The RNA-binding protein Puf5 contributes to buffering of mRNA upon chromatin-mediated changes in nascent transcription

Gene expression involves regulation of chromatin structure and transcription, as well as processing of the transcribed mRNA. While there are feedback mechanisms, it is not clear whether these include crosstalk between chromatin architecture and mRNA decay. To address this, we performed a genome-wide genetic screen using a yeast strain harbouring the H3K56A mutation known to perturb chromatin structure and nascent transcription. We identified Puf5 as essential in an H3K56A background. Depletion of Puf5 in this background leads to downregulation of Puf5 targets. We suggest that Puf5 plays a role in post-transcriptional buffering of mRNAs and support this by transcriptional shutoff experiments in which Puf5 mRNA targets are degraded slower in H3K56A compared to wildtype. Finally, we show that post-transcriptional buffering of Puf5 targets is widespread and does not occur only in an H3K56A mutant, but also in an H3K4R background, which leads to a global increase in nascent transcription. Our data suggest that Puf5 determines the fate of its mRNA targets in a context-dependent manner acting as an mRNA surveillance hub balancing de-regulated nascent transcription to maintain physiological mRNA levels.

Download Full-text

The RNA-binding protein Puf5 buffers mRNA levels against chromatin-mediated changes in nascent transcription

10.1101/2020.08.13.249912 ◽

2020 ◽

Author(s):

David Z. Kochan ◽

Julia S. P. Mawer ◽

Kiril Tishinov ◽

Swati Parekh ◽

Jennifer Massen ◽

...

Keyword(s):

Rna Binding ◽

Genetic Interaction ◽

Synthetic Lethality ◽

Mrna Export ◽

Mrna Levels ◽

Dependent Manner ◽

Ribosomal Protein Genes ◽

Feedback Mechanisms ◽

Mrna Targets ◽

Widespread Phenomenon

ABSTRACTGene expression is a dynamic process regulated at all stages, starting with opening of chromatin, transcription, and continuing with mRNA export, translation and, finally, degradation. While there are feedback mechanisms within the system, it is not clear whether these extend to crosstalk between chromatin architecture and mRNA decay. Here, we show that changes in nascent transcription, mediated by mutating H3K56 to alanine, are post-transcriptionally buffered by the Pumilio protein Puf5, which stabilizes transcripts in a context-dependent manner. Depleting Puf5 in an H3K56A background leads to synthetic lethality. This genetic interaction can be explained by a decrease in translation due to downregulation of its direct mRNA targets, largely consisting of ribosomal protein genes. Importantly, we show that this post-transcriptional buffering is not only linked to H3K56A, but may be a more widespread phenomenon that also buffers against an increase in nascent RNA transcription in order to maintain physiological mRNA levels and cellular homeostasis.

Download Full-text

Genome-wide identification and characterization of transcripts translationally regulated by bacterial lipopolysaccharide in macrophage-like J774.1 cells

Physiological Genomics ◽

10.1152/physiolgenomics.00095.2007 ◽

2008 ◽

Vol 33 (1) ◽

pp. 121-132 ◽

Cited By ~ 18

Author(s):

Hiroshi Kitamura ◽

Masatoshi Ito ◽

Tomoko Yuasa ◽

Chisato Kikuguchi ◽

Atsushi Hijikata ◽

...

Keyword(s):

Nitric Oxide ◽

Respiratory Chain ◽

Mrna Level ◽

Critical Role ◽

Mitochondrial Respiratory Chain ◽

Mrna Levels ◽

Dependent Manner ◽

Genome Wide ◽

A Genome ◽

Mitochondrial Respiratory

Although Escherichia coli LPS is known to elicit various proinflammatory responses in macrophages, its effect on the translational states of transcripts has not yet been explored on a genome-wide scale. To address this, we investigated the mRNA profiles in polysomal and free messenger ribonucleoprotein particle (mRNP) fractions of mouse macrophage-like J774.1 cells, using Affymetrix Mouse Genome 430 2.0 GeneChips. Comparison of the mRNA profiles in total cellular, polysomal, and free mRNP fractions enabled us to identify transcripts that were modulated at the translational level by LPS: among 19,791 transcripts, 115 and 418 were up- and downregulated at 1, 2, or 4 h after LPS stimulation (100 ng/ml) in a translation-dependent manner. Interestingly, gene ontology-based analysis suggested that translation-dependent downregulated genes frequently include those encoding proteins in the mitochondrial respiratory chain. In fact, the mRNA levels of some transcripts for complexes I, IV, and V in the mitochondrial respiratory chain were translationally downregulated, eventually contributing to the decline of their protein levels. Moreover, the amount of metabolically labeled cytochrome oxidase subunit Va in complex IV was decreased without any change of its mRNA level in total cellular fraction after LPS stimulation. Consistently, the total amounts and activities of complexes I and IV were attenuated by LPS stimulation, and the attenuation was independent of nitric oxide. These results demonstrated that translational suppression may play a critical role in the LPS-mediated attenuation of mitochondrial oxidative phosphorylation in a nitric oxide-independent manner in J774.1 cells.

Download Full-text

The Nuclear Exosome Contributes to Autogenous Control of NAB2 mRNA Levels

Molecular and Cellular Biology ◽

10.1128/mcb.25.5.1577-1585.2005 ◽

2005 ◽

Vol 25 (5) ◽

pp. 1577-1585 ◽

Cited By ~ 37

Author(s):

Kelly M. Roth ◽

Maria K. Wolf ◽

Marie Rossi ◽

J. Scott Butler

Keyword(s):

Rna Processing ◽

Untranslated Region ◽

Rna Binding ◽

Noncoding Rnas ◽

Mrna Levels ◽

Dependent Manner ◽

Autogenous Control ◽

Regulatory Circuit ◽

Mrna Surveillance ◽

Nuclear Exosome

ABSTRACT The RNA-processing exosome is a complex of riboexonucleases required for 3′-end formation of some noncoding RNAs and for the degradation of mRNAs in eukaryotes. The nuclear form of the exosome functions in an mRNA surveillance pathway that retains and degrades improperly processed precursor mRNAs within the nucleus. We report here that the nuclear exosome controls the level of NAB2 mRNA, encoding the nuclear poly(A)+-RNA-binding protein Nab2p. Mutations affecting the activity of the nuclear, but not the cytoplasmic, exosome cause an increase in the amount of NAB2 mRNA. Cis- and trans-acting mutations that inhibit degradation by the nuclear-exosome subunit Rrp6p result in elevated levels of NAB2 mRNA. Control of NAB2 mRNA levels occurs posttranscriptionally and requires a sequence of 26 consecutive adenosines (A26) in the NAB2 3′ untranslated region, which represses NAB2 3′-end formation and sensitizes the transcript to degradation by Rrp6p. Analysis of NAB2 mRNA levels in a nab2-1 mutant and in the presence of excess Nab2p indicates that Nab2p activity negatively controls NAB2 mRNA levels in an A26- and Rrp6p-dependent manner. These findings suggest a novel regulatory circuit in which the nuclear exosome controls the level of NAB2 mRNA in response to changes in the activity of Nab2 protein.

Download Full-text

Abstract 12192: MBNL1 Directly Regulates the Alternative Splicing of mRNAs That Promote Myofibroblast Differentiation

Circulation ◽

10.1161/circ.130.suppl_2.12192 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Jennifer Davis ◽

Michelle Sargent ◽

Jianjian Shi ◽

Lei Wei ◽

Maurice S Swanson ◽

...

Keyword(s):

Alternative Splicing ◽

Molecular Mechanisms ◽

Rna Binding ◽

Transforming Growth Factor ◽

Ventricular Remodeling ◽

Cardiac Injury ◽

Myofibroblast Differentiation ◽

Genome Wide ◽

A Genome

Rationale: During the cardiac injury response fibroblasts differentiate into myofibroblasts, a cell type that enhances extracellular matrix production and facilitates ventricular remodeling. To better understand the molecular mechanisms whereby myofibroblasts are generated in the heart we performed a genome-wide screen with 18,000 cDNAs, which identified the RNA-binding protein muscleblind-like splicing regulator 1 (MBNL1), suggesting a novel association between mRNA alternative splicing and the regulation of myofibroblast differentiation. Objective: To determine the mechanism whereby MBNL1 regulates myofibroblast differentiation and the cardiac fibrotic response. Methods and Results: Confirming the results from our genome wide screen, adenoviral-mediated overexpression of MBNL1 promoted transformation of rat cardiac fibroblasts and mouse embryonic fibroblasts (MEFs) into myofibroblasts, similar to the level of conversion obtained by the profibrotic agonist transforming growth factor β (TGFβ). Antithetically, Mbnl1 -/- MEFs were refractory to TGFβ-induced myofibroblast differentiation. MBNL1 expression is induced in transforming fibroblasts in response to TGFβ and angiotensin II. These results were extended in vivo by analysis of dermal wound healing, a process dependent on myofibroblast differentiation and their proper activity. By day 6 control mice had achieved 82% skin wound closure compared with only 40% in Mbnl1 -/- mice. Moreover, Mbnl1 -/- mice had reduced survival following myocardial infarction injury due to defective fibrotic scar formation and healing. High throughput RNA sequencing (RNAseq) and RNA immunoprecipitation revealed that MBNL1 directly regulates the alternative splicing of transcripts for myofibroblast signaling factors and cytoskeletal-assembly elements. Functional analysis of these factors as mediators of MBNL1 activity is also described here. Conclusions: Collectively, our data suggest that MBNL1 coordinates myofibroblast transformation by directly mediating the alternative splicing of an array of mRNAs encoding differentiation-specific signaling transcripts, which then alter the fibroblast proteome for myofibroblast structure and function.

Download Full-text

Chromatin architectural proteins regulate flowering time by precluding gene looping

Science Advances ◽

10.1126/sciadv.abg3097 ◽

2021 ◽

Vol 7 (24) ◽

pp. eabg3097

Author(s):

Bo Zhao ◽

Yanpeng Xi ◽

Junghyun Kim ◽

Sibum Sung

Keyword(s):

Chromatin Structure ◽

Cellular Processes ◽

Genome Wide ◽

A Genome ◽

Evolutionarily Conserved ◽

Architectural Proteins ◽

Floral Repressor ◽

Flanking Regions ◽

Genome Wide Study

Chromatin structure is critical for gene expression and many other cellular processes. In Arabidopsis thaliana, the floral repressor FLC adopts a self-loop chromatin structure via bridging of its flanking regions. This local gene loop is necessary for active FLC expression. However, the molecular mechanism underlying the formation of this class of gene loops is unknown. Here, we report the characterization of a group of linker histone-like proteins, named the GH1-HMGA family in Arabidopsis, which act as chromatin architecture modulators. We demonstrate that these family members redundantly promote the floral transition through the repression of FLC. A genome-wide study revealed that this family preferentially binds to the 5′ and 3′ ends of gene bodies. The loss of this binding increases FLC expression by stabilizing the FLC 5′ to 3′ gene looping. Our study provides mechanistic insights into how a family of evolutionarily conserved proteins regulates the formation of local gene loops.

Download Full-text

Function of Pumilio Genes in Human Embryonic Stem Cells and Their Effect in Stemness and Cardiomyogenesis

10.1101/751537 ◽

2019 ◽

Author(s):

Isabelle Leticia Zaboroski Silva ◽

Anny Waloski Robert ◽

Guillermo Cabrera Cabo ◽

Lucia Spangenberg ◽

Marco Augusto Stimamiglio ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Rna Binding ◽

Rna Binding Proteins ◽

Embryonic Stem ◽

Mrna Levels ◽

Human Escs ◽

Mrna Targets ◽

Cardiomyogenic Differentiation

AbstractPosttranscriptional regulation plays a fundamental role in the biology of embryonic stem cells (ESCs). Many studies have demonstrated that multiple mRNAs are coregulated by one or more RNA binding proteins (RBPs) that orchestrate the expression of these molecules. A family of RBPs, known as PUF (Pumilio-FBF), is highly conserved among species and has been associated with the undifferentiated and differentiated states of different cell lines. In humans, two homologs of the PUF family have been found: Pumilio 1 (PUM1) and Pumilio 2 (PUM2). To understand the role of these proteins in human ESCs (hESCs), we first demonstrated the influence of the silencing of PUM1 and PUM2 on pluripotency genes. OCT4 and NANOG mRNA levels decreased significantly with the knockdown of Pumilio, suggesting that PUMILIO proteins play a role in the maintenance of pluripotency in hESCs. Furthermore, we observed that the hESCs silenced for PUM1 and 2 exhibited an improvement in efficiency of in vitro cardiomyogenic differentiation. Using in silico analysis, we identified mRNA targets of PUM1 and PUM2 expressed during cardiomyogenesis. With the reduction of PUM1 and 2, these target mRNAs would be active and could be involved in the progression of cardiomyogenesis.

Download Full-text

Star-PAP RNA Binding Landscape Reveals Novel Role of Star-PAP in mRNA Metabolism That Requires RBM10-RNA Association

International Journal of Molecular Sciences ◽

10.3390/ijms22189980 ◽

2021 ◽

Vol 22 (18) ◽

pp. 9980

Author(s):

Ganesh R. Koshre ◽

Feba Shaji ◽

Neeraja K. Mohanan ◽

Nimmy Mohan ◽

Jamshaid Ali ◽

...

Keyword(s):

Rna Binding ◽

Coding Region ◽

High Association ◽

Target Mrnas ◽

Mrna Metabolism ◽

Mrna Targets ◽

Genome Wide ◽

Global Profile ◽

Specific Mrna

Star-PAP is a non-canonical poly(A) polymerase that selects mRNA targets for polyadenylation. Yet, genome-wide direct Star-PAP targets or the mechanism of specific mRNA recognition is still vague. Here, we employ HITS-CLIP to map the cellular Star-PAP binding landscape and the mechanism of global Star-PAP mRNA association. We show a transcriptome-wide association of Star-PAP that is diminished on Star-PAP depletion. Consistent with its role in the 3′-UTR processing, we observed a high association of Star-PAP at the 3′-UTR region. Strikingly, there is an enrichment of Star-PAP at the coding region exons (CDS) in 42% of target mRNAs. We demonstrate that Star-PAP binding de-stabilises these mRNAs indicating a new role of Star-PAP in mRNA metabolism. Comparison with earlier microarray data reveals that while UTR-associated transcripts are down-regulated, CDS-associated mRNAs are largely up-regulated on Star-PAP depletion. Strikingly, the knockdown of a Star-PAP coregulator RBM10 resulted in a global loss of Star-PAP association on target mRNAs. Consistently, RBM10 depletion compromises 3′-end processing of a set of Star-PAP target mRNAs, while regulating stability/turnover of a different set of mRNAs. Our results establish a global profile of Star-PAP mRNA association and a novel role of Star-PAP in the mRNA metabolism that requires RBM10-mRNA association in the cell.

Download Full-text

Genome-wide CRISPR screen identifies ELP5 as a determinant of gemcitabine sensitivity in gallbladder cancer

Nature Communications ◽

10.1038/s41467-019-13420-x ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 8

Author(s):

Sunwang Xu ◽

Ming Zhan ◽

Cen Jiang ◽

Min He ◽

Linhua Yang ◽

...

Keyword(s):

Gallbladder Cancer ◽

Locally Advanced ◽

Internal Ribosomal Entry Site ◽

Dependent Manner ◽

Entry Site ◽

P53 Expression ◽

Elongator Complex ◽

Genome Wide ◽

A Genome ◽

Crispr Screen

AbstractGemcitabine is the first-line treatment for locally advanced and metastatic gallbladder cancer (GBC), but poor gemcitabine response is universal. Here, we utilize a genome-wide CRISPR screen to identify that loss of ELP5 reduces the gemcitabine-induced apoptosis in GBC cells in a P53-dependent manner through the Elongator complex and other uridine 34 (U34) tRNA-modifying enzymes. Mechanistically, loss of ELP5 impairs the integrity and stability of the Elongator complex to abrogate wobble U34 tRNA modification, and directly impedes the wobble U34 modification-dependent translation of hnRNPQ mRNA, a validated P53 internal ribosomal entry site (IRES) trans-acting factor. Downregulated hnRNPQ is unable to drive P53 IRES-dependent translation, but rescuing a U34 modification-independent hnRNPQ mutant could restore P53 translation and gemcitabine sensitivity in ELP5-depleted GBC cells. GBC patients with lower ELP5, hnRNPQ, or P53 expression have poor survival outcomes after gemcitabine chemotherapy. These results indicate that the Elongator/hnRNPQ/P53 axis controls gemcitabine sensitivity in GBC cells.

Download Full-text

Identification and function of hypoxia-response genes in Drosophila melanogaster

Physiological Genomics ◽

10.1152/physiolgenomics.00262.2005 ◽

2006 ◽

Vol 25 (1) ◽

pp. 134-141 ◽

Cited By ~ 46

Author(s):

Guowen Liu ◽

Julianne Roy ◽

Eric A. Johnson

Keyword(s):

Recovery Time ◽

Normal Activity ◽

Mrna Levels ◽

Low Oxygen ◽

Hypoxia Response ◽

Oxygen Treatment ◽

Genome Wide ◽

A Genome ◽

And Function ◽

Genome Wide Study

Hypoxia, an insufficient level of oxygen in the cell, occurs during normal activity and also in pathological conditions such as ischemia and tumorigenesis. Although many hypoxia-response genes have been identified, an understanding of the functional role for these genes in the living animal is lacking. Here we present a genome-wide study of gene expression changes during hypoxia and then functionally test a subset of these genes for roles in survival and recovery from hypoxia. We found 79 genes with increased mRNA levels when adult flies were treated with 0.5% O2 for 6 h. A subset of these genes had detectably increased levels in as short as 1 h of low-oxygen treatment. Mild hypoxia levels resulted in an increase in transcription levels for only 20 genes. Viability during hypoxia and recovery time from hypoxia-induced paralysis was examined in flies with a reduction in activity in hypoxia-response genes. The observed decreased viability and increased recovery time from paralysis in many of the lines demonstrate that the increased transcript levels seen after hypoxia are important for the response to low oxygen.

Download Full-text