Biology subjects

Padgett, L. R.

Publications and source records attributed to Padgett, L. R..

3 recordsLinked to original sources

bioRxiv2023

Deoxyhypusine synthase is required for the translational regulation of pancreatic beta cell maturation

As professional secretory cells, beta cells require adaptable mRNA translation to facilitate a rapid synthesis of proteins, including insulin, in response to changing metabolic cues. Specialized mRNA translation programs are essential drivers of cellular development and differentiation. However, in the pancreatic beta cell, the majority of factors identified to promote growth and development function primarily at the level of transcription. Therefore, despite its importance, the regulatory role of mRNA translation in the formation and maintenance of functional beta cells is not well defined. In this study, we have identified a translational regulatory mechanism in the beta cell driven by the specialized mRNA translation factor, eukaryotic initiation factor 5A (eIF5A), which facilitates beta cell maturation. The mRNA translation function of eIF5A is only active when it is post-translationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS). We have discovered that the absence of beta cell DHPS in mice reduces the synthesis of proteins critical to beta cell identity and function at the stage of beta cell maturation, leading to a rapid and reproducible onset of diabetes. Therefore, our work has revealed a gatekeeper of specialized mRNA translation that permits the beta cell, a metabolically responsive secretory cell, to maintain the integrity of protein synthesis necessary during times of induced or increased demand. ARTICLE HIGHLIGHTSO_LIPancreatic beta cells are professional secretory cells that require adaptable mRNA translation for the rapid, inducible synthesis of proteins, including insulin, in response to changing metabolic cues. Our previous work in the exocrine pancreas showed that development and function of the acinar cells, which are also professional secretory cells, is regulated at the level of mRNA translation by a specialized mRNA translation factor, eIF5AHYP. We hypothesized that this translational regulation, which can be a response to stress such as changes in growth or metabolism, may also occur in beta cells. C_LIO_LIGiven that the mRNA translation function of eIF5A is only active when the factor is post-translationally modified ("hypusinated") by the enzyme deoxyhypusine synthase (DHPS), we asked the question: does DHPS/eIF5AHYP regulate the formation and maintenance of functional beta cells? C_LIO_LIWe discovered that in the absence of beta cell DHPS in mice, eIF5A is not hypusinated (activated), which leads to a reduction in the synthesis of critical beta cell proteins that interrupts pathways critical for identity and function. This translational regulation occurs at weaning age, which is a stage of cellular stress and maturation for the beta cell. Therefore without DHPS/eIF5AHYP, beta cells do not mature and mice progress to hyperglycemia and diabetes. C_LIO_LIOur findings suggest that secretory cells have a mechanism to regulate mRNA translation during times of cellular stress. Our work also implies that driving an increase in mRNA translation in the beta cell might overcome or possibly reverse the beta cell defects that contribute to early dysfunction and the progression to diabetes. C_LI

developmental biology↗

bioRxiv2021

m6A RNA methylation facilitates pre-mRNA 3-end formation and is essential for viability of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3-boundary of mRNAs within the consensus sequence, YGCAUGCR. Knockdown of the m6A writer enzyme METTL3 resulted in diminished m6A marks, loss of a target transcript, and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3-end processing of pre-mRNAs. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3-ends. Author SummaryToxoplasma gondii is a parasite of medical importance that causes disease upon immuno-suppression. Uncovering essential pathways that the parasite uses for its basic biological processes may reveal opportunities for new anti-parasitic drug therapies. Here, we describe the machinery that Toxoplasma uses to modify specific adenosine residues within its messenger RNAs (mRNA) by N6-adenosine methylation (m6A). We discovered that m6A mRNA methylation is prevalent in multiple stages of the parasite life cycle and is required for parasite replication. We also establish that m6A plays a major role in the proper maturation of mRNA. Two proteins that bind m6A modifications on mRNA associate with factors responsible for the cleavage and final processing steps of mRNA maturation. Since all of the machinery is conserved from plants to Toxoplasma and other related parasites, we propose that this system operates similarly in these organisms.

microbiology↗

bioRxiv2019

Hypusinated eIF5A is expressed in pancreas and spleen of individuals with type 1 and type 2 diabetes

Eukaryotic initiation factor 5A (EIF5A) is found in diabetes-susceptibility loci in mouse and human. eIF5A is the only protein known to contain hypusine (hydroxyputrescine lysine), a polyamine-derived amino acid formed post-translationally in a reaction catalyzed by deoxyhypusine synthase (DHPS). Previous studies showed pharmacologic blockade of DHPS in type 1 diabetic NOD mice and type 2 diabetic db/db mice improved glucose tolerance and preserved beta-cell mass, which suggests that hypusinated eIF5A (eIF5AHyp) may play a role in diabetes pathogenesis by direct action on the beta cells and/or altering the adaptive or innate immune responses. To translate these findings to human, we examined tissue from individuals with and without type 1 and type 2 diabetes to determine the expression of eIF5AHyp. We detected eIF5AHyp in beta cells, exocrine cells and immune cells; however, there was also unexpected enrichment of eIF5AHyp in pancreatic polypeptide-expressing PP cells. Interestingly, the presence of eIF5AHyp co-expressing PP cells was not enhanced with disease. These data identify new aspects of eIF5A biology and highlight the need to examine human tissue to understand disease.

cell biology↗