Biology subjects

Kowalska, J.

Publications and source records attributed to Kowalska, J..

6 recordsLinked to original sources

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↗