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The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated.

Timmerman SL, Pfingsten JS, Kieft JS, Krushel LA - PLoS ONE (2007)

Bottom Line: Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active.However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1).Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, Colorado, USA.

ABSTRACT
A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.

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The mouse TrkB 5′ leaders bind PTB1 protein.Purified PTB1 was added in increasing amounts to radiolabeled RNA containing Ex1a, Ex2, or the negative control CrPV IRES. A Langmuir plot was created using the calculated fraction bound. Disassociation constants of 85 nM and 46 nM were determined for the PTB1 interaction with Ex1a and Ex2, respectively.
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pone-0003242-g008: The mouse TrkB 5′ leaders bind PTB1 protein.Purified PTB1 was added in increasing amounts to radiolabeled RNA containing Ex1a, Ex2, or the negative control CrPV IRES. A Langmuir plot was created using the calculated fraction bound. Disassociation constants of 85 nM and 46 nM were determined for the PTB1 interaction with Ex1a and Ex2, respectively.

Mentions: PTB activates or enhances IRES activity from a number of cellular mRNAs [6], [9], [35]. As mentioned previously, unr and PTB bind to the Apaf-1 IRES, inducing conformational changes and allowing for internal initiation to occur [5]. PTB binds to the Apaf-1 IRES at two polypyrimidine tracts located 74 and 118 nucleotides from the initiator codon, respectively. Sequence comparison revealed that the mouse TrkB 5′ leader has two polypyrimidine tracts in similar locations of 75 and 124 nucleotides from the initiaton codon suggesting that PTB may also influence RNA secondary structure, and ultimately the IRES activity, of the mouse TrkB 5′ leader. To determine the ability of PTB1 to directly bind the Ex1a and Ex2 IRESes, we performed a filter binding assay. The cricket paralysis virus (CrPV) IRES was used as a negative control since it has been established that the CrPV IRES does not require protein factors to initiate translation [36]. Radiolabeled RNA consisting of Ex1a, Ex2, or CrPV was incubated in the presence of increasing amounts of recombinant PTB1 protein and passed through a dot blot apparatus. The resulting binding curve was fit using the Langmuir equation. As expected, the CrPV IRES did not bind to PTB1 with a significant affinity (Fig 8). However, Ex1a and Ex2 bound to PTB1 with Kd values equaling 85 nM and 46 nM, respectively. Although PTB1 binding to both IRESes falls within the same order of magnitude, the two-fold difference may reflect a biologically significant difference.


The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated.

Timmerman SL, Pfingsten JS, Kieft JS, Krushel LA - PLoS ONE (2007)

The mouse TrkB 5′ leaders bind PTB1 protein.Purified PTB1 was added in increasing amounts to radiolabeled RNA containing Ex1a, Ex2, or the negative control CrPV IRES. A Langmuir plot was created using the calculated fraction bound. Disassociation constants of 85 nM and 46 nM were determined for the PTB1 interaction with Ex1a and Ex2, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2531235&req=5

pone-0003242-g008: The mouse TrkB 5′ leaders bind PTB1 protein.Purified PTB1 was added in increasing amounts to radiolabeled RNA containing Ex1a, Ex2, or the negative control CrPV IRES. A Langmuir plot was created using the calculated fraction bound. Disassociation constants of 85 nM and 46 nM were determined for the PTB1 interaction with Ex1a and Ex2, respectively.
Mentions: PTB activates or enhances IRES activity from a number of cellular mRNAs [6], [9], [35]. As mentioned previously, unr and PTB bind to the Apaf-1 IRES, inducing conformational changes and allowing for internal initiation to occur [5]. PTB binds to the Apaf-1 IRES at two polypyrimidine tracts located 74 and 118 nucleotides from the initiator codon, respectively. Sequence comparison revealed that the mouse TrkB 5′ leader has two polypyrimidine tracts in similar locations of 75 and 124 nucleotides from the initiaton codon suggesting that PTB may also influence RNA secondary structure, and ultimately the IRES activity, of the mouse TrkB 5′ leader. To determine the ability of PTB1 to directly bind the Ex1a and Ex2 IRESes, we performed a filter binding assay. The cricket paralysis virus (CrPV) IRES was used as a negative control since it has been established that the CrPV IRES does not require protein factors to initiate translation [36]. Radiolabeled RNA consisting of Ex1a, Ex2, or CrPV was incubated in the presence of increasing amounts of recombinant PTB1 protein and passed through a dot blot apparatus. The resulting binding curve was fit using the Langmuir equation. As expected, the CrPV IRES did not bind to PTB1 with a significant affinity (Fig 8). However, Ex1a and Ex2 bound to PTB1 with Kd values equaling 85 nM and 46 nM, respectively. Although PTB1 binding to both IRESes falls within the same order of magnitude, the two-fold difference may reflect a biologically significant difference.

Bottom Line: Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active.However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1).Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver School of Medicine, Aurora, Colorado, USA.

ABSTRACT
A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.

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