scholarly journals 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

2009 ◽  
Vol 29 (10) ◽  
pp. 2899-2912 ◽  
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.

scholarly journals Transient Expression of Cellular Polypyrimidine-Tract Binding Protein Stimulates Cap-Independent Translation Directed by Both Picornaviral and Flaviviral Internal Ribosome Entry Sites In Vivo

2000 ◽  
Vol 20 (5) ◽  
pp. 1583-1595 ◽  
Author(s):  
Rainer Gosert ◽  
Ki Ha Chang ◽  
Rene Rijnbrand ◽  
MinKyung Yi ◽  
David V. Sangar ◽  
...  
Keyword(s):  
Binding Protein ◽  
Fold Increase ◽  
Null Mutant ◽  
Polypyrimidine Tract ◽  
Reporter Protein ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Independent Translation

ABSTRACT The regulation of cap-independent translation directed by the internal ribosome entry sites (IRESs) present in some viral and cellular RNAs is poorly understood. Polypyrimidine-tract binding protein (PTB) binds specifically to several viral IRESs. IRES-directed translation may be reduced in cell-free systems that are depleted of PTB and restored by reconstitution of lysates with recombinant PTB. However, there are no data concerning the effects of PTB on IRES-directed translation in vivo. We transfected cells with plasmids expressing dicistronic transcripts in which the upstream cistron encoded PTB or PTB deletion mutants (including a null mutant lacking amino acid residues 87 to 531). The downstream cistron encoded a reporter protein (chloramphenicol acetyltransferase [CAT]) under translational control of the poliovirus IRES which was placed within the intercistronic space. In transfected BS-C-1 cells, transcripts expressing wild-type PTB produced 12-fold more reporter protein than similar transcripts encoding the PTB null mutant. There was a 2.4-fold difference in CAT produced from these transcripts in HeLa cells, which contain a greater natural abundance of PTB. PTB similarly stimulated CAT production from transcripts containing the IRES of hepatitis A virus or hepatitis C virus in BS-C-1 cells and Huh-7 cells (37- to 44-fold increase and 5 to 5.3-fold increase, respectively). Since PTB had no quantitative or qualitative effect on transcription from these plasmids, we conclude that PTB stimulates translation of representative picornaviral and flaviviral RNAs in vivo. This is likely to reflect the stabilization of higher ordered RNA structures within the IRES and was not observed with PTB mutants lacking RNA recognition motifs located in the C-terminal third of the molecule.

scholarly journals Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Proteinase Inhibits Viral Internal Ribosome Entry Site-Mediated Translation

2008 ◽  
Vol 82 (19) ◽  
pp. 9389-9399 ◽  
Author(s):  
Jennifer M. Bonderoff ◽  
Jennifer L. LaRey ◽  
Richard E. Lloyd
Keyword(s):  
Internal Ribosome Entry Site ◽  
Binding Protein ◽  
Virus Protein ◽  
Dependent Manner ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
Polypyrimidine Tract Binding Protein ◽  
In Cells

ABSTRACT The two enteroviral proteinases, 2A proteinase (2Apro) and 3C proteinase (3Cpro), induce host cell translation shutoff in enterovirus-infected cells by cleaving canonical translation initiation factors. Cleavage of poly(A)-binding protein (PABP) by 3Cpro has been shown to be a necessary component for host translation shutoff. Here we show that 3Cpro inhibits cap-independent translation mediated by the poliovirus internal ribosome entry site (IRES) in a dose-dependent manner in HeLa translation extracts displaying cap-poly(A) synergy. This effect is independent of the stimulatory effect of 2Apro on IRES translation, and 3Cpro-induced translation inhibition can be partially rescued by addition of recombinant PABP in vitro. 3Cpro inhibits IRES translation on transcripts containing or lacking poly(A) tails, suggesting that cleavage of PABP and IRES trans-activating factors polypyrimidine tract-binding protein and poly r(C)-binding protein 2 may also be important for inhibition. Expression of 3Cpro cleavage-resistant PABP in cells increased translation of nonreplicating viral minigenome reporter RNAs during infection and also delayed and reduced virus protein synthesis from replicating RNA. Further, expression of cleavage-resistant PABP in cells reduced the accumulation of viral RNA and the output of infectious virus. These results suggest that cleavage of PABP contributes to viral translation shutoff that is required for the switch from translation to RNA replication.

scholarly journals Hepatitis C Virus Internal Ribosome Entry Site-Dependent Translation in Saccharomyces cerevisiae Is Independent of Polypyrimidine Tract-Binding Protein, Poly(rC)-Binding Protein 2, and La Protein

2005 ◽  
Vol 79 (16) ◽  
pp. 10126-10137 ◽  
Author(s):  
Amy B. Rosenfeld ◽  
Vincent R. Racaniello
Keyword(s):  
Mammalian Cells ◽  
Binding Protein ◽  
Polypyrimidine Tract ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Reading Frame ◽  
Polypyrimidine Tract Binding Protein ◽  
La Protein ◽  
Internal Initiation ◽  
Hcv Ires

ABSTRACT Translation initiation of some viral and cellular mRNAs occurs by ribosome binding to an internal ribosome entry site (IRES). Internal initiation mediated by the hepatitis C virus (HCV) IRES in Saccharomyces cerevisiae was shown by translation of the second open reading frame in a bicistronic mRNA. Introduction of a single base change in the HCV IRES, known to abrogate internal initiation in mammalian cells, abolished translation of the second open reading frame. Internal initiation mediated by the HCV IRES was independent of the nonsense-mediated decay pathway and the cap binding protein eIF4E, indicating that translation is not a result of mRNA degradation or 5′-end-dependent initiation. Human La protein binds the HCV IRES and is required for efficient internal initiation. Disruption of the S. cerevisiae genes that encode La protein orthologs and synthesis of wild-type human La protein in yeast had no effect on HCV IRES-dependent translation. Polypyrimidine tract-binding protein (Ptb) and poly-(rC)-binding protein 2 (Pcbp2), which may be required for HCV IRES-dependent initiation in mammalian cells, are not encoded within the S. cerevisiae genome. HCV IRES-dependent translation in S. cerevisiae was independent of human Pcbp2 protein and stimulated by the presence of human Ptb protein. These findings demonstrate that the genome of S. cerevisiae encodes all proteins necessary for internal initiation of translation mediated by the HCV IRES.

Internal Ribosome Entry Site-Mediated Translation in Neuronal Protein Synthesis

2018 ◽  
Author(s):  
Martin Holcik
Keyword(s):  
Protein Synthesis ◽  
Internal Ribosome Entry Site ◽  
Normal Function ◽  
Entry Site ◽  
Alternative Mode ◽  
Internal Ribosome Entry ◽  
Neuronal Protein ◽  
Global Protein Synthesis ◽  
Cellular Mrnas ◽  
Specific Mrnas

While the majority of cellular mRNAs are translated by a cap-dependent mechanism, a subset of mRNAs can use an alternative mode of translation that, instead of cap, relies on discreet RNA elements that help to recruit the ribosome. This mode of translation, termed Internal Ribosome Entry Site (IRES)–dependent translation, is particularly important during conditions of compromised global protein synthesis or for a local, precisely timed translation of specific mRNAs. This latter purpose is of considerable importance in cells of the CNS for their normal function. Recently, the disruption of the IRES-mediated translation has also been linked to pathological processes, suggesting that full understanding and targeting of this peculiar mechanism could be used for therapeutic intervention.

scholarly journals Mechanisms involved in the nutritional regulation of mRNA translation: features of the avian model

2006 ◽  
Vol 19 (1) ◽  
pp. 104-116 ◽  
Author(s):  
Sophie Tesseraud ◽  
Mourad Abbas ◽  
Sophie Duchene ◽  
Karine Bigot ◽  
Pascal Vaudin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Muscle Growth ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Nutritional Regulation ◽  
Translational Repressor

Abstract:Insulin and amino acids are key factors in regulating protein synthesis. The mechanisms of their action have been widely studied for several years. The insulin signal is mediated by the activation of intracellular kinases such as phosphatidylinositol–3'kinase and the mammalian target of rapamycin (mTOR), affecting the phosphorylation of some major effectors involved in the regulation of translation initiation, i.e. p70 S6 kinase (p70S6K) and the translational repressor eukaryotic initiation factor 4E binding protein (4E-BP1). The amino acid–induced signalling cascade also originates from mTOR and promotes p70S6K and 4E–BP1 activation. However, the mechanisms of regulation are complex and little understood, especially in vivo. Elucidating these mechanisms is important for both fundamental physiology and nutritional applications, i.e. better control of the use of nutrients and optimisation of dietary amino acid supplies in various physiological and physiopathological situations. In comparative physiology, the chicken is an interesting model to gain better understanding of the nutritional regulation of mRNA translation because of the very high rates of muscle growth and protein synthesis, and the unusual features compared with mammals. In the present review we provide an overview of the roles of insulin and amino acids as regulators of protein synthesis in both mammals and avian species.

scholarly journals Identification of Internal Ribosome Entry Segment (IRES)-trans-Acting Factors for the Myc Family of IRESs

2007 ◽  
Vol 28 (1) ◽  
pp. 40-49 ◽  
Author(s):  
Laura C. Cobbold ◽  
Keith A. Spriggs ◽  
Stephen J. Haines ◽  
Helen C. Dobbyn ◽  
Christopher Hayes ◽  
...  
Keyword(s):  
Binding Protein ◽  
Structural Element ◽  
Internal Ribosome Entry ◽  
Polypyrimidine Tract Binding Protein ◽  
Binding Factor ◽  
Internal Ribosome Entry Segment ◽  
Complex Structural ◽  
Rna Chaperones

ABSTRACT The proto-oncogenes c-, L-, and N-myc can all be translated by the alternative method of internal ribosome entry whereby the ribosome is recruited to a complex structural element (an internal ribosome entry segment [IRES]). Ribosome recruitment is dependent upon the presence of IRES-trans-acting factors (ITAFs) that act as RNA chaperones and allow the mRNA to attain the correct conformation for the interaction of the 40S subunit. One of the major challenges for researchers in this area is to determine whether there are groups of ITAFs that regulate the IRES-mediated translation of subsets of mRNAs. We have identified four proteins, termed GRSF-1 (G-rich RNA sequence binding factor 1), YB-1 (Y-box binding protein 1), PSF (polypyrimidine tract binding protein-associated splicing factor), and its binding partner, p54nrb, that bind to the myc family of IRESs. We show that these proteins positively regulate the translation of the Myc family of oncoproteins (c-, L-, and N-Myc) in vivo and in vitro. Interestingly, synthesis from the unrelated IRESs, BAG-1 and Apaf-1, was not affected by YB-1, GRSF-1, or PSF levels in vivo, suggesting that these three ITAFs are specific to the myc IRESs. Myc proteins play a role in cell proliferation; therefore, these results have important implications regarding the control of tumorigenesis.

scholarly journals Efficient Cleavage of Ribosome-Associated Poly(A)-Binding Protein by Enterovirus 3C Protease

2002 ◽  
Vol 76 (5) ◽  
pp. 2062-2074 ◽  
Author(s):  
N. Muge Kuyumcu-Martinez ◽  
Michelle Joachims ◽  
Richard E. Lloyd
Keyword(s):  
Translation Initiation ◽  
Binding Protein ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Factors ◽  
3C Protease

ABSTRACT Poliovirus (PV) causes a rapid and drastic inhibition of host cell cap-dependent protein synthesis during infection while preferentially allowing cap-independent translation of its own genomic RNA via an internal ribosome entry site element. Inhibition of cap-dependent translation is partly mediated by cleavage of an essential translation initiation factor, eIF4GI, during PV infection. In addition to cleavage of eIF4GI, cleavage of eIF4GII and poly(A)-binding protein (PABP) has been recently proposed to contribute to complete host translation shutoff; however, the relative importance of eIF4GII and PABP cleavage has not been determined. At times when cap-dependent translation is first blocked during infection, only 25 to 35% of the total cellular PABP is cleaved; therefore, we hypothesized that the pool of PABP associated with polysomes may be preferentially targeted by viral proteases. We have investigated what cleavage products of PABP are produced in vivo and the substrate determinants for cleavage of PABP by 2A protease (2Apro) or 3C protease (3Cpro). Our results show that PABP in ribosome-enriched fractions is preferentially cleaved in vitro and in vivo compared to PABP in other fractions. Furthermore, we have identified four N-terminal PABP cleavage products produced during PV infection and have shown that viral 3C protease generates three of the four cleavage products. Also, 3Cpro is more efficient in cleaving PABP in ribosome-enriched fractions than 2Apro in vitro. In addition, binding of PABP to poly(A) RNA stimulates 3Cpro-mediated cleavage and inhibits 2Apro-mediated cleavage. These results suggest that 3Cpro plays a major role in processing PABP during virus infection and that the interaction of PABP with translation initiation factors, ribosomes, or poly(A) RNA may promote its cleavage by viral 2A and 3C proteases.

scholarly journals IRES-dependent regulation of FGF-2 mRNA translation in pathophysiological conditions in the mouse

2006 ◽  
Vol 34 (1) ◽  
pp. 17-21 ◽  
Author(s):  
I.G. Gonzalez-Herrera ◽  
L. Prado-Lourenco ◽  
S. Teshima-Kondo ◽  
K. Kondo ◽  
F. Cabon ◽  
...  
Keyword(s):  
Translational Control ◽  
Vascular Complications ◽  
Mrna Translation ◽  
Adult Brain ◽  
Angiogenic Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Nuclear Ribonucleoprotein

The mRNA coding for FGF-2 (fibroblast growth factor 2), a major angiogenic factor, is translated by an IRES (internal ribosome entry site)-dependent mechanism. We have studied the role of the IRES in the regulation of FGF-2 expression in vivo, under pathophysiological conditions, by creating transgenic mice lines expressing bioluminescent bicistronic transgenes. Analysis of FGF-2 IRES activity indicates strong tissue specificity in adult brain and testis, suggesting a role of the IRES in the activation of FGF-2 expression in testis maturation and brain function. We have explored translational control of FGF-2 mRNA under diabetic hyperglycaemic conditions, as FGF-2 is implied in diabetes-related vascular complications. FGF-2 IRES is specifically activated in the aorta wall in streptozotocin-induced diabetic mice, in correlation with increased expression of endogenous FGF-2. Thus, under hyperglycaemic conditions, where cap-dependent translation is blocked, IRES activation participates in FGF-2 overexpression, which is one of the keys of diabetes-linked atherosclerosis aggravation. IRES activation under such pathophysiological conditions may involve ITAFs (IRES trans-acting factors), such as p53 or hnRNP AI (heterogeneous nuclear ribonucleoprotein AI), recently identified as inhibitory or activatory ITAFs respectively for FGF-2 IRES.

scholarly journals Comprehensive assessment of post-prandial protein handling by the application of intrinsically labelled protein in vivo in human subjects

2021 ◽  
pp. 1-9
Author(s):  
Jorn Trommelen ◽  
Andrew M. Holwerda ◽  
Philippe J. M. Pinckaers ◽  
Luc J. C. van Loon
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Stable Isotope ◽  
Dietary Protein ◽  
Protein Metabolism ◽  
Muscle Protein ◽  
Human Subjects ◽  
Whole Body ◽  
Body Protein

All human tissues are in a constant state of remodelling, regulated by the balance between tissue protein synthesis and breakdown rates. It has been well-established that protein ingestion stimulates skeletal muscle and whole-body protein synthesis. Stable isotope-labelled amino acid methodologies are commonly applied to assess the various aspects of protein metabolism in vivo in human subjects. However, to achieve a more comprehensive assessment of post-prandial protein handling in vivo in human subjects, intravenous stable isotope-labelled amino acid infusions can be combined with the ingestion of intrinsically labelled protein and the collection of blood and muscle tissue samples. The combined application of ingesting intrinsically labelled protein with continuous intravenous stable isotope-labelled amino acid infusion allows the simultaneous assessment of protein digestion and amino acid absorption kinetics (e.g. release of dietary protein-derived amino acids into the circulation), whole-body protein metabolism (whole-body protein synthesis, breakdown and oxidation rates and net protein balance) and skeletal muscle metabolism (muscle protein fractional synthesis rates and dietary protein-derived amino acid incorporation into muscle protein). The purpose of this review is to provide an overview of the various aspects of post-prandial protein handling and metabolism with a focus on insights obtained from studies that have applied intrinsically labelled protein under a variety of conditions in different populations.

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