Biology subjects

Tu, B. P.

Publications and source records attributed to Tu, B. P..

9 recordsLinked to original sources

bioRxiv2026

Metabolic stress reveals widespread accumulation of cap-unmethylated RNAs

RNA polymerase II transcripts are capped with N7-methylguanosine (mG), a conserved modification essential for mRNA function. Although traditionally viewed as constitutive, we developed a mass spectrometry-based method to demonstrate that in both yeast and mammalian cells, a substantial population of mRNAs lack cap methylation in response to SAM-limiting conditions and oxidative stress, which may be frequently encountered across organisms. Through developing two transcriptome-wide approaches, we found that methylation is enriched on specific transcripts and uncovered an unexpected connection between histone H3K36me3 and cap methylation, with both marks preferentially associated with stress-responsive MAPK signaling pathways. Strikingly, cap-unmethylated mRNAs exhibit features of canonical mRNAs--they are polyadenylated, exported to the cytosol, and translated. Enforced cap methylation reduces cell growth under SAM limitation, suggesting that unmethylated mRNAs confer an adaptive advantage during stress. These findings establish mRNA cap methylation as a dynamic, regulated modification and a previously underappreciated layer of gene expression control.

cell biology↗

bioRxiv2023

Exonuclease Xrn1 regulates TORC1 signaling in response to SAM availability

Autophagy is a conserved process of cellular self-digestion that promotes survival during nutrient stress. In yeast, methionine starvation is sufficient to induce autophagy. One pathway of autophagy induction is governed by the SEACIT complex, which regulates TORC1 activity in response to amino acids through the Rag GTPases Gtr1 and Gtr2. However, the precise mechanism by which SEACIT senses amino acids and regulates TORC1 signaling remains incompletely understood. Here, we identify the conserved 5-3 RNA exonuclease Xrn1 as a surprising and novel regulator of TORC1 activity in response to methionine starvation. This role of Xrn1 is dependent on its catalytic activity, but not on degradation of any specific class of mRNAs. Instead, Xrn1 modulates the nucleotide-binding state of the Gtr1/2 complex, which is critical for its interaction with and activation of TORC1. This work identifies a critical role for Xrn1 in nutrient sensing and growth control that extends beyond its canonical housekeeping function in RNA degradation and indicates an avenue for RNA metabolism to function in amino acid signaling into TORC1.

cell biology↗

bioRxiv2022

Pbp1 stabilizes and promotes the translation of Puf3-target mRNAs involved in mitochondrial biogenesis

Pbp1 (poly(A)-binding protein - binding protein 1) is a cytoplasmic stress granule marker that is capable of forming condensates that function in the negative regulation of TORC1 signaling under respiratory conditions. How mutations in its mammalian ortholog ataxin-2 are linked to neurodegenerative conditions remains unclear. Here, we show that loss of Pbp1 leads to decreases in amounts of mitochondrial proteins whose encoding mRNAs are targets of Puf3, a member of the PUF (Pumilio and FBF) family of RNA-binding proteins. We found that Pbp1 stabilizes and promotes the translation of Puf3-target mRNAs in respiratory conditions, such as those involved in the assembly of cytochrome c oxidase. We further show that Pbp1 and Puf3 interact through their respective low complexity domains, which is required for Puf3-target mRNA stabilization and translation. Our findings reveal a key role for Pbp1-containing assemblies in enabling the translation of mRNAs critical for mitochondrial biogenesis and respiration. They may further begin to explain prior associations of Pbp1/ataxin-2 with RNA, stress granule biology, mitochondrial function, and neuronal health.

cell biology↗

bioRxiv2021

TMEM120 is a coenzyme A-binding membrane protein with structural similarities to ELOVL fatty acid elongase

TMEM120A, also named as TACAN, is a novel membrane protein highly conserved in vertebrates and was recently proposed to be a mechanosensitive channel involved in sensing mechanical pain. Here we present the single particle cryo-EM structure of human TMEM120A which forms a tightly packed dimer with extensive interactions mediate by the N-terminal coiled coil domain (CCD), the C-terminal transmembrane domain (TMD), and the re-entrant loop between the two domains. The TMD of each TMEM120A subunit contains six transmembrane helices (TMs) and has no clear structural feature of a channel protein. Instead, the six TMs form an -barrel with a deep pocket where a coenzyme A (CoA) molecule is bound. Intriguingly, some structural features of TMEM120A resemble those of elongase for very long-chain fatty acid (ELOVL) despite low sequence homology between them, pointing to the possibility that TEME120A may function as an enzyme for fatty acid metabolism, rather than a mechanosensitive channel.

biophysics↗

bioRxiv2021

The SCFMet30 ubiquitin ligase senses cellular redox state to regulate the transcription of sulfur metabolism genes

In yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor which activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCFMet30 E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the transsulfuration pathway to regulate the MET gene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4 in vivo. Additionally, we identify important cysteine residues in Met30s WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCFMet30 dynamically senses the flow of sulfur metabolites through the transsulfuration pathway to regulate synthesis of these special amino acids.

biochemistry↗

bioRxiv2020

SAM homeostasis is regulated by CFIm-mediated splicing of MAT2A

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of the MAT2A RNA, which encodes only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) to be a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing, independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in detained intron and 3' UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal previously undescribed roles for CFIm in splicing and SAM metabolism.

molecular biology↗

bioRxiv2019

Redox-mediated regulation of a labile, evolutionarily conserved cross-β structure formed by the TDP43 low complexity domain

An evolutionarily conserved low complexity (LC) domain is found within a 152 residue segment localized to the carboxyl-terminal region of the TDP43 RNA-binding protein. This TDP43 LC domain contains ten conserved methionine residues. Self-association of this domain leads to the formation of liquid-like droplets composed of labile, cross-{beta} polymers. Exposure of polymers to low concentrations of H2O2 leads to a phenomenon of droplet melting that can be reversed upon exposure of the oxidized protein to the MsrA and MsrB methionine sulfoxide reductase enzymes, thioredoxin, thioredoxin reductase and NADPH. Morphological features of the cross-{beta} polymers were revealed by a method of H2O2-mediated footprinting. Similar TDP43 LC domain footprints were observed in highly polymerized, hydrogel samples, liquid-like droplet samples, and living cells. The ability of H2O2 to impede cross-{beta} polymerization was abrogated by a prominent ALS-causing mutation that changes methionine residue 337 to valine. These observations offer potentially useful insight into the biological role of TDP43 in facilitating synapse-localized translation, as well as aberrant aggregation of the protein in neurodegenerative disease.

biochemistry↗

bioRxiv2019

Methyl-Metabolite Depletion Elicits Adaptive Responses to Support Heterochromatin Stability and Epigenetic Persistence

S-adenosylmethionine (SAM) is the methyl-donor substrate for DNA and histone methyltransferases that regulate cellular epigenetic states. This metabolism-epigenome link enables the sensitization of chromatin methylation to altered SAM abundance. However, a chromatin-wide understanding of the adaptive/responsive mechanisms that allow cells to actively protect epigenetic information during life-experienced fluctuations in SAM availability are unknown. We identified a robust response to SAM depletion that is highlighted by preferential cytoplasmic and nuclear de novo mono-methylation of H3 Lys 9 (H3K9) at the expense of global losses in histone di- and tri-methylation. Under SAM-depleted conditions, de novo H3K9 mono-methylation preserves heterochromatin stability and supports global epigenetic persistence upon metabolic recovery. This unique chromatin response was robust across the mouse lifespan and correlated with improved metabolic health, supporting a significant role for epigenetic adaptation to SAM depletion in vivo. Together, these studies provide the first evidence for active epigenetic adaptation and persistence to metabolic stress.

biochemistry↗

bioRxiv2017

Human to yeast pathway transplantation: cross-species dissection of the adenine de novo pathway regulatory node

Pathway transplantation from one organism to another represents a means to a more complete understanding of a biochemical or regulatory process. The purine biosynthesis pathway, a core metabolic function, was transplanted from human to yeast. We replaced the entire Saccharomyces cerevisiae adenine de novo pathway with the cognate human pathway components. A yeast strain was \"humanized\" for the full pathway by deleting all relevant yeast genes completely and then providing the human pathway in trans using a neochromosome expressing the human protein coding regions under the transcriptional control of their cognate yeast promoters and terminators. The \"humanized\" yeast strain grows in the absence of adenine, indicating complementation of the yeast pathway by the full set of human proteins. While the strain with the neochromosome is indeed prototrophic, it grows slowly in the absence of adenine. Dissection of the phenotype revealed that the human ortholog of ADE4, PPAT, shows only partial complementation. We have used several strategies to understand this phenotype, that point to PPAT/ADE4 as the central regulatory node. Pathway metabolites are responsible for regulating PPATs protein abundance through transcription and proteolysis as well as its enzymatic activity by allosteric regulation in these yeast cells. Extensive phylogenetic analysis of PPATs from diverse organisms hints at adaptations of the enzyme-level regulation to the metabolite levels in the organism. Finally, we isolated specific mutations in PPAT as well as in other genes involved in the purine metabolic network that alleviate incomplete complementation by PPAT and provide further insight into the complex regulation of this critical metabolic pathway.

synthetic biology↗