Biology subjects

Sikorski, P. J.

Publications and source records attributed to Sikorski, P. J..

5 recordsLinked to original sources

bioRxiv2026

RNA length and receptor usage define innate immune recognition across species.

Recognition of viral double-stranded RNA (dsRNA) is a central trigger of innate antiviral immunity, yet the role of RNA length in shaping immune sensing across species remains poorly defined. Here, we identify RNA length as a key determinant of innate immune activation and demonstrate that this parameter is differentially tuned across vertebrate hosts. Using defined dsRNA molecules, we compared immune responses in human, duck, and chicken cells. Short 5-triphosphorylated dsRNAs, including influenza-derived mini viral RNAs, robustly activated antiviral responses in human and duck cells but failed to do so in chicken cells. In contrast, longer dsRNAs induced immune activation across all species, revealing a species-specific threshold for dsRNA recognition. Mechanistically, this threshold is defined by differential engagement of RNA sensors. In human cells, sensing of short and intermediate dsRNA is strictly dependent on RIG-I, whereas in chicken cells, which lack RIG-I, MDA5 mediates dsRNA recognition and imposes a higher length requirement for activation. Consistent with this, duck cells, which retain RIG-I, respond to short dsRNA similarly to human cells. These differences in dsRNA sensing are accompanied by differential activation of downstream antiviral pathways, including PKR-dependent translational control and OAS/RNase L activation. Together, our findings identify RNA length as a fundamental parameter governing innate immune recognition and reveal that species-specific usage of RNA sensors constrains antiviral sensing, with implications for host susceptibility and virus-host interactions. Significance StatementRecognition of viral RNA is central to antiviral immunity, yet the rules governing which viral RNAs are sensed across species remain poorly understood. Here, we show that RNA length defines a species-specific threshold for innate immune recognition. Human and duck cells efficiently detect short viral double-stranded RNAs generated during influenza infection, whereas chicken cells respond only to substantially longer RNA duplexes. Mechanistically, this difference reflects the usage of distinct antiviral RNA sensors: RIG-I enables sensing of short viral RNAs, while MDA5 imposes a higher length requirement for activation. Our findings reveal that host species differ in the spectrum of viral RNA ligands accessible to immune detection, providing a framework for understanding species-specific antiviral responses and host susceptibility.

microbiology↗

bioRxiv2024

Effective recognition of double-stranded RNA does not require activation of cellular inflammation

Excess double-stranded RNA (dsRNA) is present in the cytoplasm of human cells, usually following viral infections. Recognition of dsRNAs activates innate immune pathways, leading to cellular inflammation and inhibition of cell growth. Here, we show that an effective dsRNA response may occur without the onset of inflammation. Interestingly, pro-inflammatory (RLR-dependent pathway) and cell growth inhibitory mechanisms (OAS/RNase L- and PKR-dependent pathways) can act independently. We found that the 5' ends of dsRNA direct the onset of cellular inflammation, whereas RNA duplex activates the OAS/RNase L and PKR pathways. Unexpectedly, three of the most common human RNA epitranscriptomic markers - i.e. N6-methyladenosine, 5-methylcytosine, and pseudouridine - did not affect the immunogenicity of dsRNA; however, the presence of N6-methyladenosine inhibited the OAS/RNase L pathway. Our observations demonstrate how precisely innate immunity is fine-tuned in cells to take appropriate countermeasures when a specific threat arises.

immunology↗

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↗

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↗

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↗