Jelen, S. | bioRxiv Science

bioRxiv2024

The polyadenylation landscape after in vivo long-term potentiation in the rat brain

Local protein synthesis in neurons is vital for synaptic maintenance and plasticity, yet the regulatory mechanisms, particularly cytoplasmic polyadenylation, are not fully understood. This study employed nanopore sequencing to examine transcriptomic responses in rat hippocampi during in vivo long-term potentiation (LTP) and in synaptoneurosomes after in vitro stimulation. Our long-read transcriptomic dataset allows for detailed analysis of mRNA 3'-ends, poly(A) tail lengths, and nucleotide composition. We observed dynamic shifts in polyadenylation site preference post-LTP induction, with significant poly(A) tail lengthening restricted to transcriptionally induced mRNAs. The poly(A) tails of these genes showed increased non-adenosine abundance. In synaptoneurosomes, chemical stimulation led to shortening of poly(A) tails on preexisting mRNAs, indicating translation-induced deadenylation. Additionally, we discovered a group of neuronal transcripts with poly(A) tails abundant in non-adenosine residues. These tails are semi-templated and derived from extremely adenosine-rich 3'UTRs. This study provides a comprehensive overview of mRNA 3'-end dynamics during LTP, offering insights into post-transcriptional regulation following synaptic activation of plasticity in neurons.

neuroscience↗

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↗