Post-Transcriptional Regulation of Cardiac Sarcomere Protein Titin Through 3’ Untranslated Regions

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Striated Muscle ◽  
Binding Capacity ◽  
Structural Features ◽  
Neonatal Rat ◽  
Regulatory Control ◽  
Untranslated Regions ◽  
Luciferase Reporter ◽  
Regulatory Effects

Titin is the largest known protein in the human body, and forms the backbone of all striated muscle sarcomeres. The elastic nature of titin is an important component of muscle compliance and functionality. A significant amount of energy is expended to synthesize titin, thus we postulate that titin gene expression is under strict regulatory control in order to conserve cellular resources. In general, gene expression is mediated in part by post-transcriptional control elements located within the 5’ and 3’ untranslated regions (UTRs) of mature mRNA. The 3’UTR in particular contains structural features that affect binding capacity to other RNA components, such as MicroRNA, which control mRNA localization, translation, and degradation. The degree and significance of the regulatory effects mediated by two determined variants of titin’s 3’ UTR were evaluated in Neonatal Rat Ventricular Myocyte and Human Embryonic Kidney cell lines. Recombinant plasmids to transfect these cells lines were engineered by insertion of the variant titin 3’UTR 431- and 1047-base pairs sequences into luciferase reporter vectors. Expression due to an unaltered reporter vector served as the control. Quantitative changes in luciferase activity due to the recombinants proportionally represented the effect titin’s respective 3’UTR conferred on downstream post-transcriptional expression relative to the control. The effect due to titin’s shorter 3’UTR sequence was inconclusive; however, results illustrated that titin’s longer 3’UTR sequence caused a 35 percent decrease in protein expression. Secondary structural analysis of the two sequences revealed differential folding patterns that affect the stability and degree of MicroRNA-binding within titin’s variant 3’UTR sequences.

scholarly journals Arginine Methyltransferases as Regulators of RNA-Binding Protein Activities in Pathogenic Kinetoplastids

2021 ◽  
Vol 8 ◽  
Author(s):  
Gustavo D. Campagnaro ◽  
Edward Nay ◽  
Michael J. Plevin ◽  
Angela K. Cruz ◽  
Pegine B. Walrad
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Rna Binding ◽  
Methylation Status ◽  
Regulation Of Gene Expression ◽  
Structural Features ◽  
Arginine Methylation ◽  
Diverse Range ◽  
Host Interaction ◽  
Intronless Genes

A large number of eukaryotic proteins are processed by single or combinatorial post-translational covalent modifications that may alter their activity, interactions and fate. The set of modifications of each protein may be considered a “regulatory code”. Among the PTMs, arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), can affect how a protein interacts with other macromolecules such as nucleic acids or other proteins. In fact, many RNA-binding (RBPs) proteins are targets of PRMTs. The methylation status of RBPs may affect the expression of their bound RNAs and impact a diverse range of physiological and pathological cellular processes. Unlike most eukaryotes, Kinetoplastids have overwhelmingly intronless genes that are arranged within polycistronic units from which mature mRNAs are generated by trans-splicing. Gene expression in these organisms is thus highly dependent on post-transcriptional control, and therefore on the action of RBPs. These genetic features make trypanosomatids excellent models for the study of post-transcriptional regulation of gene expression. The roles of PRMTs in controlling the activity of RBPs in pathogenic kinetoplastids have now been studied for close to 2 decades with important advances achieved in recent years. These include the finding that about 10% of the Trypanosoma brucei proteome carries arginine methylation and that arginine methylation controls Leishmania:host interaction. Herein, we review how trypanosomatid PRMTs regulate the activity of RBPs, including by modulating interactions with RNA and/or protein complex formation, and discuss how this impacts cellular and biological processes. We further highlight unique structural features of trypanosomatid PRMTs and how it contributes to their singular functionality.

scholarly journals Regulatory signals in messenger RNA: determinants of nutrient–gene interaction and metabolic compartmentation

1998 ◽  
Vol 80 (4) ◽  
pp. 307-321
Author(s):  
John E. Hesketh ◽  
M. Helena Vasconcelos ◽  
Giovanna Bermano
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Mrna Stability ◽  
Transcriptional Control ◽  
Regulation Of Gene Expression ◽  
Untranslated Regions ◽  
Nutritional Requirements ◽  
Control Of Gene Expression ◽  
Metabolic Compartmentation ◽  
Post Transcriptional Regulation

Nutrition has marked influences on gene expression and an understanding of the interaction between nutrients and gene expression is important in order to provide a basis for determining the nutritional requirements on an individual basis. The effects of nutrition can be exerted at many stages between transcription of the genetic sequence and production of a functional protein. This review focuses on the role of post-transcriptional control, particularly mRNA stability, translation and localization, in the interactions of nutrients with gene expression. The effects of both macronutrients and micronutrients on regulation of gene expression by post-transcriptional mechanisms are presented and the post-transcriptional regulation of specific genes of nutritional relevance (glucose transporters, transferrin, selenoenzymes, metallothionein, lipoproteins) is described in detail. The function of the regulatory signals in the untranslated regions of the mRNA is highlighted in relation to control of mRNA stability, translation and localization and the importance of these mRNA regions to regulation by nutrients is illustrated by reference to specific examples. The localization of mRNA by signals in the untranslated regions and its function in the spatial organization of protein synthesis is described; the potential of such mechanisms to play a key part in nutrient channelling and metabolic compartmentation is discussed. It is concluded that nutrients can influence gene expression through control of the regulatory signals in these untranslated regions and that the post-transcriptional regulation of gene expression by these mechanisms may influence nutritional requirements. It is emphasized that in studies of nutritional control of gene expression it is important not to focus only on regulation through gene promoters but also to consider the possibility of post-transcriptional control.

Endothelin ETA receptor regulates signaling and ANF gene expression via multiple G protein-linked pathways

1997 ◽  
Vol 272 (1) ◽  
pp. H130-H137 ◽  
Author(s):  
R. Hilal-Dandan ◽  
M. T. Ramirez ◽  
S. Villegas ◽  
A. Gonzalez ◽  
Y. Endo-Mochizuki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adenylyl Cyclase ◽  
Hormonal Regulation ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Mrna Levels ◽  
Binding Studies ◽  
Eta Receptor

We have characterized the interaction of endothelin (ET) with cultured neonatal rat ventricular myocytes. Binding studies indicate a single population of ETA receptors [53,000 sites/cell, apparent dissociation constant (Kd) for ET-1 approximately 0.07 nM]. Analysis of mRNA levels for ET receptors using 35 cycles of reverse transcriptase-polymerase chain reaction demonstrates the presence of only ETA-receptor message. Studies with ET-1 and a variety of congeners and antagonists indicate that ETA receptors couple to both the stimulation of phosphoinositide turnover and the inhibition of adenylyl cyclase. In myocytes transfected with an atrial natriuretic factor (ANF) promoter linked to a luciferase reporter gene, ET-1 stimulates luciferase expression through an ETA receptor. These data indicate that the ETA receptor is the exclusive receptor on neonatal ventricular myocytes and that this receptor couples to both phosphoinositide hydrolysis and adenylyl cyclase. ET-1 also induces a threefold increase in mitogen-activated protein kinase (MAPK) activity, an effect that is not sensitive to pertussis toxin (PTx). By contrast, ET-stimulated ANF-luciferase expression is partially inhibited by treatment of cells with PTx, suggesting that both PTx-sensitive (Gi) and PTx-insensitive (Gq) pathways mediate the effects of ET-1 on ANF gene expression in neonatal myocytes and that hormonal regulation of ANF expression may utilize pathways in addition to the activation of MAPK.

scholarly journals BMP type I receptor ALK2 is required for angiotensin II-induced cardiac hypertrophy

2016 ◽  
Vol 310 (8) ◽  
pp. H984-H994 ◽  
Author(s):  
Mohd Shahid ◽  
Ester Spagnolli ◽  
Laura Ernande ◽  
Robrecht Thoonen ◽  
Starsha A. Kolodziej ◽  
...  
Keyword(s):  
Gene Expression ◽  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Left Ventricular ◽  
Bmp Signaling ◽  
Neonatal Rat ◽  
Luciferase Reporter ◽  
Type I ◽  
Nuclear Factor ◽  
Rat Cardiomyocytes

Bone morphogenetic protein (BMP) signaling contributes to the development of cardiac hypertrophy. However, the identity of the BMP type I receptor involved in cardiac hypertrophy and the underlying molecular mechanisms are poorly understood. By using quantitative PCR and immunoblotting, we demonstrated that BMP signaling increased during phenylephrine-induced hypertrophy in cultured neonatal rat cardiomyocytes (NRCs), as evidenced by increased phosphorylation of Smads 1 and 5 and induction of Id1 gene expression. Inhibition of BMP signaling with LDN193189 or noggin, and silencing of Smad 1 or 4 using small interfering RNA diminished the ability of phenylephrine to induce hypertrophy in NRCs. Conversely, activation of BMP signaling with BMP2 or BMP4 induced hypertrophy in NRCs. Luciferase reporter assay further showed that BMP2 or BMP4 treatment of NRCs repressed atrogin-1 gene expression concomitant with an increase in calcineurin protein levels and enhanced activity of nuclear factor of activated T cells, providing a mechanism by which BMP signaling contributes to cardiac hypertrophy. In a model of cardiac hypertrophy, C57BL/6 mice treated with angiotensin II (A2) had increased BMP signaling in the left ventricle. Treatment with LDN193189 attenuated A2-induced cardiac hypertrophy and collagen deposition in left ventricles. Cardiomyocyte-specific deletion of BMP type I receptor ALK2 (activin-like kinase 2), but not ALK1 or ALK3, inhibited BMP signaling and mitigated A2-induced cardiac hypertrophy and left ventricular fibrosis in mice. The results suggest that BMP signaling upregulates the calcineurin/nuclear factor of activated T cell pathway via BMP type I receptor ALK2, contributing to cardiac hypertrophy and fibrosis.

scholarly journals The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element

2000 ◽  
Vol 278 (6) ◽  
pp. H1769-H1774 ◽  
Author(s):  
Michael R. Morissette ◽  
Valerie P. Sah ◽  
Christopher C. Glembotski ◽  
Joan Heller Brown
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Dominant Negative ◽  
Neonatal Rat ◽  
Common Element ◽  
Luciferase Reporter ◽  
Serum Response Element ◽  
Transcriptional Responses ◽  
Response Element ◽  
Serum Response

The low-molecular-weight GTP-binding protein RhoA mediates hypertrophic growth and atrial natriuretic factor (ANF) gene expression in neonatal rat ventricular myocytes. Neither the effector nor the promoter elements through which Rho exerts its regulatory effects on ANF gene expression have been elucidated. When constitutively activated forms of Rho kinase and two protein kinase C-related kinases, PKN (PRK1) and PRK2, were compared, only PKN generated a robust stimulation of a luciferase reporter gene driven by a 638-bp fragment on the ANF promoter. This ANF promoter fragment contains a proximal serum response element (SRE) and an Sp-1-like element required for the transcriptional response to phenylephrine (PE). This response was inhibited by dominant negative Rho. The ability of dominant negative Rho to inhibit the response to PE and the ability of PKN to stimulate ANF reporter gene expression were both lost when the SRE was mutated. Mutation of the Sp-1-like element also attenuated the response to PKN. A minimal promoter driven by ANF SRE sequences was sufficient to confer Rho- and PKN-mediated gene expression. Interestingly, PKN preferentially stimulated the ANF versus the c- fos SRE reporter gene. Thus PKN and Rho are able to regulate transcriptional activation of the ANF SRE by a common element that could implicate PKN as a downstream effector of Rho in transcriptional responses associated with hypertrophy.

scholarly journals Effects of HTR1B 3' region polymorphisms and functional regions on gene expression regulation

2020 ◽  
Author(s):  
xi xia ◽  
Mei Ding ◽  
Jin-feng Xuan ◽  
Jia-xin Xing ◽  
Hao Pang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Regulation ◽  
Inhibitory Effect ◽  
Expression Level ◽  
Luciferase Reporter ◽  
Negative Effects ◽  
Hek 293 ◽  
Functional Regions ◽  
Regulatory Effects ◽  
Post Transcriptional Regulation

Abstract Background The HTR1B gene encodes the 5-hydroxytryptamine (5-HT1B) receptor, which is involved in a variety of brain activities and mental disorders. The regulatory effects of non-coding regions on genomic DNA are one of many reasons for the cause of genetic-related diseases. Post-transcriptional regulation that depends on the function of 3' regulatory regions plays a particularly important role. This study investigated the effects, on reporter gene expression, of several haplotypes of the HTR1B gene (rs6297, rs3827804, rs140792648, rs9361234, rs76194807, rs58138557, and rs13212041) and truncated fragments in order to analyze the function of the 3' region of HTR1B. Methods Seven haplotypes, consisting of rs6297, rs3827804, rs140792648, rs9361234, rs76194807, rs58138557, and rs13212041, and truncated fragments of the HTR1B gene 3' region were transfected into SK-N-SH, HEK-293, and U87 cell lines. The relative fluorescence intensities were detected by a dual luciferase reporter assay system.Results We found that the haplotype, AG_CT_A, enhanced the expression level compared to the main haplotype; AG_CG_A; GG_CG_G decreased the expression level. Two alleles, rs76194807T and rs6297G, exhibited different relative luciferase intensities compared to their counterparts at each locus. We also found that +2440 ~ +2769 bp and +1953 ~ +2311 bp regions both had negative effects on gene expression.ConclusionsThe 3' region of HTR1B has a regulatory effect on gene expression, which is likely closely associated with the interpretation of HTR1B-related disorders. In addition, the HTR1B 3' region includes several effector binding sites that induce an inhibitory effect on gene expression.

scholarly journals The Regulatory Effects of Purα on PARP1 Gene Expression and its functions on DNA repair

2020 ◽  
Vol 1 ◽  
Author(s):  
Wenxin He He ◽  
Jianqi Cui ◽  
Yongling Li ◽  
Xiaoguang Shi ◽  
Juan Chai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Damage ◽  
Expression Vector ◽  
Eukaryotic Expression ◽  
Luciferase Assay ◽  
Luciferase Reporter ◽  
Physical Interaction ◽  
Eukaryotic Expression Vector ◽  
Regulatory Effects ◽  
Damaged Dna

Abstract: The luciferase reporter construct, which contains a poly ADP-ribose polymerase 1 gene promoter, was transfected into U87MG cells with the Purα eukaryotic expression vector, and the activities of PARP1 promoter were assessed by luciferase assay to evaluate the regulatory effects of Purα on PARP1 gene expression. The Purα eukaryotic expression vector was transfected into U87MG cells, and the cell total RNA and protein were extracted to determine the effects of Purα on PARP1 gene expression at transcriptional and translational levels by real-time PCR and Western blotting assay. The results demonstrated that Purα can positively regulate PARP1 promoter activity and promote PARP1 gene expression both at transcriptional and translational levels. The further study illustrated that Purα can collaborate with PARP1 in the repair of damaged DNA; the results of a pull-down assay suggested that there is a physical interaction between Purα and PARP1. The overexpression of Purα can increase endogenous PARP1 expression and alleviate the expression of the DNA damage signal protein γH2AX. Above all, we believe that Purα possesses a positive regulatory effect on PARP1 gene expression and collaborates with PARP1 to repair damaged DNA. Keywords: Purα; poly ADP-ribose polymerase 1; regulation of gene expression, DNA damage, and repair.

scholarly journals Sp3 is involved in the regulation of SOCS3 gene expression

2005 ◽  
Vol 387 (3) ◽  
pp. 737-745 ◽  
Author(s):  
Christian EHLTING ◽  
Dieter HÄUSSINGER ◽  
Johannes G. BODE
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Transcriptional Activation ◽  
Transcriptional Control ◽  
Gene Promoter ◽  
Janus Kinase ◽  
Regulatory Control ◽  
Upstream Region ◽  
Socs3 Expression ◽  
Socs3 Mrna

Cytokine-induced expression of SOCS (suppressor of cytokine signalling) molecules is important for the negative regulatory control of STAT (signal transduction and activators of transcription)-dependent cytokine signalling, e.g. for the signal transduction of IL-6 (interleukin-6)-type cytokines through the JAK (Janus kinase)/STAT cascade. STAT activation itself represents an important step in the transcriptional activation of SOCS3 gene expression. However, downstream of the STAT-responsive element, the SOCS3 gene contains a GC-rich element in its 5′-upstream region. The aim of the present study was to investigate the implications of this GC-rich element in the transcriptional control of SOCS3 gene expression. In the present study, we show that mutation of this GC-rich element abolishes IL-6-dependent transcriptional activation of the SOCS3 promoter and that Sp3 (specificity protein 3), a ubiquitously expressed transcription factor, but not Sp1 binds to this GC-rich motif, suggesting that Sp3 is involved in the regulation of SOCS3 expression. The results suggest that Sp3 is important for IL-6-induced transcriptional activation of the SOCS3 (gene) promoter and acts as an enhancer of basal as well as induced transcriptional activity, resulting in enhanced SOCS3 mRNA and protein expression. Mutation of Lys-483, a potential target for Sp3 acetylation, inhibited Sp3-mediated enhancement of SOCS3 mRNA expression and SOCS3 promoter activation, indicating that the acetylation of this lysine residue of Sp3 is important for the enhancing effect of Sp3 on SOCS3 expression.

Identification of ATF2 as a transcriptional regulator of renin gene

2012 ◽  
Vol 393 (1-2) ◽  
pp. 93-100 ◽  
Author(s):  
Michael Desch ◽  
Gerit Hackmayer ◽  
Vladimir T. Todorov
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Transcriptional Control ◽  
Regulatory Region ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Distal Enhancer ◽  
Reporter System ◽  
Renin Gene ◽  
Factor Α

Abstract The cAMP response element (enhCRE) in the distal enhancer regulatory region of renin gene is believed to play a major role in the control of renin transcription. enhCRE binds the CRE-binding protein (CREB), which is the main transcription factor target of cAMP signaling. Using the mouse renin-producing cell line As4.1 we found that activating transcription factor-2 (ATF2) also binds to enhCRE. N-terminal phosphorylation of ATF2, which controls its transactivation, is associated with downregulation of renin gene expression by the cytokine tumor necrosis factor-α (TNFα). The ubiquitin proteasome inhibitor MG132 also phosphorylates ATF2 and inhibits renin expression. Knockdown of ATF2 attenuated the suppression of renin gene expression by MG132, thus demonstrating that ATF2 mediates the inhibitory effect of MG132. In addition, MG132 increased the DNA-binding of ATF2 as well as the ratio of bound ATF2 to CREB. Using ATF2- and CREB-Gal4 fusion protein constructs coupled with luciferase reporter system we showed that ATF2 has a weaker transactivating capacity than CREB. These data suggest that ATF2 represses renin expression by drifting the transcriptional control of renin gene away from CREB. Accordingly, TNFα completely abrogated the cAMP-dependent stimulation of renin gene expression.

Nutrient-regulated gene expression in eukaryotes

2006 ◽  
Vol 73 ◽  
pp. 85-96 ◽  
Author(s):  
Richard J. Reece ◽  
Laila Beynon ◽  
Stacey Holden ◽  
Amanda D. Hughes ◽  
Karine Rébora ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcriptional Control ◽  
Direct Interaction ◽  
Signalling Pathways ◽  
A Cell ◽  
One Step ◽  
Higher Eukaryotes ◽  
Regulated Gene Expression

The recognition of changes in environmental conditions, and the ability to adapt to these changes, is essential for the viability of cells. There are numerous well characterized systems by which the presence or absence of an individual metabolite may be recognized by a cell. However, the recognition of a metabolite is just one step in a process that often results in changes in the expression of whole sets of genes required to respond to that metabolite. In higher eukaryotes, the signalling pathway between metabolite recognition and transcriptional control can be complex. Recent evidence from the relatively simple eukaryote yeast suggests that complex signalling pathways may be circumvented through the direct interaction between individual metabolites and regulators of RNA polymerase II-mediated transcription. Biochemical and structural analyses are beginning to unravel these elegant genetic control elements.

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