Biology subjects

Paddison, P.

Publications and source records attributed to Paddison, P..

3 recordsLinked to original sources

bioRxiv2025

N6-methyladenosine regulation of mRNA translation is essential for early human erythropoiesis

N6-methyladenosine (m6A) is an abundant modification of mRNA with important regulatory roles in normal and malignant hematopoiesis. We previously reported that in human erythroid leukemia (HEL) cells, m6A mRNA marking selectively regulates translation of essential erythropoiesis genes required for in vitro differentiation and human erythroid colony formation. Here, we further investigated the timing and nature of requirement for m6A-methyltransferase (MTase) activity during human erythropoiesis, using a standardized in vitro erythroid differentiation assay for hHSPCs. We identified two critical m6A regulated developmental windows in BFU-E and during the transition from CFU-E to proerythroblasts. These windows of m6A-MTase requirement coincide with rising global m6A levels, which peak in proerythroblasts. After proerythroblast formation, however, m6A -MTase activity is dispensable for differentiation, proliferation, and survival. In BFU-E, m6A-MTase promotes proliferation but is dispensable for differentiation, while, in CFU-E, both m6A -MTase and the YTHDF family of m6A readers are essential for differentiation to proerythroblasts. Mechanistically, in CFU-E, m6A MTase activity enhances translation of ribosomal and oxidative phosphorylation (OXPHOS) genes, thereby elevating global protein synthesis rates and enabling efficient erythroblast formation. We propose that this form of translational regulation by m6A emerged as an evolutionary adaptation to meet the high translational demands of human erythropoiesis.

developmental biology↗

bioRxiv2021

Functional dissection of human mitotic proteins using CRISPR-Cas9 tiling screens

Kinetochores are large protein complexes that assemble at the centromere and bind to mitotic spindle microtubules to ensure accurate chromosome segregation. Like most protein-coding genes, the full multifunctional nature of kinetochore factors remains uncharacterized due to the limited experimental tools for unbiased dissection of human protein sequences. We developed a method that leverages CRISPR-Cas9 induced mutations to identify key functional regions within protein sequences required for cellular outgrowth. Our analysis of 48 human mitotic genes revealed hundreds of regions required for cell proliferation, including known domains and uncharacterized ones. We validated the essential nature for 15 of these regions, including amino acids 387-402 of Mad1, which identified an unknown domain that contributes to Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR-Cas9-based tiling mutagenesis identifies key functional domains in protein-coding genes de novo, which elucidates separation of function mutants and allows functional annotation across the human proteome.

cell biology↗

bioRxiv2018

N6-methyladenosine mRNA marking promotes selective translation of regulons required for human erythropoiesis

Many of the regulatory features governing erythrocyte specification, maturation, and associated disorders remain enigmatic. To identify new regulators of erythropoiesis, we performed a functional genomic screen for genes affecting expression of the erythroid marker CD235a/GYPA. Among validating hits were genes coding for the N6-methyladenosine (m6A) mRNA methyltransferase (MTase) complex, including, METTL14, METTL3, and WTAP. We found that m6A MTase activity promotes erythroid gene expression programs and lineage specification through selective translation of >200 m6A marked mRNAs, including those coding for SETD methyltransferase, ribosome, and polyA RNA binding proteins. Remarkably, loss of m6A marks resulted in dramatic loss of H3K4me3 across key erythroid-specific KLF1 transcriptional targets (e.g., Heme biosynthesis genes). Further, each m6A MTase subunit and a subset of their mRNAs targets, including BRD7, CXXC1, PABPC1, PABPC4, STK40, and TADA2B, were required for erythroid specification. Thus, m6A mRNA marks promote the translation of a network of genes required for human erythropoiesis.

cell biology↗