Transcriptional and post-transcriptional mechanisms regulating the rat pituitary vasopressin V1b receptor gene

The number of V1b vasopressin receptors (V1bR) in the anterior pituitary plays an important role during adaptation of the hypothalamic-pituitary-adrenal axis to stress in rats. Regulation of V1bR expression involves transcriptional and translational mechanisms. One of the elements mediating transcriptional activation of the rat V1bR gene is a long stretch of GAGA repeats (GAGA box) in the promoter located near the transcription start point capable of binding a protein complex of 127 kDa present in pituitary nuclear extracts. There is a lack of correlation between changes in V1bR mRNA and the number of VP binding sites, suggesting that V1bR expression depends on the efficiency of V1b R mRNA translation into protein. Two mechanisms by which the 5' untranslated region (5'-UTR) of the rat V1bR mRNA can mediate either inhibition or activation of V1bR mRNA translation have been identified. First, upstream open reading frames (ORF) present in the 5'-UTR repress translation of the major ORF encoding the V1b receptor, and secondly, an internal ribosome entry site (IRES) activates V1bR translation. Stimulation of IRES activity through protein kinase C-mediated pathways results in V1bR mRNA translation increasing V1bR protein levels. The existence of multiple loci of regulation for the V1bR at transcriptional and translational levels provides a mechanism to facilitate plasticity of regulation of the number of pituitary vasopressin receptors according to physiological demand.

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Augmentation of Epithelial Resistance to Invading Bacteria by Using mRNA Transfections

Infection and Immunity ◽

10.1128/iai.00539-13 ◽

2013 ◽

Vol 81 (11) ◽

pp. 3975-3983 ◽

Cited By ~ 12

Author(s):

Xianqiong Zou ◽

Brent S. Sorenson ◽

Karen F. Ross ◽

Mark C. Herzberg

Keyword(s):

Epithelial Cells ◽

Open Reading Frames ◽

Antimicrobial Protein ◽

Entry Site ◽

Internal Ribosome Entry ◽

Content Type ◽

Protein Levels ◽

Oral Epithelial Cells ◽

Mrna Transfection ◽

Oral Epithelial

ABSTRACTTo protect against invading bacteria, oral epithelial cells appear to use two effector antimicrobial peptides (AMPs): calprotectin (S100A8-S100A9 heterodimer [S100A8/A9]) in the cytosol and cathelicidin antimicrobial protein (CAMP) in endosomes. We sought to learn whether innate immunity might be augmented benignly to increase resistance against invasive bacteria. Epithelial cells were transiently transfected with mRNA constructs containing either theCAMP,S100A8, andS100A9open reading frames,A8-IRES-A9(fusion sequence), orA8-nIRES-A9(fusion with native internal ribosome entry site [IRES] sequence). CAMP, S100A8, and S100A9 protein levels generally peaked between 16 and 44 h after mRNA transfection, depending on the construct; CAMP was processed to LL-37 over time. Following transfection with the respective mRNAs, CAMP and S100A8/A9 each independently increased resistance of epithelial cells to invasion byListeriaandSalmonellafor up to 48 h; tandem S100A8/A9 constructs were also effective. Cotransfection to express S100A8/A9 and CAMP together augmented resistance, but synergy was not seen. Independent of the new proteins produced, transfection reduced cell viability after 48 h by 20%, with only 2% attributable to apoptosis. Taken together, these results suggest that epithelial cell resistance to invasive pathogens can be augmented by transient transfection of antimicrobial mRNAs into epithelial cells.

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Human Cytomegalovirus Induces the Endoplasmic Reticulum Chaperone BiP through Increased Transcription and Activation of Translation by Using the BiP Internal Ribosome Entry Site

Journal of Virology ◽

10.1128/jvi.01330-10 ◽

2010 ◽

Vol 84 (21) ◽

pp. 11479-11486 ◽

Cited By ~ 29

Author(s):

Nicholas J. Buchkovich ◽

Yongjun Yu ◽

Francis J. Pierciey ◽

James C. Alwine

Keyword(s):

Endoplasmic Reticulum ◽

Transcriptional Activation ◽

Internal Ribosome Entry Site ◽

Hcmv Infection ◽

Mrna Translation ◽

Entry Site ◽

Internal Ribosome Entry ◽

Unfolded Protein ◽

Infected Cells ◽

Protein Response

ABSTRACT The endoplasmic reticulum (ER) chaperone BiP (immunoglobulin binding protein) plays a major role in the control of the unfolded protein response. We have previously shown that BiP levels are dramatically increased during human cytomegalovirus (HCMV) infection, where BiP performs unique roles in viral assembly and egress. We show that BiP mRNA levels increase during infection due to activation of the BiP promoter by the major immediate-early (MIE) proteins. The BiP promoter, like other ER stress-activated promoters, contains endoplasmic reticulum stress elements (ERSEs), which are activated by unfolded protein response (UPR)-induced transcription factors. However, these elements are not needed for MIE protein-mediated transcriptional activation; thus, a virus-specific transcriptional activation mechanism is used. Transcriptional activation results in only a 3- to 4-fold increase in BiP mRNA, suggesting that additional mechanisms for BiP production are utilized. The BiP mRNA contains an internal ribosome entry site (IRES) which increases the level of BiP mRNA translation. We show that utilization of the BiP IRES is dramatically increased in HCMV-infected cells. Utilization of the BiP IRES can be activated by the La autoantigen, also called Sjögren's syndrome antigen B (SSB). We show that SSB/La levels are significantly increased during HCMV infection, and SSB/La depletion causes the loss of BiP IRES utilization and lowers endogenous BiP levels in infected cells. Our data show that BiP levels increase in HCMV-infected cells through the combination of increased BiP gene transcription mediated by the MIE proteins and increased BiP mRNA translation due to SSB/La-induced utilization of the BiP IRES.

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The hnRNA-Binding Proteins hnRNP L and PTB Are Required for Efficient Translation of the Cat-1 Arginine/Lysine Transporter mRNA during Amino Acid Starvation

Molecular and Cellular Biology ◽

10.1128/mcb.01774-08 ◽

2009 ◽

Vol 29 (10) ◽

pp. 2899-2912 ◽

Cited By ~ 63

Author(s):

Mithu Majumder ◽

Ibrahim Yaman ◽

Francesca Gaccioli ◽

Vladimir V. Zeenko ◽

Chuanping Wang ◽

...

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Binding Protein ◽

Mrna Translation ◽

Amino Acid Starvation ◽

Entry Site ◽

Internal Ribosome Entry ◽

Polypyrimidine Tract Binding Protein ◽

Global Protein Synthesis

ABSTRACT The response to amino acid starvation involves the global decrease of protein synthesis and an increase in the translation of some mRNAs that contain an internal ribosome entry site (IRES). It was previously shown that translation of the mRNA for the arginine/lysine amino acid transporter Cat-1 increases during amino acid starvation via a mechanism that utilizes an IRES in the 5′ untranslated region of the Cat-1 mRNA. It is shown here that polypyrimidine tract binding protein (PTB) and an hnRNA binding protein, heterogeneous nuclear ribonucleoprotein L (hnRNP L), promote the efficient translation of Cat-1 mRNA during amino acid starvation. Association of both proteins with Cat-1 mRNA increased during starvation with kinetics that paralleled that of IRES activation, although the levels and subcellular distribution of the proteins were unchanged. The sequence CUUUCU within the Cat-1 IRES was important for PTB binding and for the induction of translation during amino acid starvation. Binding of hnRNP L to the IRES or the Cat-1 mRNA in vivo was independent of PTB binding but was not sufficient to increase IRES activity or Cat-1 mRNA translation during amino acid starvation. In contrast, binding of PTB to the Cat-1 mRNA in vivo required hnRNP L. A wider role of hnRNP L in mRNA translation was suggested by the decrease of global protein synthesis in cells with reduced hnRNP L levels. It is proposed that PTB and hnRNP L are positive regulators of Cat-1 mRNA translation via the IRES under stress conditions that cause a global decrease of protein synthesis.

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miR-181a Regulates Cap-Dependent Translation of p27kip1 mRNA in Myeloid Cells

Molecular and Cellular Biology ◽

10.1128/mcb.01971-08 ◽

2009 ◽

Vol 29 (10) ◽

pp. 2841-2851 ◽

Cited By ~ 52

Author(s):

Rafael Cuesta ◽

Aida Martínez-Sánchez ◽

Fátima Gebauer

Keyword(s):

Cell Cycle ◽

Cancer Progression ◽

Translational Control ◽

Cell Cycle Progression ◽

Mrna Translation ◽

Myeloid Cell ◽

Entry Site ◽

Internal Ribosome Entry ◽

Hl60 Cells ◽

Block Cell Cycle Progression

ABSTRACT p27kip1 (p27) is a cell cycle inhibitor and tumor suppressor whose expression is tightly regulated in the cell. Translational control of p27 mRNA has emerged as a prominent mechanism to regulate p27 expression during differentiation, quiescence, and cancer progression. The microRNAs miR-221 and miR-222 repress p27 expression in various cancer cells, and this repression promotes tumor cell proliferation. In addition, the presence of an internal ribosome entry site in the 5′ untranslated region (UTR) of p27 mRNA has been reported. Here, we show that p27 mRNA is translated via a cap-dependent mechanism in HeLa and HL60 cells and that the previously reported IRES activity can be attributed to cryptic promoters in the sequence corresponding to the p27 5′ UTR. Furthermore, cap-dependent translation of p27 mRNA is repressed by miR-181a in undifferentiated HL60 cells. Repression by miR-181a is relieved during differentiation of HL60 into monocyte-like cells, allowing the accumulation of p27, which is necessary to fully block cell cycle progression and reach terminal differentiation. These results identify miR-181a as a regulator of p27 mRNA translation during myeloid cell differentiation.

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TransCirc: an interactive database for translatable circular RNAs based on multi-omics evidence

Nucleic Acids Research ◽

10.1093/nar/gkaa823 ◽

2020 ◽

Vol 49 (D1) ◽

pp. D236-D242 ◽

Cited By ~ 1

Author(s):

Wendi Huang ◽

Yunchao Ling ◽

Sirui Zhang ◽

Qiguang Xia ◽

Ruifang Cao ◽

...

Keyword(s):

Translation Initiation ◽

Open Reading Frames ◽

Mass Spectrometry Data ◽

Circular Rnas ◽

Entry Site ◽

Additional Species ◽

Internal Ribosome Entry ◽

Sequence Composition ◽

User Friendly ◽

Reading Frames

Abstract TransCirc (https://www.biosino.org/transcirc/) is a specialized database that provide comprehensive evidences supporting the translation potential of circular RNAs (circRNAs). This database was generated by integrating various direct and indirect evidences to predict coding potential of each human circRNA and the putative translation products. Seven types of evidences for circRNA translation were included: (i) ribosome/polysome binding evidences supporting the occupancy of ribosomes onto circRNAs; (ii) experimentally mapped translation initiation sites on circRNAs; (iii) internal ribosome entry site on circRNAs; (iv) published N-6-methyladenosine modification data in circRNA that promote translation initiation; (v) lengths of the circRNA specific open reading frames; (vi) sequence composition scores from a machine learning prediction of all potential open reading frames; (vii) mass spectrometry data that directly support the circRNA encoded peptides across back-splice junctions. TransCirc provides a user-friendly searching/browsing interface and independent lines of evidences to predicte how likely a circRNA can be translated. In addition, several flexible tools have been developed to aid retrieval and analysis of the data. TransCirc can serve as an important resource for investigating the translation capacity of circRNAs and the potential circRNA-encoded peptides, and can be expanded to include new evidences or additional species in the future.

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Bcr-Abl Regulates IRES-Dependent Translation of Lymphoid Enhancer Factor-1 in Chronic Myelogenous Leukemia.

Blood ◽

10.1182/blood.v108.11.2124.2124 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2124-2124

Author(s):

Judy Jimenez ◽

Min Zhang ◽

Marian L. Waterman ◽

Tiong Ong

Keyword(s):

Translational Control ◽

Myelogenous Leukemia ◽

Mrna Levels ◽

Entry Site ◽

Aberrant Expression ◽

Internal Ribosome Entry ◽

Protein Levels ◽

Clinical Resistance ◽

Bona Fide ◽

Enhancer Factor

Abstract The constitutive tyrosine kinase activity of Bcr-Abl leads to aberrant expression of multiple genes by several mechanisms, including dysregulation of transcription. Recently though, increasing attention has been focused on the effect of Bcr-Abl in dysregulating translation. Our group has previously documented the effects of Bcr-Abl on key regulators of cap-dependent translation and the role that this mechanism plays in transformation (Ly et al, Cancer Research, 2003; Prabhu et al., Oncogene, in press). Here we describe a novel form of translational control by Bcr-Abl. Specifically, we show how Bcr-Abl regulates cap-independent translation of Lymphoid Enhancer Factor-1 (LEF-1) via a bona fide internal ribosome entry site (IRES) in the 5′ untranslated region (UTR) of LEF1. Lymphoid Enhancer Factor-1 (LEF-1), a transcription factor that mediates Wnt signals via interaction with β-catenin, is often expressed in cancers derived from aberrant Wnt signaling. Lately, it has been reported that LEF1 transcripts are elevated in CML. We examined LEF-1 expression in primary CML cells and cell lines (K562 and Ba/F3-Bcr-Abl) and show that LEF-1 protein is detected in all patient-derived cells. Treatment of these cells with the Bcr-Abl imatinib mesylate (imatinib) inhibits LEF-1 expression in imatinib-sensitive cancers, but not in cancers that exhibit clinical resistance even though such cancers express imatinib-sensitive Bcr-Abl. For those cancers that are sensitive, inhibition of Bcr-Abl has a partial effect on LEF1 mRNA levels, and a significant effect on LEF-1 protein levels. LEF-1 protein is produced via two IRESs in it’s 5′ UTR. IRES-driven translation of LEF-1 was highly sensitive to Bcr-Abl as treatment with imatinib reduced IRES activity 5 fold. Transfection of CML cells with dicistronic mRNAs suggests that Bcr-Abl stimulates LEF-1 protein production through steps in the nucleus and cytoplasm. We propose that, in addition to its strong effects on cap-dependent translation in CML, Bcr-Abl is an important regulator of alternative translation pathways. mRNAs that are translated via IRES-dependent mechanisms are particularly relevant to cancer since they encode proteins that regulate cell proliferation and survival. Together, these observations underscore the important role which dysregulated translation plays in transformation, and suggest novel approaches with which to counteract the transforming properties of Bcr-Abl.

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Identification of minimal sequences of the Rhopalosiphum padi virus 5′ untranslated region required for internal initiation of protein synthesis in mammalian, plant and insect translation systems

Journal of General Virology ◽

10.1099/vir.0.82682-0 ◽

2007 ◽

Vol 88 (5) ◽

pp. 1583-1588 ◽

Cited By ~ 17

Author(s):

Elisabetta Groppelli ◽

Graham J. Belsham ◽

Lisa O. Roberts

Keyword(s):

Untranslated Region ◽

Rhopalosiphum Padi ◽

Open Reading Frames ◽

Distinct Region ◽

Entry Site ◽

Internal Ribosome Entry ◽

Internal Initiation ◽

Minimal Sequences ◽

Translation Systems

Rhopalosiphum padi virus (RhPV) is a member of the family Dicistroviridae. The genomes of viruses in this family contain two open reading frames, each preceded by distinct internal ribosome entry site (IRES) elements. The RhPV 5′ IRES is functional in mammalian, insect and plant translation systems and can form 48S initiation complexes in vitro with just the mammalian initiation factors eIF2, eIF3 and eIF1. Large regions of the 5′ untranslated region (UTR) can be deleted without affecting initiation-complex formation. The minimal sequences required for directing internal initiation in mammalian (rabbit reticulocyte lysate), plant (wheatgerm extract) and insect (Sf21 cells) translation systems have now been defined. A fragment (nt 426–579) from the 3′ portion of the 5′ UTR can direct translation in each of these translation systems. In addition, a distinct region (nt 300–429) is also active. Thus, unstructured regions within the 5′ UTR seem to be critical for IRES function.

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Activation of a GPCR leads to eIF4G phosphorylation at the 5′ cap and to IRES-dependent translation

Journal of Molecular Endocrinology ◽

10.1530/jme-14-0009 ◽

2014 ◽

Vol 52 (3) ◽

pp. 373-382 ◽

Cited By ~ 6

Author(s):

Kelly León ◽

Thomas Boulo ◽

Astrid Musnier ◽

Julia Morales ◽

Christophe Gauthier ◽

...

Keyword(s):

Mammalian Target Of Rapamycin ◽

Mrna Translation ◽

Hek293 Cells ◽

Initiation Factor ◽

Entry Site ◽

Internal Ribosome Entry ◽

Translational Machinery ◽

Phosphorylation Event ◽

Eukaryotic Initiation Factor 4G ◽

Molecular Bases

The control of mRNA translation has been mainly explored in response to activated tyrosine kinase receptors. In contrast, mechanistic details on the translational machinery are far less available in the case of ligand-bound G protein-coupled receptors (GPCRs). In this study, using the FSH receptor (FSH-R) as a model receptor, we demonstrate that part of the translational regulations occurs by phosphorylation of the translation pre-initiation complex scaffold protein, eukaryotic initiation factor 4G (eIF4G), in HEK293 cells stably expressing the FSH-R. This phosphorylation event occurred when eIF4G was bound to the mRNA 5′ cap, and probably involves mammalian target of rapamycin. This regulation might contribute to cap-dependent translation in response to FSH. The cap-binding protein eIF4E also had its phosphorylation level enhanced upon FSH stimulation. We also show that FSH-induced signaling not only led to cap-dependent translation but also to internal ribosome entry site (IRES)-dependent translation of some mRNA. These data add detailed information on the molecular bases underlying the regulation of selective mRNA translation by a GPCR, and a topological model recapitulating these mechanisms is proposed.

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Naturally Occurring Dicistronic Cricket Paralysis Virus RNA Is Regulated by Two Internal Ribosome Entry Sites

Molecular and Cellular Biology ◽

10.1128/mcb.20.14.4990-4999.2000 ◽

2000 ◽

Vol 20 (14) ◽

pp. 4990-4999 ◽

Cited By ~ 199

Author(s):

Joan E. Wilson ◽

Marguerite J. Powell ◽

Susan E. Hoover ◽

Peter Sarnow

Keyword(s):

Mutational Analysis ◽

Mechanistic Explanation ◽

Virus Genome ◽

Open Reading Frames ◽

Structural Proteins ◽

Entry Site ◽

Internal Ribosome Entry ◽

Naturally Occurring ◽

Eukaryotic Mrnas ◽

Paralysis Virus

ABSTRACT Cricket paralysis virus is a member of a group of insect picorna-like viruses. Cloning and sequencing of the single plus-strand RNA genome revealed the presence of two nonoverlapping open reading frames, ORF1 and ORF2, that encode the nonstructural and structural proteins, respectively. We show that each ORF is preceded by one internal ribosome entry site (IRES). The intergenic IRES is located 6,024 nucleotides from the 5′ end of the viral RNA and is more active than the IRES located at the 5′ end of the RNA, providing a mechanistic explanation for the increased abundance of structural proteins relative to nonstructural proteins in infected cells. Mutational analysis of this intergenic-region IRES revealed that ORF2 begins with a noncognate CCU triplet. Complementarity of this CCU triplet with sequences in the IRES is important for IRES function, pointing to an involvement of RNA-RNA interactions in translation initiation. Thus, the cricket paralysis virus genome is an example of a naturally occurring, functionally dicistronic eukaryotic mRNA whose translation is controlled by two IRES elements located at the 5′ end and in the middle of the mRNA. This finding argues that eukaryotic mRNAs can express multiple proteins not only by polyprotein processing, reinitiation and frameshifting but also by using multiple IRES elements.

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