Bag-1 Internal Ribosome Entry Segment Activity Is Promoted by Structural Changes Mediated by Poly(rC) Binding Protein 1 and Recruitment of Polypyrimidine Tract Binding Protein 1

ABSTRACT We have shown previously that an internal ribosome entry segment (IRES) directs the synthesis of the p36 isoform of Bag-1 and that polypyrimidine tract binding protein 1 (PTB-1) and poly(rC) binding protein 1 (PCBP1) stimulate IRES-mediated translation initiation in vitro and in vivo. Here, a secondary structural model of the Bag-1 IRES has been derived by using chemical and enzymatic probing data as constraints on the RNA folding algorithm Mfold. The ribosome entry window has been identified within this structural model and is located in a region in which many residues are involved in base-pairing interactions. The interactions of PTB-1 and PCBP1 with their cognate binding sites on the IRES disrupt many of the RNA-RNA interactions, and this creates a largely unstructured region of approximately 40 nucleotides that could permit ribosome binding. Mutational analysis of the PTB-1 and PCBP1 binding sites suggests that PCBP1 acts as an RNA chaperone to open the RNA in the vicinity of the ribosome entry window while PTB-1 is probably an essential part of the preinitiation complex.

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Identification of a motif that mediates polypyrimidine tract-binding protein-dependent internal ribosome entry

Genes & Development ◽

10.1101/gad.339105 ◽

2005 ◽

Vol 19 (13) ◽

pp. 1556-1571 ◽

Cited By ~ 75

Author(s):

S. A. Mitchell

Keyword(s):

Binding Protein ◽

Polypyrimidine Tract ◽

Internal Ribosome Entry ◽

Polypyrimidine Tract Binding ◽

Polypyrimidine Tract Binding Protein

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

Molecular and Cellular Biology ◽

10.1128/mcb.20.5.1583-1595.2000 ◽

2000 ◽

Vol 20 (5) ◽

pp. 1583-1595 ◽

Cited By ~ 88

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.

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Protein Factor Requirements of the Apaf-1 Internal Ribosome Entry Segment: Roles of Polypyrimidine Tract Binding Protein and upstream of N-ras

Molecular and Cellular Biology ◽

10.1128/mcb.21.10.3364-3374.2001 ◽

2001 ◽

Vol 21 (10) ◽

pp. 3364-3374 ◽

Cited By ~ 92

Author(s):

Sally A. Mitchell ◽

Emma C. Brown ◽

Mark J. Coldwell ◽

Richard J. Jackson ◽

Anne E. Willis

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Cell Types ◽

Polypyrimidine Tract ◽

Internal Ribosome Entry ◽

Polypyrimidine Tract Binding ◽

Polypyrimidine Tract Binding Protein ◽

Different Cell Types

ABSTRACT It has been reported previously that the 5′ untranslated region of the mRNA encoding Apaf-1 (apoptotic protease-activating factor 1) has an internal ribosome entry site (IRES), whose activity varies widely among different cell types. Here it is shown that the Apaf-1 IRES is active in rabbit reticulocyte lysates, provided that the system is supplemented with polypyrimidine tract binding protein (PTB) and upstream of N-ras (unr), two cellular RNA binding proteins previously identified to be required for rhinovirus IRES activity. In UV cross-linking assays and electrophoretic mobility shift assays with individual recombinant proteins, the Apaf-1 IRES binds unr but not PTB; however, PTB binding occurs if unr is present. Over a range of different cell types there is a broad correlation between the activity of the Apaf-1 IRES and their content of PTB and unr. In cell lines deficient in these proteins, overexpression of PTB and unr stimulated Apaf-1 IRES function. This is the first example where an IRES in a cellular mRNA has been shown to be functionally dependent, both in vitro and in vivo, on specific cellular RNA binding proteins. Given the critical role of Apaf-1 in apoptosis, these results have important implications for the control of the apoptotic cascade.

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Investigation of interactions of polypyrimidine tract-binding protein with artificial internal ribosome entry segments

Biochemical Society Transactions ◽

10.1042/bst0331483 ◽

2005 ◽

Vol 33 (6) ◽

pp. 1483-1486 ◽

Cited By ~ 5

Author(s):

K.A. Spriggs ◽

S.A. Mitchell ◽

A.E. Willis

Keyword(s):

Translation Initiation ◽

Binding Protein ◽

Regulation Of Gene Expression ◽

Sequence Motif ◽

Polypyrimidine Tract ◽

Protein Motif ◽

Internal Ribosome Entry ◽

Eukaryotic Translation ◽

Polypyrimidine Tract Binding ◽

Polypyrimidine Tract Binding Protein

Most eukaryotic translation initiation is thought to be dependent on the 5′-cap structure of the mRNA. It is becoming apparent, however, that the mRNAs of many genes contain IRESs (internal ribosome entry segments) within the 5′-UTR (5′-untranslated region) that allow ribosomes to initiate translation independently of the 5′-cap. IRESs can enable the expression of these genes under conditions (such as viral infection, cellular stress and apoptosis) when cap-dependent translation initiation is compromised, and also provide a target for regulation of gene expression. Recent results from our laboratory and others suggest that 10% of mRNAs (∼4000 genes) use this mechanism to initiate translation. One of the central goals of those working in the field of translation is to identify the sequence motif(s) and proteins that are required for internal ribosome entry. We have identified recently a unique PTB (polypyrimidine tract-binding protein) motif (CCU)n that is present in a large subset of cellular IRESs, and the results suggest that PTB itself is involved either directly or indirectly in ribosome recruitment. Here, we describe further investigations of PTB with artificial sequences that harbour this motif.

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