Biology subjects

Tooley, J. G.

Publications and source records attributed to Tooley, J. G..

3 recordsLinked to original sources

bioRxiv2026

Characterization of novel cytoplasmic roles for the N-terminal methyltransferase NRMT1

N-terminal methylation of proteins by the trimethylase NRMT1 plays important roles in oncogenesis, development, and aging. As N-terminal methylation has frequently been shown to regulate protein-DNA interactions, and many NRMT1 substrates are transcription factors or regulators of chromatin structure, previous research has focused on how transcriptional regulation by NRMT1 affects cell growth and differentiation. However, we have recently identified a new, cytoplasmic role for NRMT1, inhibiting the eukaryotic elongation factor 1 alpha (eEF1A) methyltransferase METTL13, which indicates NRMT1 could also be acting as a translational regulator. Here we further explore NRMT1 cytoplasmic functions and show that, unlike previously thought, NRMT1 can methylate substrates in the cytoplasm. We also show that while many of these substrates remain bound to NRMT1, it can also interact with a number of non-target ribosomal proteins and proteins associated with the endoplasmic reticulum (ER). To confirm NRMT1 interaction with the ribosome, we performed polysome profiling, which showed a portion of NRMT1 co-migrates with the 40S and 60S subunits but not with actively translating polysomes, indicating NRMT1 may play an early role in translation. To see if NRMT1 was affecting target mRNA selection of ribosomes, we also performed ribosome-sequencing experiments in proliferating and differentiating C2C12 mouse myoblasts. These results show a striking upregulation of translation of soluble proteins with NRMT1 loss and corresponding decrease in translation of transmembrane and signal sequence-containing proteins. We now propose a model where NRMT1 regulates the translation of transmembrane and secreted proteins by facilitating interactions between the ribosome and the ER.

biochemistry↗

bioRxiv2025

Loss of NRMT1 allows expression of multiple differentiation pathways and alters transcription of secreted proteins in C2C12 myoblasts

Muscle stem cells (satellite cells) retain their identity and function through expression of the paired homeobox transcription factor PAX7. PAX7 is able to both stimulate satellite cell proliferation and activate target genes involved in establishing myogenic identity, including myogenic factor 5 (MYF5) and the other myogenic regulatory factors (MRFs). Upregulation of the MRFs promotes commitment to the muscle lineage by initiating withdrawal from the cell cycle, upregulating expression of muscle-specific transcripts, and directing myoblast fusion. We have previously shown that knockout of the N-terminal methyltransferase NRMT1 in C2C12 mouse myoblasts results in significantly decreased Pax7 expression, an inability of the cells to differentiate into myotubes, and abnormal upregulation of osteogenic markers. Here, we use RNA-sequencing to more comprehensively determine how loss of NRMT1 affects the transcriptional profile of proliferating and differentiating C2C12 myoblasts. We see that upon inducing differentiation, NRMT1 knockout cells can downregulate cell cycle, DNA replication, and histone gene expression. Though they also have significantly downregulated Pax7 and Myf5 expression, other muscle-specific transcripts are significantly increased over wild type, indicating the muscle transcriptional program is not completely inhibited. However, signaling pathways involved in the differentiation of other types of mesenchymal and hematopoietic lineages are also increased with NRMT1 loss and expression of chemotactic genes is downregulated. Together, these data indicate that NRMT1 knockout cells can upregulate genes needed for cell cycle withdrawal and muscle specification but fail to suppress markers of other lineages and activate normal chemotactic signaling, which may lead to the observed differentiation phenotypes.

genomics↗

bioRxiv2021

CREB-mediated transcriptional activation of NRMT1 drives muscle differentiation

The N-terminal methyltransferase NRMT1 is an important regulator of protein-DNA interactions and plays a role in many cellular processes, including mitosis, cell cycle progression, chromatin organization, DNA damage repair, and transcriptional regulation. Accordingly, loss of NRMT1 results in both developmental pathologies and oncogenic phenotypes. Though NRMT1 plays such important and diverse roles in the cell, little is known about its own regulation. To better understand the mechanisms governing NRMT1 expression, we first identified its predominant transcriptional start site and minimal promoter region with predicted transcription factor motifs. We then used a combination of luciferase and binding assays to confirm CREB1 as the major regulator of NRMT1 transcription. We tested which conditions known to activate CREB1 also activated NRMT1 transcription, and found CREB1-mediated NRMT1 expression was increased during recovery from serum starvation and muscle cell differentiation. To determine how NRMT1 expression affects myoblast differentiation, we used CRISPR/Cas9 technology to knock out NRMT1 expression in immortalized C2C12 mouse myoblasts. C2C12 cells depleted of NRMT1 lacked Pax7 expression and were unable to proceed down the muscle differentiation pathway. Instead, they took on characteristics of C2C12 cells that have transdifferentiated into osteoblasts, including increased alkaline phosphatase and type I collagen expression and decreased proliferation. These data implicate NRMT1 as an important downstream target of CREB1 during muscle cell differentiation.

biochemistry↗