scholarly journals Regulation of the cell-cycle-dependent internal ribosome entry site of the PITSLRE protein kinase: roles of Unr (upstream of N-ras) protein and phosphorylated translation initiation factor eIF-2α

2004 ◽  
Vol 385 (1) ◽  
pp. 155-163 ◽  
Author(s):  
Sandrine A. TINTON ◽  
Bert SCHEPENS ◽  
Yanik BRUYNOOGHE ◽  
Rudi BEYAERT ◽  
Sigrid CORNELIS
Keyword(s):  
Cell Cycle ◽  
Internal Ribosome Entry Site ◽  
Encephalomyocarditis Virus ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Α Subunit ◽  
Initiation Of Translation ◽  
Cycle Dependent

The PITSLRE kinases belong to the large family of cyclin-dependent protein kinases. Their function has been related to cell-cycle regulation, splicing and apoptosis. We have previously shown that the open reading frame of the p110PITSLRE transcript contains an IRES (internal ribosome entry site) that allows the expression of a smaller p58PITSLRE isoform during the G2/M stage of the cell cycle. In the present study we investigated further the role of cis- and trans-acting factors in the regulation of the PITSLRE IRES. Progressive deletion analysis showed that both a purine-rich sequence and a Unr (upstream of N-ras) consensus binding site are essential for PITSLRE IRES activity. In line with these observations, we demonstrate that the PITSLRE IRES interacts with the Unr protein, which is more prominently expressed at the G2/M stage of the cell cycle. We also show that phosphorylation of the α-subunit of the canonical initiation factor eIF-2 is increased at G2/M. Interestingly, phosphorylation of eIF-2α has a permissive effect on the efficiency of both the PITSLRE IRES and the ornithine decarboxylase IRES, two cell cycle-dependent IRESs, in mediating internal initiation of translation, whereas this was not observed with the viral EMCV (encephalomyocarditis virus) and HRV (human rhinovirus) IRESs.

scholarly journals Conserved Nucleotides within the J Domain of the Encephalomyocarditis Virus Internal Ribosome Entry Site Are Required for Activity and for Interaction with eIF4G

2003 ◽  
Vol 77 (23) ◽  
pp. 12441-12449 ◽  
Author(s):  
Angela T. Clark ◽  
Morwenna E. M. Robertson ◽  
Graeme L. Conn ◽  
Graham J. Belsham
Keyword(s):  
Internal Ribosome Entry Site ◽  
Single Point ◽  
Encephalomyocarditis Virus ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Initiation Of Translation ◽  
Mouth Disease Virus ◽  
J Domain

ABSTRACT The internal ribosome entry site (IRES) elements of cardioviruses (e.g., encephalomyocarditis virus [EMCV] and foot-and-mouth disease virus) are predicted to have very similar secondary structures. Among these complex RNA structures there is only rather limited complete sequence conservation. Within the J domain of the EMCV IRES there are four highly conserved nucleotides (A704, C705, G723, and A724)., which are predicted to be unpaired and have been targeted for mutagenesis. Using an IRES-dependent cell selection system, we have isolated functional IRES elements from a pool of up to 256 mutants. All changes to these conserved nucleotides resulted in IRES elements that were less efficient at directing internal initiation of translation than the wild-type element, and even some of the single point mutants were highly defective. Each of the mutations adversely affected the ability of the RNAs to interact with the translation initiation factor eIF4G.

scholarly journals Selection and Analysis of Mutations in an Encephalomyocarditis Virus Internal Ribosome Entry Site That Improve the Efficiency of a Bicistronic Flavivirus Construct

2007 ◽  
Vol 81 (22) ◽  
pp. 12619-12629 ◽  
Author(s):  
Klaus K. Orlinger ◽  
Regina M. Kofler ◽  
Franz X. Heinz ◽  
Verena M. Hoenninger ◽  
Christian W. Mandl
Keyword(s):  
Internal Ribosome Entry Site ◽  
Rna Replication ◽  
Particle Formation ◽  
Encephalomyocarditis Virus ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Expression Vectors ◽  
Nucleotide Substitutions ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT Flaviviruses have a positive-stranded RNA genome, which simultaneously serves as an mRNA for translation of the viral proteins. All of the structural and nonstructural proteins are translated from a cap-dependent cistron as a single polyprotein precursor. In an earlier study (K. K. Orlinger, V. M. Hoenninger, R. M. Kofler, and C. W. Mandl, J. Virol. 80:12197-12208, 2006), it was demonstrated that an artificial bicistronic flavivirus genome, TBEV-bc, in which the region coding for the viral surface glycoproteins prM and E from tick-borne encephalitis virus (TBEV) had been removed from its natural context and inserted into the 3′ noncoding region under the control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) produces viable, infectious virus when cells are transfected with this RNA. The rates of RNA replication and infectious particle formation were significantly lower with TBEV-bc, however, than with wild-type TBEV. In this study, we have identified two types of mutations, selected by passage in BHK-21 cells, that enhance the growth properties of TBEV-bc. The first type occurred in the E protein, and these most likely increase the affinity of the virus for heparan sulfate on the cell surface. The second type occurred in the inserted EMCV IRES, in the oligo(A) loop of the J-K stem-loop structure, a binding site for the eukaryotic translation initiation factor 4G. These included single-nucleotide substitutions as well as insertions of additional adenines in this loop. An A-to-C substitution in the oligo(A) loop decreased the efficiency of the IRES itself but nevertheless resulted in improved rates of virus particle formation and overall replication efficiency. These results demonstrate the need for proper balance in the competition for free template RNA between the viral RNA replication machinery and the cellular translation machinery at the two different start sites and also identify specific target sites for the improvement of bicistronic flavivirus expression vectors.

scholarly journals A Cell Cycle–Dependent Internal Ribosome Entry Site

2000 ◽  
Vol 5 (4) ◽  
pp. 607-616 ◽  
Author(s):  
Stéphane Pyronnet ◽  
Lucien Pradayrol ◽  
Nahum Sonenberg
Keyword(s):  
Cell Cycle ◽  
Internal Ribosome Entry Site ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
A Cell ◽  
Cycle Dependent

scholarly journals A Novel Form of DAP5 Protein Accumulates in Apoptotic Cells as a Result of Caspase Cleavage and Internal Ribosome Entry Site-Mediated Translation

2000 ◽  
Vol 20 (2) ◽  
pp. 496-506 ◽  
Author(s):  
Sivan Henis-Korenblit ◽  
Naomi Levy Strumpf ◽  
Dan Goldstaub ◽  
Adi Kimchi
Keyword(s):  
Internal Ribosome Entry Site ◽  
Dominant Negative ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Apoptotic Cells ◽  
Entry Site ◽  
Translation Rate ◽  
Internal Ribosome Entry ◽  
Caspase Cleavage ◽  
Induced Apoptosis

ABSTRACT Death-associated protein 5 (DAP5) (also named p97 and NAT1) is a member of the translation initiation factor 4G (eIF4G) family that lacks the eIF4E binding site. It was previously implicated in apoptosis, based on the finding that a dominant negative fragment of the protein protected against cell death. Here we address its function and two distinct levels of regulation during apoptosis that affect the protein both at translational and posttranslational levels. DAP5 protein was found to be cleaved at a single caspase cleavage site at position 790, in response to activated Fas or p53, yielding a C-terminal truncated protein of 86 kDa that is capable of generating complexes with eIF4A and eIF3. Interestingly, while the overall translation rate in apoptotic cells was reduced by 60 to 70%, in accordance with the simultaneous degradation of the two major mediators of cap-dependent translation, eIF4GI and eIF4GII, the translation rate of DAP5 protein was selectively maintained. An internal ribosome entry site (IRES) element capable of directing the translation of a reporter gene when subcloned into a bicistronic vector was identified in the 5′ untranslated region of DAP5 mRNA. While cap-dependent translation from this transfected vector was reduced during Fas-induced apoptosis, the translation via the DAP5 IRES was selectively maintained. Addition of recombinant DAP5/p97 or DAP5/p86 to cell-free systems enhanced preferentially the translation through the DAP5 IRES, whereas neutralization of the endogenous DAP5 in reticulocyte lysates by adding a dominant negative DAP5 fragment interfered with this translation. The DAP5/p86 apoptotic form was more potent than DAP5/p97 in these functional assays. Altogether, the data suggest that DAP5 is a caspase-activated translation factor which mediates cap-independent translation at least from its own IRES, thus generating a positive feedback loop responsible for the continuous translation of DAP5 during apoptosis.

scholarly journals Eukaryotic Initiation Factors 4G and 4A Mediate Conformational Changes Downstream of the Initiation Codon of the Encephalomyocarditis Virus Internal Ribosomal Entry Site

2003 ◽  
Vol 23 (2) ◽  
pp. 687-698 ◽  
Author(s):  
Victoria G. Kolupaeva ◽  
Ivan B. Lomakin ◽  
Tatyana V. Pestova ◽  
Christopher U. T. Hellen
Keyword(s):  
Conformational Changes ◽  
Internal Ribosomal Entry Site ◽  
Encephalomyocarditis Virus ◽  
Initiation Factor ◽  
Initiation Codon ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Central Domain ◽  
C Terminus ◽  
Initiation Of Translation

ABSTRACT Initiation of translation of encephalomyocarditis virus mRNA is mediated by an internal ribosome entry site (IRES) comprising structural domains H, I, J-K, and L immediately upstream of the initiation codon AUG at nucleotide 834 (AUG834). Assembly of 48S ribosomal complexes on the IRES requires eukaryotic initiation factor 2 (eIF2), eIF3, eIF4A, and the central domain of eIF4G to which eIF4A binds. Footprinting experiments confirmed that eIF4G binds a three-way helical junction in the J-K domain and showed that it interacts extensively with RNA duplexes in the J-K and L domains. Deletion of apical hairpins in the J and K domains synergistically impaired the binding of eIF4G and IRES function. Directed hydroxyl radical probing, done by using Fe(II) tethered to surface residues in eIF4G's central domain, indicated that it is oriented with its N terminus towards the base of domain J and its C terminus towards the apex. eIF4G recruits eIF4A to a defined location on the IRES, and the eIF4G/eIF4A complex caused localized ATP-independent conformational changes in the eIF4G-binding region of the IRES. This complex also induced more extensive conformational rearrangements at the 3′ border of the ribosome binding site that required ATP and active eIF4A. We propose that these conformational changes prepare the region flanking AUG834 for productive binding of the ribosome.

scholarly journals Involvement of Proteasome α-Subunit PSMA7 in Hepatitis C Virus Internal Ribosome Entry Site-Mediated Translation

2001 ◽  
Vol 21 (24) ◽  
pp. 8357-8364 ◽  
Author(s):  
Martin Krüger ◽  
Carmela Beger ◽  
Peter J. Welch ◽  
Jack R. Barber ◽  
Michael P. Manns ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Inhibitory Effect ◽  
Encephalomyocarditis Virus ◽  
Luciferase Reporter ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Α Subunit ◽  
Hcv Ires

ABSTRACT Ribozymes are small catalytic RNA molecules that can be engineered to enzymatically cleave RNA transcripts in a sequence-specific fashion and thereby inhibit expression and function of the corresponding gene product. With their simple structures and site-specific cleavage activity, they have been exploited as potential therapeutic agents in a variety of human disorders, including hepatitis C virus (HCV) infection. We have designed a hairpin ribozyme (Rz3′X) targeting the HCV minus-strand replication intermediate at position 40 within the 3′X tail. Surprisingly, Rz3′X was found to induce ganciclovir (GCV)-resistant colonies in a bicistronic cellular reporter system with HCV internal ribosome entry site (IRES)-dependent translation of herpes simplex virus thymidine kinase (TK). Rz3′X-transduced GCV-resistant HeLa reporter cells showed substantially reduced IRES-mediated HCV core protein translation compared with control vector-transduced cells. Since these reporter systems do not contain the HCV 3′X tail sequences, the results indicate that Rz3′X probably exerted an inhibitory effect on HCV IRES activity fortuitously through another gene target. A novel technique of ribozyme cleavage-based target gene identification (cleavage-specific amplification of cDNA ends) (M. Krüger, C. Beger, P. J. Welch, J. R. Barber, and F. Wong-Staal, Nucleic Acids Res. 29:e94, 2001) revealed that human 20S proteasome α-subunit PSMA7 mRNA was a target RNA recognized and cleaved by Rz3′X. We then showed that additional ribozymes directed against PSMA7 RNA inhibited HCV IRES activity in two assay systems: GCV resistance in the HeLa IRES TK reporter cell system and a transient transfection assay performed with a bicistronicRenilla-HCV IRES-firefly luciferase reporter in Huh7 cells. In contrast, ribozymes were inactive against IRES of encephalomyocarditis virus and human rhinovirus. Additionally, proteasome inhibitor MG132 exerted a dose-dependent inhibitory effect on HCV IRES-mediated translation but not on cap-dependent translation. These data suggest a principal role for PSMA7 in regulating HCV IRES activity, a function essential for HCV replication.

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