Biology subjects

Mamot, A.

Publications and source records attributed to Mamot, A..

4 recordsLinked to original sources

bioRxiv2026

Remodeling of mRNA by eIF4F in human translation initiation

For the ribosome to load onto an mRNA during the early steps of translation initiation, the mRNA must be activated by the eIF4F complex. The mechanism of this activation step has remained elusive. Here we employ multi-perspective real-time single-molecule assays to observe directly mRNA-eIF4F binding near the 5' end, mRNA conformational remodeling, and 40S ribosomal subunit loading. eIFs 4E, 4G, and 4B play distinct roles in promoting eIF4F association and stabilizing eIF4A binding. Binding of eIF4F is the rate-limiting step in mRNA activation: once bound, mRNA conformation is rapidly extended in an ATP-dependent manner. The mRNA extended state is the necessary substrate for 43S PIC loading and perturbations to extension delay loading. Features of the mRNA, such as the 7-methylguanosine cap at the 5' end and secondary structures, modulate these steps and regulate ribosome loading. Our results establish a kinetic and mechanistic framework for the early steps in translation initiation.

biophysics↗

bioRxiv2026

Co-transcriptional Phase Separation of Nucleic Acids at Membrane Surfaces

Transcription is usually framed as information transfer, yet it also injects a new polymer into a crowded, confined environment. Here we demonstrate how spatial confinement to surfaces in a minimal membrane-bound transcription (MBT) system displays the physical consequences of RNA synthesis. Within a dense membrane-tethered DNA network, transcription drives co-transcriptional RNA phase separation: nascent RNA oligomerizes, gels and demixes from a surrounding fluid DNA phase, generating stable spatial patterns while mechanically remodeling the DNA layer. RNA gelation sequesters T7 RNA polymerase, whereas RNA-binding and translation-associated factors reverse gelation and restore fluidity. Thus, in the absence of downstream regulatory machinery, transcription under confinement is sufficient to trigger RNA condensation and nucleic-acid phase separation. The membrane as confining interface catalyzes the onset of DNA-RNA demixing and modulates the morphology of the resulting patterns. Since such large-scale spatial unmixing may be detrimental to cellular physiology, we suggest that one fundamental role of translation is to actively prevent condensation effects created by continuous RNA production.

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↗

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↗