Title: Post-transcriptional regulation shapes the transcriptome of quiescent budding yeast
Abstract
To facilitate long-term survival, cells must exit the cell cycle and enter quiescence, a reversible non-replicative state. Budding yeast cells reprogram their gene expression during quiescence entry to silence transcription, but how the nascent transcriptome changes in quiescence is unknown. By investigating the nascent transcriptome, we identified over a thousand noncoding RNAs in quiescent and G1 yeast cells, and found noncoding transcription represented a larger portion of the quiescent transcriptome than in G1. Additionally, both mRNA and ncRNA are subject to increased post-transcriptional regulation in quiescence compared to G1. We found that, in quiescence, the nuclear exosome-NNS pathway suppresses over one thousand mRNAs, in addition to canonical noncoding RNAs. RNA sequencing through quiescent entry revealed two distinct time points at which the nuclear exosome controls the abundance of mRNAs involved in protein production, cellular organization, and metabolism, thereby facilitating efficient quiescence entry. Our work identified a previously unknown key biological role for the nuclear exosome-NNS pathway in mRNA regulation and uncovered a novel layer of gene-expression control in quiescence.
Next-generation sequencing (NGS) technologies - Illumina RNA-seq, Pacific Biosciences isoform sequencing (PacBio Iso-seq), and Oxford Nanopore direct RNA sequencing (DRS) - have revealed the complexity of plant transcriptomes and their regulation at the co-/post-transcriptional level. Global analysis of mature mRNAs, transcripts from nuclear run-on assays, and nascent chromatin-bound mRNAs using short as well as full-length and single-molecule DRS reads have uncovered potential roles of different forms of RNA polymerase II during the transcription process, and the extent of co-transcriptional pre-mRNA splicing and polyadenylation. These tools have also allowed mapping of transcriptome-wide start sites in cap-containing RNAs, poly(A) site choice, poly(A) tail length, and RNA base modifications. The emerging theme from recent studies is that reprogramming of gene expression in response to developmental cues and stresses at the co-/post-transcriptional level likely plays a crucial role in eliciting appropriate responses for optimal growth and plant survival under adverse conditions. Although the mechanisms by which developmental cues and different stresses regulate co-/post-transcriptional splicing are largely unknown, a few recent studies indicate that the external cues target spliceosomal and splicing regulatory proteins to modulate alternative splicing. In this review, we provide an overview of recent discoveries on the dynamics and complexities of plant transcriptomes, mechanistic insights into splicing regulation, and discuss critical gaps in co-/post-transcriptional research that need to be addressed using diverse genomic and biochemical approaches.
INTRODUCTION Eukaryotes contain a highly conserved signaling pathway that becomes rapidly activated when adenosine triphosphate (ATP) levels decrease, as happens during conditions of nutrient shortage or mitochondrial dysfunction. The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is activated within minutes of energetic stress and phosphorylates a limited number of substrates to biochemically rewire metabolism from an anabolic state to a catabolic state to restore metabolic homeostasis. AMPK also promotes prolonged metabolic adaptation through transcriptional changes, decreasing biosynthetic genes while increasing expression of genes promoting lysosomal and mitochondrial biogenesis. The transcription factor EB (TFEB) is a well-appreciated effector of AMPK-dependent signals, but many of the molecular details of how AMPK controls these processes remain unknown. RATIONALE The requirement of AMPK and its specific downstream targets that control aspects of the transcriptional adaptation of metabolism remain largely undefined. We performed time courses examining gene expression changes after various mitochondrial stresses in wild-type (WT) or AMPK knockout cells. We hypothesized that a previously described interacting protein of AMPK, folliculin-interacting protein 1 (FNIP1), may be involved in how AMPK promotes increases in gene expression after metabolic stress. FNIP1 forms a complex with the protein folliculin (FLCN), together acting as a guanosine triphosphate (GTP)–activating protein (GAP) for RagC. The FNIP1-FLCN complex has emerged as an amino acid sensor to the mechanistic target of rapamycin complex 1 (mTORC1), involved in how amino acids control TFEB activation. We therefore examined whether AMPK may regulate FNIP1 to dominantly control TFEB independently of amino acids. RESULTS AMPK was found to govern expression of a core set of genes after various mitochondrial stresses. Hallmark features of this response were activation of TFEB and increases in the transcription of genes specifying lysosomal and mitochondrial biogenesis. AMPK directly phosphorylated five conserved serine residues in FNIP1, suppressing the function of the FLCN-FNIP1 GAP complex, which resulted in dissociation of RagC and mTOR from the lysosome, promoting nuclear translocation of TFEB even in the presence of amino acids. FNIP1 phosphorylation was required for AMPK to activate TFEB and for subsequent increases in peroxisome proliferation–activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) mRNAs. Cells in which the five serines in FNIP1 were mutated to alanine were unable to increase lysosomal and mitochondrial gene expression programs after treatment with mitochondrial poisons or AMPK activators despite the presence and normal regulation of all other substrates of AMPK. By contrast, neither AMPK nor its control of FNIP1 were needed for activation of TFEB after amino acid withdrawal, illustrating the specificity to energy-limited conditions. CONCLUSION Our data establish FNIP1 as the long-sought substrate of AMPK that controls TFEB translocation to the nucleus, defining AMPK phosphorylation of FNIP1 as a singular event required for increased lysosomal and mitochondrial gene expression programs after metabolic stresses. This study also illuminates the larger biological question of how mitochondrial damage triggers a temporal response of repair and replacement of damaged mitochondria: Within early hours, AMPK-FNIP1–activated TFEB induces a wave of lysosome and autophagy genes to promote degradation of damaged mitochondria, and a few hours later, TFEB–up-regulated PGC1⍺ and ERR⍺ promote expression of a second wave of genes specifying mitochondrial biogenesis. These insights open therapeutic avenues for several common diseases associated with mitochondrial dysfunction, ranging from neurodegeneration to type 2 diabetes to cancer. Mitochondrial damage activates AMPK to phosphorylate FNIP1, stimulating TFEB translocation to the nucleus and sequential waves of lysosomal and mitochondrial biogenesis. After mitochondrial damage, activated AMPK phosphorylates FNIP1 (1), causing inhibition of FLCN-FNIP1 GAP activity (2). This leads to accumulation of RagC in its GTP-bound form, causing dissociation of RagC, mTORC1, and TFEB from the lysosome (3). TFEB is therefore not phosphorylated and translocates to the nucleus, inducing transcription of lysosomal or autophagy genes, with parallel increases in NT-PGC1α mRNA (4), which, in concert with ERRα (5), subsequently induces mitochondrial biogenesis (6). CCCP, carbonyl cyanide m-chlorophenylhydrazone; CLEAR, coordinated lysosomal expression and regulation; GDP, guanosine diphosphate; P, phosphorylation. [Figure created using BioRender]
Aherrahrou, Rédouane; Lue, Dillon; Perry, R. Noah; Aberra, Yonathan Tamrat; Khan, Mohammad Daud; Soh, Joon Yuhl; Örd, Tiit; Singha, Prosanta; Yang, Qianyi; Gilani, Huda; et al(
, Circulation Research)
Background: Coronary artery disease (CAD) is the leading cause of death worldwide. Recent meta-analyses of genome-wide association studies have identified over 175 loci associated with CAD. The majority of these loci are in noncoding regions and are predicted to regulate gene expression. Given that vascular smooth muscle cells (SMCs) play critical roles in the development and progression of CAD, we aimed to identify the subset of the CAD loci associated with the regulation of transcription in distinct SMC phenotypes. Methods: We measured gene expression in SMCs isolated from the ascending aortas of 151 heart transplant donors of various genetic ancestries in quiescent or proliferative conditions and calculated the association of their expression and splicing with ~6.3 million imputed single-nucleotide polymorphism markers across the genome. Results: We identified 4910 expression and 4412 splicing quantitative trait loci (sQTLs) representing regions of the genome associated with transcript abundance and splicing. A total of 3660 expression quantitative trait loci (eQTLs) had not been observed in the publicly available Genotype-Tissue Expression dataset. Further, 29 and 880 eQTLs were SMC-specific and sex-biased, respectively. We made these results available for public query on a user-friendly website. To identify the effector transcript(s) regulated by CAD loci, we used 4 distinct colocalization approaches. We identified 84 eQTL and 164 sQTL that colocalized with CAD loci, highlighting the importance of genetic regulation of mRNA splicing as a molecular mechanism for CAD genetic risk. Notably, 20% and 35% of the eQTLs were unique to quiescent or proliferative SMCs, respectively. One CAD locus colocalized with a sex-specific eQTL ( TERF2IP ), and another locus colocalized with SMC-specific eQTL ( ALKBH8 ). The most significantly associated CAD locus, 9p21, was an sQTL for the long noncoding RNA CDKN2B-AS1 , also known as ANRIL , in proliferative SMCs. Conclusions: Collectively, our results provide evidence for the molecular mechanisms of genetic susceptibility to CAD in distinct SMC phenotypes.
Folate is an essential B-group vitamin and a key methyl donor with important biological functions including DNA methylation regulation. Normal neurodevelopment and physiology are sensitive to the cellular folate levels. Either deficiency or excess of folate may lead to neurological disorders. Recently, folate has been linked to tRNA cytosine-5 methylation (m5C) and translation in mammalian mitochondria. However, the influence of folate intake on neuronal mRNA m5C modification and translation remains largely unknown. Here, we provide transcriptome-wide landscapes of m5C modification in poly(A)-enriched RNAs together with mRNA transcription and translation profiles for mouse neural stem cells (NSCs) cultured in three different concentrations of folate.
Results
NSCs cultured in three different concentrations of folate showed distinct mRNA methylation profiles. Despite uncovering only a few differentially expressed genes, hundreds of differentially translated genes were identified in NSCs with folate deficiency or supplementation. The differentially translated genes induced by low folate are associated with cytoplasmic translation and mitochondrial function, while the differentially translated genes induced by high folate are associated with increased neural stem cell proliferation. Interestingly, compared to total mRNAs, polysome mRNAs contained high levels of m5C. Furthermore, an integrative analysis indicated a transcript-specific relationship between RNA m5C methylation and mRNA translation efficiency.
Conclusions
Altogether, our study reports a transcriptome-wide influence of folate on mRNA m5C methylation and translation in NSCs and reveals a potential link between mRNA m5C methylation and mRNA translation.
Garg, Angad; Sanchez, Ana M.; Miele, Matthew; Schwer, Beate; Shuman, Stewart(
, Nucleic Acids Research)
Abstract
Inorganic phosphate is an essential nutrient acquired by cells from their environment. Here, we characterize the adaptative responses of fission yeast to chronic phosphate starvation, during which cells enter a state of quiescence, initially fully reversible upon replenishing phosphate after 2 days but resulting in gradual loss of viability during 4 weeks of starvation. Time-resolved analyses of changes in mRNA levels revealed a coherent transcriptional program in which phosphate dynamics and autophagy were upregulated, while the machineries for rRNA synthesis and ribosome assembly, and for tRNA synthesis and maturation, were downregulated in tandem with global repression of genes encoding ribosomal proteins and translation factors. Consistent with the transcriptome changes, proteome analysis highlighted global depletion of 102 ribosomal proteins. Concomitant with this ribosomal protein deficit, 28S and 18S rRNAs became vulnerable to site-specific cleavages that generated temporally stable rRNA fragments. The finding that Maf1, a repressor of RNA polymerase III transcription, was upregulated during phosphate starvation prompted a hypothesis that its activity might prolong lifespan of the quiescent cells by limiting production of tRNAs. Indeed, we found that deletion of maf1 results in precocious death of phosphate-starved cells via a distinctive starvation-induced pathway associated with tRNA overproduction and dysfunctional tRNA biogenesis.
Greenlaw, Alison C., Alavattam, Kris G., and Tsukiyama, Toshio.
"Post-transcriptional regulation shapes the transcriptome of quiescent budding yeast". Nucleic Acids Research 52 (3). Country unknown/Code not available: Oxford University Press. https://doi.org/10.1093/nar/gkad1147.https://par.nsf.gov/biblio/10477769.
@article{osti_10477769,
place = {Country unknown/Code not available},
title = {Post-transcriptional regulation shapes the transcriptome of quiescent budding yeast},
url = {https://par.nsf.gov/biblio/10477769},
DOI = {10.1093/nar/gkad1147},
abstractNote = {Abstract To facilitate long-term survival, cells must exit the cell cycle and enter quiescence, a reversible non-replicative state. Budding yeast cells reprogram their gene expression during quiescence entry to silence transcription, but how the nascent transcriptome changes in quiescence is unknown. By investigating the nascent transcriptome, we identified over a thousand noncoding RNAs in quiescent and G1 yeast cells, and found noncoding transcription represented a larger portion of the quiescent transcriptome than in G1. Additionally, both mRNA and ncRNA are subject to increased post-transcriptional regulation in quiescence compared to G1. We found that, in quiescence, the nuclear exosome-NNS pathway suppresses over one thousand mRNAs, in addition to canonical noncoding RNAs. RNA sequencing through quiescent entry revealed two distinct time points at which the nuclear exosome controls the abundance of mRNAs involved in protein production, cellular organization, and metabolism, thereby facilitating efficient quiescence entry. Our work identified a previously unknown key biological role for the nuclear exosome-NNS pathway in mRNA regulation and uncovered a novel layer of gene-expression control in quiescence.},
journal = {Nucleic Acids Research},
volume = {52},
number = {3},
publisher = {Oxford University Press},
author = {Greenlaw, Alison C. and Alavattam, Kris G. and Tsukiyama, Toshio},
}
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