Biology subjects

Mestre-Fos, S.

Publications and source records attributed to Mestre-Fos, S..

4 recordsLinked to original sources

bioRxiv2023

eIF3 engages with 3'-UTR termini of highly translated mRNAs in neural progenitor cells

Stem cell differentiation involves a global increase in protein synthesis to meet the demands of specialized cell types. However, the molecular mechanisms underlying this translational burst and the involvement of initiation factors remains largely unknown. Here, we investigate the role of eukaryotic initiation factor 3 (eIF3) in early differentiation of human pluripotent stem cell (hPSC)-derived neural progenitor cells (NPCs). Using Quick-irCLIP and alternative polyadenylation (APA) Seq, we show eIF3 crosslinks predominantly with 3 untranslated region (3-UTR) termini of multiple mRNA isoforms, adjacent to the poly(A) tail. Furthermore, we find that eIF3 engagement at 3-UTR ends is dependent on polyadenylation. High eIF3 crosslinking at 3-UTR termini of mRNAs correlates with high translational activity, as determined by ribosome profiling, but not with translational efficiency. The results presented here show that eIF3 engages with 3-UTR termini of highly translated mRNAs, likely reflecting a general rather than specific regulatory function of eIF3, and supporting a role of mRNA circularization in the mechanisms governing mRNA translation.

molecular biology↗

bioRxiv2022

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin and finally elution and identification by mass spectrometry. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in a SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization and vesicular trafficking, many of which have been associated with heme through complimentary studies published recently. Taken together, these results provide support for the emerging role for heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.

biochemistry↗

bioRxiv2020

Human Ribosomal G-Quadruplexes Regulate Heme Bioavailability

The in vitro formation of stable G-quadruplexes (G4s) in human ribosomal RNA (rRNA) was recently reported. However, their formation in cells and their cellular roles have not been resolved. Here, by taking a chemical biology approach that integrates results from immunofluorescence, G4 ligands, heme affinity reagents, and a genetically encoded fluorescent heme sensor, we report that human ribosomes can form G4s in vivo that regulate heme bioavailability. Immunofluorescence experiments indicate that the vast majority of extra-nuclear G4s are associated with rRNA. Moreover, titrating human cells with a G4 ligand alters the ability of ribosomes to bind heme and disrupts cellular heme bioavailability as measured by a genetically encoded fluorescent heme sensor. Overall, these results suggest ribosomes are central hubs of heme metabolism.

biochemistry↗

bioRxiv2018

Dynamic G-quadruplexes on the surface of the human ribosome

Profound similarities and critical differences mark ribosomes across phylogeny. The ribosomal core, approximated by the prokaryotic ribosome, is universal, yet mammalian ribosomes are nearly twice as large as those of prokaryotes. Differences in size are due in part to rRNA expansion segments. Here we show rRNA tentacles of Expansion Segment 7 (ES7) of Homo sapiens can form G-quadruplexes in vitro. G-quadruplex-forming regions are located on the most surface-exposed regions of the ribosome, near the termini of rRNA tentacles. We characterized rRNA of the large ribosomal subunit by computation, circular dichroism, gel mobility, fluorescent probes, nuclease accessibility, electrophoretic mobility shifts and blotting. We investigated ES7 and oligomers derived from ES7, intact 28S rRNA, and 80S ribosomes and polysomes. We used mass spectrometry to identify proteins that bind to rRNA G-quadruplexes in cell lysates. Proteins that associate with rRNA G-quadruplexes include helicases (DDX3, CNBP, DDX21, DDX17) and heterogeneous nuclear ribonucleoproteins (hnRNPs). And finally, by multiple sequence alignments, we observed that G-quadruplex-forming sequences appear to be a general feature LSU rRNA of the phylum Chordata but not in other phyla. It is known that G-quadruplexes form in telomeres, promoters, and untranslated regions of mRNA but, to our knowledge, they have not been reported previously in ribosomes.

biochemistry↗