularly inoculated mRNA-1273 undergoes more complex modifications. Notably, mRNA-1273 molecules are re-adenylated after m{Psi}Cm{Psi}AG removal. Detailed analysis of immune cells involved in antigen production revealed that in macrophages, after m{Psi}Cm{Psi}AG removal, vaccine mRNA is very efficiently re-adenylated, and poly(A) tails can reach up to 200A. In contrast, in dendritic cells, vaccine mRNA undergoes slow deadenylation-dependent decay. We further demonstrate that enhancement of mRNA stability in macrophages is mediated by TENT5 poly(A) polymerases, whose expression is induced by the vaccine itself. Lack of TENT5-mediated re-adenylation results in lower antigen production and severely compromises specific immunoglobulin production following vaccination. Together, our findings provide an unexpected principle for the high efficacy of mRNA vaccines and open new possibilities for their improvement. They also emphasize that, in addition to targeting a protein of interest, the design of mRNA therapeutics should be customized to its cellular destination.

immunology↗

bioRxiv2022

HIV-1 infection reduces NAD capping of host cell snRNA and snoRNA

Nicotinamide adenine dinucleotide (NAD) is a critical component of the cellular metabolism and also serves as an alternative 5' cap on various RNAs. However, the function of the NAD RNA cap is still under investigation. We studied NAD capping of RNAs in HIV-1-infected cells because HIV-1 is responsible for the depletion of the NAD/NADH cellular pool and causing intracellular pellagra. By applying the NAD captureSeq protocol to HIV-1-infected and uninfected cells, we revealed that four snRNAs (e.g. U1) and four snoRNAs lost their NAD cap when infected with HIV-1. Here, we provide evidence that the presence of the NAD cap decreases the stability of the U1/HIV-1 pre-mRNA duplex. Additionally, we demonstrate that reducing the quantity of NAD-capped RNA by overexpressing the NAD RNA decapping enzyme DXO results in an increase in HIV-1 infectivity. This suggests that NAD capping is unfavorable for HIV-1 and plays a role in its infectivity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/515957v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@16d5cf0org.highwire.dtl.DTLVardef@f0bc60org.highwire.dtl.DTLVardef@df83bborg.highwire.dtl.DTLVardef@41a2db_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

2'-O -methylation of the second transcribed nucleotide within mRNA 5' cap impacts protein production level in a cell specific manner and contributes to RNA immune evasion

In higher eukaryotes, m7G-adjacent nucleotides undergo extensive modifications. Ribose of the first or first and second transcribed nucleotides can be subjected to 2-O-methylation to form cap1 or cap2, respectively. Additionally, when the first transcribed nucleotide is adenosine, it can not only undergo 2-O-methylation but can also be methylated at position N6 forming N6,2-O-dimethyladenosine (m6Am). Recent studies have shed some light on the functions of cap1, showing that cap1 in mammalian cells plays a crucial role in distinguishing between self and non-self RNA during viral infection. Here, we attempted to understand the impact of other cap methylations on RNA-related processes. Therefore, we synthesized tetranucleotide cap analogs and used them for efficient co-transcriptional RNA capping during in vitro transcription. Using this tool, we found that 2-O-methylation of the second transcribed nucleotide within the mRNA 5 cap influences protein production levels in a cell-specific manner. The presence of this modification can strongly hamper protein biosynthesis or do not influence protein production levels. Interestingly, 2-O-methylation of the second transcribed nucleotide as well as the presence of N6,2-O-dimethyladenosine as the first transcribed nucleotide serve as determinants that define transcripts as self and contribute to transcript escape from the host innate immune response. Additionally, cap methylation status does not influence transcript affinity towards translation initiation factor 4E or in vitro susceptibility to decapping by DCP2; however what we observe is resistance of RNA capped with cap2 to DXO-mediated decapping and degradation. Significance StatementMethylation of mRNA cap structure regulates protein biosynthesis in a cell-dependent manner. Among the three known m7G cap modifications, the 2-O-methylation is dominant. 2-O-methylation of the first transcribed nucleotide can boost protein production, whereas the same modification of the second transcribed nucleotide can strongly decrease translation. Interestingly, we show that in the JAWS II cell line, 2-O-methylation of mRNA cap had a prominent impact on the composition of the protein interactome associated with the RNA bearing mentioned modifications. Further analysis revealed that 2-O-methylation of the second transcribed nucleotide and N6-methylation of adenosine as the first transcribed nucleotide serve as determinants defining transcripts as self and contribute to transcript escape from the host innate immune response.

biochemistry↗

bioRxiv2020

Substrate specificity of SARS-CoV-2 nsp10-nsp16 methyltransferase

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2) encodes all viral enzymes. In this work, we focus on one particular methyltransferase (MTase), nsp16, which in complex with nsp10 is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered five types of capped RNA (m7Gp3A(G)-, Gp3A(G)- and Gp4A-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin (286 {+/-} 66 nM) to illustrate the value of our approach for inhibitor screening. In the future, this approach can be used for screening inhibitors of any type of 2'-O-MTase.

molecular biology↗

bioRxiv2020

Functional and LC-MS/MS analysis of in vitro transcribed mRNAs carrying phosphorothioate or boranophosphate moieties reveal polyA tail modifications that prevent deadenylation without compromising protein expression

ABSTRACTChemical 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 polyA tail influence the biological properties of eukaryotic mRNA. To obtain modified and unmodified in vitro transcribed mRNAs, we used ATP and ATP analogues modified at the α-phosphate (containing either O-to-S or O-to-BH3 substitutions) and three different RNA polymerases—SP6, T7 and polyA polymerase. To verify the efficiency of incorporation of ATP analogues 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 polyA 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 polyA 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.View Full Text

biochemistry↗

bioRxiv2019

The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5' cap modulates protein expression in living cells

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 {Phi}6.5 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). In all cases the highest expression was achieved for mRNAs carrying 5-terminal A and m6A, whereas the lowest was observed for G and Gm. The 2-O-methylation of the first transcribed nucleotide (cap 1) significantly increased expression compared to 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.

biochemistry↗