Brouze, A. | Search bioRxiv

bioRxiv2024

Efficient globin production during terminal erythropoiesis depends on the synergistic action of TENT5C poly(A) polymerase and LARP4/5

Red blood cell development is a unique process where reduced transcriptome and proteome complexity facilitates vast hemoglobin production. Here, we describe the cooperative role of cytoplasmic poly(A) polymerase TENT5C and the poly(A) tail-protecting LARP4/5 RNA-binding proteins in ensuring proper hemoglobin production. TENT5C catalytic mutant knock-in mice display microcytic hypochromic anemia resembling constitutive knockout. TENT5C counteracts gradual globin mRNA deadenylation during erythropoiesis. In the late stages, TENT5C dysfunction leads to globin poly(A) tail shortening and a drastic reduction of mRNA levels in reticulocytes. Proteomic experiments revealed transient but specific association of TENT5C with LARP4/5. Indeed, LARP4/5 depletion leads to downregulation and poly(A) tail shortening of globin mRNAs. Furthermore, lack of TENT5C catalytic activity is accompanied by compensatory upregulation of LARP4/5. Finally, the importance of precise regulation of globin poly(A) tails by deadenylation and re-adenylation is highlighted by the destabilization of TENT5C by CCR4-NOT deadenylase complex-associated E3 ubiquitin ligase CNOT4. O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/623596v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@82c736org.highwire.dtl.DTLVardef@1e4ea16org.highwire.dtl.DTLVardef@1c482b7org.highwire.dtl.DTLVardef@61e54a_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental 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↗