5 Untranslated Region

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Jack T. Rogers - One of the best experts on this subject based on the ideXlab platform.

  • the anticholinesterase phenserine and its enantiomer posiphen as 5 Untranslated Region directed translation blockers of the parkinson s alpha synuclein expression
    Parkinson's Disease, 2012
    Co-Authors: Sohan Mikkilineni, Catherine M Cahill, Ippolita Cantuticastelvetri, Amelie Balliedier, Nigel H Greig, Jack T. Rogers
    Abstract:

    There is compelling support for limiting expression of alpha-synuclein (α-syn) in the brains of Parkinson's disease (PD) patients. An increase of SNCA gene copy number can genetically cause familial PD where increased dose of this pathogenic protein correlates with severity of symptoms (triplication of the SNCA gene causes dementia in PD patients). Gene promoter polymorphisms were shown to increase α-synuclein expression as a risk for PD. Cholinesterase inhibitors can clinically slow cognitive decline in the later stages of PD etiology similar to their widespread use in Alzheimer's disease (AD). Pertinent to this, we identified that the well-tolerated anticholinesterase, phenserine, blocked neural SNCA mRNA translation and tested for targeting via its 5Untranslated Region (5′UTR) in a manner similar to its action to limit the expression of the AD-specific amyloid precursor protein (APP). Posiphen, its better-tolerated (+) enantiomer (devoid of anticholinesterase action), repressed neural α-synuclein translation. Primary metabolic analogs of posiphen were, likewise, characterized using primary fetal neurons grown ex vivo from the brains of Parkinson's transgenic mice expressing the human SNCA gene.

  • the alpha synuclein 5 Untranslated Region targeted translation blockers anti alpha synuclein efficacy of cardiac glycosides and posiphen
    Journal of Neural Transmission, 2011
    Co-Authors: Jack T. Rogers, Xudong Huang, Catherine M Cahill, Sanghamitra Bandyopadhyay, Sohan Mikkilineni, Ippolita Cantuticastelvetri, Deborah H Smith, Maria L Maccecchini, Debomoy K Lahiri, Nigel H Greig
    Abstract:

    Increased brain α-synuclein (SNCA) protein expression resulting from gene duplication and triplication can cause a familial form of Parkinson’s disease (PD). Dopaminergic neurons exhibit elevated iron levels that can accelerate toxic SNCA fibril formation. Examinations of human post mortem brain have shown that while mRNA levels for SNCA in PD have been shown to be either unchanged or decreased with respect to healthy controls, higher levels of insoluble protein occurs during PD progression. We show evidence that SNCA can be regulated via the 5Untranslated Region (5′UTR) of its transcript, which we modeled to fold into a unique RNA stem loop with a CAGUGN apical loop similar to that encoded in the canonical iron-responsive element (IRE) of L- and H-ferritin mRNAs. The SNCA IRE-like stem loop spans the two exons that encode its 5′UTR, whereas, by contrast, the H-ferritin 5′UTR is encoded by a single first exon. We screened a library of 720 natural products (NPs) for their capacity to inhibit SNCA 5′UTR driven luciferase expression. This screen identified several classes of NPs, including the plant cardiac glycosides, mycophenolic acid (an immunosuppressant and Fe chelator), and, additionally, posiphen was identified to repress SNCA 5′UTR conferred translation. Western blotting confirmed that Posiphen and the cardiac glycoside, strophanthidine, selectively blocked SNCA expression (~1 μM IC50) in neural cells. For Posiphen this inhibition was accelerated in the presence of iron, thus providing a known APP-directed lead with potential for use as a SNCA blocker for PD therapy. These are candidate drugs with the potential to limit toxic SNCA expression in the brains of PD patients and animal models in vivo.

  • the 5 Untranslated Region of parkinson s disease α synuclein messengerrna contains a predicted iron responsive element
    Molecular Psychiatry, 2007
    Co-Authors: Avi L. Friedlich, Rudolph E. Tanzi, Jack T. Rogers
    Abstract:

    The 5′-Untranslated Region of Parkinson's disease α -synuclein messengerRNA contains a predicted iron responsive element

  • a high throughput drug screen targeted to the 5 Untranslated Region of alzheimer amyloid precursor protein mrna
    Journal of Biomolecular Screening, 2006
    Co-Authors: Sanghamitra Bandyopadhyay, Amy Ruggiero, Karen Walshe, Mark F Rogers, Naibedya Chattopadhyay, Marcie A Glicksman, Jack T. Rogers
    Abstract:

    The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5Untranslated Region (5′UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Aβ) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5′UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5′UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5′UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5′UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5′UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Aβ production for AD. (Journal of Biomolecular Screening 2006:469-480)

  • a high throughput drug screen targeted to the 5 Untranslated Region of alzheimer amyloid precursor protein mrna
    Journal of Biomolecular Screening, 2006
    Co-Authors: Sanghamitra Bandyopadhyay, Amy Ruggiero, Karen Walshe, Naibedya Chattopadhyay, Marcie A Glicksman, Mark Rogers, Jack T. Rogers
    Abstract:

    The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5'Untranslated Region (5'UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Abeta) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5'UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5'UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5'UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5'UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5'UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Abeta production for AD.

Catherine M Cahill - One of the best experts on this subject based on the ideXlab platform.

  • the anticholinesterase phenserine and its enantiomer posiphen as 5 Untranslated Region directed translation blockers of the parkinson s alpha synuclein expression
    Parkinson's Disease, 2012
    Co-Authors: Sohan Mikkilineni, Catherine M Cahill, Ippolita Cantuticastelvetri, Amelie Balliedier, Nigel H Greig, Jack T. Rogers
    Abstract:

    There is compelling support for limiting expression of alpha-synuclein (α-syn) in the brains of Parkinson's disease (PD) patients. An increase of SNCA gene copy number can genetically cause familial PD where increased dose of this pathogenic protein correlates with severity of symptoms (triplication of the SNCA gene causes dementia in PD patients). Gene promoter polymorphisms were shown to increase α-synuclein expression as a risk for PD. Cholinesterase inhibitors can clinically slow cognitive decline in the later stages of PD etiology similar to their widespread use in Alzheimer's disease (AD). Pertinent to this, we identified that the well-tolerated anticholinesterase, phenserine, blocked neural SNCA mRNA translation and tested for targeting via its 5Untranslated Region (5′UTR) in a manner similar to its action to limit the expression of the AD-specific amyloid precursor protein (APP). Posiphen, its better-tolerated (+) enantiomer (devoid of anticholinesterase action), repressed neural α-synuclein translation. Primary metabolic analogs of posiphen were, likewise, characterized using primary fetal neurons grown ex vivo from the brains of Parkinson's transgenic mice expressing the human SNCA gene.

  • the alpha synuclein 5 Untranslated Region targeted translation blockers anti alpha synuclein efficacy of cardiac glycosides and posiphen
    Journal of Neural Transmission, 2011
    Co-Authors: Jack T. Rogers, Xudong Huang, Catherine M Cahill, Sanghamitra Bandyopadhyay, Sohan Mikkilineni, Ippolita Cantuticastelvetri, Deborah H Smith, Maria L Maccecchini, Debomoy K Lahiri, Nigel H Greig
    Abstract:

    Increased brain α-synuclein (SNCA) protein expression resulting from gene duplication and triplication can cause a familial form of Parkinson’s disease (PD). Dopaminergic neurons exhibit elevated iron levels that can accelerate toxic SNCA fibril formation. Examinations of human post mortem brain have shown that while mRNA levels for SNCA in PD have been shown to be either unchanged or decreased with respect to healthy controls, higher levels of insoluble protein occurs during PD progression. We show evidence that SNCA can be regulated via the 5Untranslated Region (5′UTR) of its transcript, which we modeled to fold into a unique RNA stem loop with a CAGUGN apical loop similar to that encoded in the canonical iron-responsive element (IRE) of L- and H-ferritin mRNAs. The SNCA IRE-like stem loop spans the two exons that encode its 5′UTR, whereas, by contrast, the H-ferritin 5′UTR is encoded by a single first exon. We screened a library of 720 natural products (NPs) for their capacity to inhibit SNCA 5′UTR driven luciferase expression. This screen identified several classes of NPs, including the plant cardiac glycosides, mycophenolic acid (an immunosuppressant and Fe chelator), and, additionally, posiphen was identified to repress SNCA 5′UTR conferred translation. Western blotting confirmed that Posiphen and the cardiac glycoside, strophanthidine, selectively blocked SNCA expression (~1 μM IC50) in neural cells. For Posiphen this inhibition was accelerated in the presence of iron, thus providing a known APP-directed lead with potential for use as a SNCA blocker for PD therapy. These are candidate drugs with the potential to limit toxic SNCA expression in the brains of PD patients and animal models in vivo.

  • the acute box cis element in human heavy ferritin mrna 5 Untranslated Region is a unique translation enhancer that binds poly c binding proteins
    Journal of Biological Chemistry, 2005
    Co-Authors: Andrew M Thomson, Catherine M Cahill, Hyun Hee Cho, Kristin D Kassachau, Michael R Epis, Kenneth R Bridges, Peter J Leedman, Jack T. Rogers
    Abstract:

    Intracellular levels of the light (L) and heavy (H) ferritin subunits are regulated by iron at the level of message translation via a modulated interaction between the iron regulatory proteins (IRP1 and IRP2) and a 5′-Untranslated Region. Iron-responsive element (IRE). Here we show that iron and interleukin-1β (IL-1β) act synergistically to increase H- and L-ferritin expression in hepatoma cells. A GC-rich cis-element, the acute box (AB), located downstream of the IRE in the H-ferritin mRNA 5′-Untranslated Region, conferred a substantial increase in basal and IL-1β-stimulated translation over a similar time course to the induction of endogenous ferritin. A scrambled version of the AB was unresponsive to IL-1. Targeted mutation of the AB altered translation; reverse orientation and a deletion of the AB abolished the wild-type stem-loop structure and abrogated translational enhancement, whereas a conservative structural mutant had little effect. Labeled AB transcripts formed specific complexes with hepatoma cell extracts that contained the poly(C)-binding proteins, iso-αCP1 and -αCP2, which have well defined roles as translation regulators. Iron influx increased the association of αCP1 with ferritin mRNA and decreased the αCP2-ferritin mRNA interaction, whereas IL-1β reduced the association of αCP1 and αCP2 with H-ferritin mRNA. In summary, the H-ferritin mRNA AB is a key cis-acting translation enhancer that augments H-subunit expression in Hep3B and HepG2 hepatoma cells, in concert with the IRE. The regulated association of H-ferritin mRNA with the poly(C)-binding proteins suggests a novel role for these proteins in ferritin translation and iron homeostasis in human liver.

  • drug discovery targeted to the alzheimer s app mrna 5 Untranslated Region the action of paroxetine and dimercaptopropanol
    Journal of Molecular Neuroscience, 2003
    Co-Authors: Sandra Payton, Jeffrey Randall, Catherine M Cahill, Steven R Gullans, Jack T. Rogers
    Abstract:

    We screened for drugs that specifically interact with the 5'-Untranslated Region of the mRNA encoding the Alzheimer's amyloid precursor protein (APP). Our goal was to use newly discovered APP 5' UTR directed compounds to limit amyloid-beta (Abeta)-peptide output in cell culture systems. The APP 5' UTR folds into a stable RNA secondary structure (Gibbs free energy: DeltaG = -54.9 kcal/mol) and is an important regulator of the amount of APP translated in response to IL-1 (Nilsson et al., 1998; Rogers et al., 1999) and iron (Rogers et al., 2002). Seventeen drug "hits" were identified from a library of 1,200 FDA preapproved drugs (Rogers et al., 2002). Six of the original 17 compounds were validated for their capacity to suppress reporter gene expression in stable neuroblastoma transfectants expressing the dicistronic reporter construct shown in Fig. 2. These six leads suppressed APP 5' UTR driven luciferase translation while causing no effect on the translation of dicistronic GFP gene translated from a viral IRES (negative control to ensure specificity during drug screens). In this report, we show that paroxetine (serotonin reuptake blocker) and dimercaptopropanol (Hg chelator) exerted significant effects on APP expression (steady-state levels of APP), whereas Azithromycin altered APP processing. None of these three compounds altered APLP-1 expression. In the future, we will identify further novel compounds that influence Abeta levels, either via translation inhibition or by changing the activity of proteins coupled between APP translation and APP processing.

  • translation of the alzheimer amyloid precursor protein mrna is up regulated by interleukin 1 through 5 Untranslated Region sequences
    Journal of Biological Chemistry, 1999
    Co-Authors: Jack T. Rogers, Catherine M Cahill, Lorene M Leiter, Jay Mcphee, Shanshan Zhan, Huntington Potter, Lars Nilsson
    Abstract:

    Abstract The amyloid precursorprotein (APP) has been associated with Alzheimer’s disease (AD) because APP is processed into the β-peptide that accumulates in amyloid plaques, and APP gene mutations can cause early onset AD. Inflammation is also associated with AD as exemplified by increased expression of interleukin-1 (IL-1) in microglia in affected areas of the AD brain. Here we demonstrate that IL-1α and IL-1β increase APP synthesis by up to 6-fold in primary human astrocytes and by 15-fold in human astrocytoma cells without changing the steady-state levels of APP mRNA. A 90-nucleotide sequence in the APP gene 5′-Untranslated Region (5′-UTR) conferred translational regulation by IL-1α and IL-1β to a chloramphenicol acetyltransferase (CAT) reporter gene. Steady-state levels of transfected APP(5′-UTR)/CAT mRNAs were unchanged, whereas both base-line and IL-1-dependent CAT protein synthesis were increased. This APP mRNA translational enhancer maps from +55 to +144 nucleotides from the 5′-cap site and is homologous to related translational control elements in the 5′-UTR of the light and and heavy ferritin genes. Enhanced translation of APP mRNA provides a mechanism by which IL-1 influences the pathogenesis of AD.

Sanghamitra Bandyopadhyay - One of the best experts on this subject based on the ideXlab platform.

  • the alpha synuclein 5 Untranslated Region targeted translation blockers anti alpha synuclein efficacy of cardiac glycosides and posiphen
    Journal of Neural Transmission, 2011
    Co-Authors: Jack T. Rogers, Xudong Huang, Catherine M Cahill, Sanghamitra Bandyopadhyay, Sohan Mikkilineni, Ippolita Cantuticastelvetri, Deborah H Smith, Maria L Maccecchini, Debomoy K Lahiri, Nigel H Greig
    Abstract:

    Increased brain α-synuclein (SNCA) protein expression resulting from gene duplication and triplication can cause a familial form of Parkinson’s disease (PD). Dopaminergic neurons exhibit elevated iron levels that can accelerate toxic SNCA fibril formation. Examinations of human post mortem brain have shown that while mRNA levels for SNCA in PD have been shown to be either unchanged or decreased with respect to healthy controls, higher levels of insoluble protein occurs during PD progression. We show evidence that SNCA can be regulated via the 5Untranslated Region (5′UTR) of its transcript, which we modeled to fold into a unique RNA stem loop with a CAGUGN apical loop similar to that encoded in the canonical iron-responsive element (IRE) of L- and H-ferritin mRNAs. The SNCA IRE-like stem loop spans the two exons that encode its 5′UTR, whereas, by contrast, the H-ferritin 5′UTR is encoded by a single first exon. We screened a library of 720 natural products (NPs) for their capacity to inhibit SNCA 5′UTR driven luciferase expression. This screen identified several classes of NPs, including the plant cardiac glycosides, mycophenolic acid (an immunosuppressant and Fe chelator), and, additionally, posiphen was identified to repress SNCA 5′UTR conferred translation. Western blotting confirmed that Posiphen and the cardiac glycoside, strophanthidine, selectively blocked SNCA expression (~1 μM IC50) in neural cells. For Posiphen this inhibition was accelerated in the presence of iron, thus providing a known APP-directed lead with potential for use as a SNCA blocker for PD therapy. These are candidate drugs with the potential to limit toxic SNCA expression in the brains of PD patients and animal models in vivo.

  • a high throughput drug screen targeted to the 5 Untranslated Region of alzheimer amyloid precursor protein mrna
    Journal of Biomolecular Screening, 2006
    Co-Authors: Sanghamitra Bandyopadhyay, Amy Ruggiero, Karen Walshe, Mark F Rogers, Naibedya Chattopadhyay, Marcie A Glicksman, Jack T. Rogers
    Abstract:

    The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5Untranslated Region (5′UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Aβ) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5′UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5′UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5′UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5′UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5′UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Aβ production for AD. (Journal of Biomolecular Screening 2006:469-480)

  • a high throughput drug screen targeted to the 5 Untranslated Region of alzheimer amyloid precursor protein mrna
    Journal of Biomolecular Screening, 2006
    Co-Authors: Sanghamitra Bandyopadhyay, Amy Ruggiero, Karen Walshe, Naibedya Chattopadhyay, Marcie A Glicksman, Mark Rogers, Jack T. Rogers
    Abstract:

    The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5'Untranslated Region (5'UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Abeta) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5'UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5'UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5'UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5'UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5'UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Abeta production for AD.

Chunghsiung Wang - One of the best experts on this subject based on the ideXlab platform.

  • the 5 Untranslated Region of perina nuda virus pnv possesses a strong internal translation activity in baculovirus infected insect cells
    FEBS Letters, 2007
    Co-Authors: Yingju Chen, Chungyung Chen, Chunghsiung Wang
    Abstract:

    A bicistronic baculovirus expression vector and fluorescent protein-based assays were used to identify the sequences that possess internal translation activity in baculovirus-infected insect cells. We demonstrated that the 5' Untranslated Region (5'UTR; 473 nucleotides) of Perina nuda virus (PnV) and the 5'UTR (579 nucleotides) of Rhopalosiphum padi virus (RhPV), but not the IRES sequence of Cricket paralysis virus, have internal translation activity in baculovirus-infected Sf21 cells. In addition, we found that including the first 22 codons of the predicted PnV open reading frame (ORF; a total of 539 nucleotides) enhanced internal translation activity by approximately 18 times. This is the first report of internal translation activity for a baculovirus expression system (BEVS) in the iflavirus 5' sequence and may facilitate the development of polycistronic baculovirus transfer vectors that can be used in BEVS for the production of multiple protein complexes.

Adri A M Thomas - One of the best experts on this subject based on the ideXlab platform.

  • control of eukaryotic protein synthesis by upstream open reading frames in the 5 Untranslated Region of an mrna
    Biochemical Journal, 2002
    Co-Authors: Hedda A Meijer, Adri A M Thomas
    Abstract:

    Control of gene expression is achieved at various levels. Translational control becomes crucial in the absence of transcription, such as occurs in early developmental stages. One of the initiating events in translation is that the 40 S subunit of the ribosome binds the mRNA at the 5'-cap structure and scans the 5'-Untranslated Region (5'-UTR) for AUG initiation codons. AUG codons upstream of the main open reading frame can induce formation of a translation-competent ribosome that may translate and (i) terminate and re-initiate, (ii) terminate and leave the mRNA, resulting in down-regulation of translation of the main open reading frame, or (iii) synthesize an N-terminally extended protein. In the present review we discuss how upstream AUGs can control the expression of the main open reading frame, and a comparison is made with other elements in the 5'-UTR that control mRNA translation, such as hairpins and internal ribosome entry sites. Recent data indicate the flexibility of controlling translation initiation, and how the mode of ribosome entry on the mRNA as well as the elements in the 5'-UTR can accurately regulate the amount of protein synthesized from a specific mRNA.

  • binding of eukaryotic initiation factor 2 and trans acting factors to the 5 Untranslated Region of encephalomyocarditis virus rna
    Biochimie, 1994
    Co-Authors: Gert C Scheper, Harry O Voorma, Adri A M Thomas
    Abstract:

    Abstract The encephalomyocarditis virus 5Untranslated Region (EMC 5 UTR) has a binding site for eukaryotic initiation factor eIF-2. Mutations in the 3′ end or deletion of the 5′ end of the internal ribosomal entry site had a negative effect on the binding of eIF-2 to the EMC 5′ UTR. The binding of eIF-2 to the mutant 5′ UTRs was completely inhibited by the addition of competitor tRNA. Cross-linking of the EMC 5′UTR with proteins from rabbit reticulocyte lysates showed binding of trans -acting factors p52 and p57. Deletions in the 5′ end of the internal ribosomal entry site resulted in a loss of the ability to bind trans -acting factor p57, in accordance with literature data, while p52 binding to these deletion mutants was weak compared to the wildtype EMC 5′UTR. Mutations in the 3′ part of the 5′UTR of EMC still resulted in binding of both trans -acting factors, as with wild type RNA, but binding was more sensitive to competitor tRNA when compared to the binding of p52/p57 to the wild type 5′UTR.