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. 2005 May 20;33(9):2929-41.
doi: 10.1093/nar/gki605. Print 2005.

Internal initiation of translation of the TrkB mRNA is mediated by multiple regions within the 5' leader

Tara Dobson  1 , Angela Minic, Kirsten Nielsen, Elizabeth Amiott, Les Krushel
Affiliations

Internal initiation of translation of the TrkB mRNA is mediated by multiple regions within the 5' leader

Tara Dobson et al. Nucleic Acids Res. .

Abstract

Translational regulation of the dendritically localized mRNA encoding for the neurotrophin receptor TrkB has important ramifications for synaptic function. We examined whether the TrkB mRNA is translated through an internal initiation entry site (IRES). The human TrkB 5' leaders are derived from the use of alternative promoters and alternative splicing, but all 5' leaders share a common exon. Insertion of a full-length 5' leader, as well as the common exon into the intercistronic region of a dicistronic luciferase construct, yielded luciferase activity generated from the second cistron that was either equivalent or higher than that observed from the encephalomyocarditis virus IRES. Moreover, inhibiting cap-dependent translation ex vivo and in in vitro lysates had only a minimal effect on the translation of mRNA containing the TrkB 5' leader. Dissecting the 5' leader showed that the IRES is located in the exon common to all TrkB 5' leaders. Moreover, six regions ranging from 2 to 25 nt were identified that either promoted or inhibited IRES activity. Taken together, these results suggest that the 5' leader of the human TrkB mRNA contains multiple cis-elements that regulate internal initiation of translation and that this mechanism may contribute significantly to the translation of the TrkB mRNA in neuronal dendrites.

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Figures

Figure 1
Figure 1
The TrkB 5′ leader exhibits IRES activity. (A) Dicistronic luciferase constructs containing the multiple cloning site (RP) of the 5′ leader from the β-globin, TrkB or mRNA or EMCV RNA inserted into the intercistronic region was transfected individually into C6 or N2a cells. Luciferase activity is shown as the ratio of Photinus luciferase to Renilla luciferase (P:R) and is normalized to the activity of the control construct RP. A P:R ratio that is above that obtained from RP indicates the presence of an IRES. (B) Northern blot analysis of transfected dicistronic luciferase constructs. A cRNA probe directed against the Photinus luciferase gene identified a single mRNA species from RNA isolated from C6 cells transfected with the dicistronic luciferase construct containing the β-globin or TrkB 5′ leader. (C) Promoterless dicistronic luciferase constructs containing the β-globin, TrkB or EMCV 5′ leader inserted into the intercistronic region were transfected individually into C6 cells. The P:R ratio is normalized to the activity obtained from the β-globin construct.
Figure 2
Figure 2
In vitro translation of dicistronic and monocistronic luciferase mRNA. (A) Dicistronic mRNA containing the β-globin or TrkB 5′ leader was translated in rabbit reticulocyte lysate in the presence of increasing concentrations of cap analog. The initial P:R ratio from the dicistronic mRNA containing the two 5′ leaders was normalized to 100. (B) Monocistronic mRNA containing the β-globin or TrkB 5′ leader was translated in rabbit reticulocyte lysate in the presence of increasing concentrations of cap analog. The initial level of Photinus luciferase activity from each monocistronic mRNA was normalized to 100.
Figure 3
Figure 3
Truncated TrkB 5′ leaders exhibit variable levels of IRES activity. Dicistronic luciferase constructs containing 5′ truncations of the TrkB 5′ leader or an internal deletion in the TrkB 5′ leader inserted into the intercistronic region were transfected into C6 cells. Luciferase activity is shown as the P:R ratio and is normalized to the activity from the control RP construct.
Figure 4
Figure 4
Inhibiting cap-dependent translation of monocistronic mRNA by overexpressing 4E-BP1. (A) Monocistronic Photinus luciferase constructs containing the β-globin, TrkB 5′ leader, 5′truncations of the TrkB 5′ leader or an internal deletion in the TrkB 5′ leader were co-transfected with either a plasmid expressing hypophosphorylated 4E-BP1 or a control plasmid and assayed for luciferase activity. The activity obtained in cells co-transfected with 4E-BP1 is represented as a percentage of the activity obtained in cells co-transfected with the control plasmid. (B) Western blot analysis using antibodies directed against 4E-BP1 or GAPDH (as a loading control) of lysates from the co-transfections in (A).
Figure 5
Figure 5
IRES activity is observed in the 5′ end of the TrkB 5′ leader in the absence of a putative inhibitory region. (A) The 5′ serial truncations were produced in a TrkB 5′ leader containing an internal deletion of 25 nt. (B) Serial truncations of 2 nt were produced in a TrkB 5′ leader containing an internal deletion of 25 nt. (C) 5′serial truncations similar to that created in (A) were produced in the 353 nt TrkB 5′ leader (containing the putative inhibitory 25 nt). All 5′ leaders were inserted into the intercistronic region of a dicistronic luciferase construct and transfected individually into C6 cells. The P:R ratios were normalized to that obtained from the control RP construct.
Figure 6
Figure 6
Serial truncations identify IRES promoting regions in the TrkB 5′ leader. Serial truncations of 4–11 nt were produced in the TrkB 5′ leader containing the 3′ 278 nt (A), 3′ 227 nt (B) or the 3′ 124 nt (C). The leaders were inserted into the intercistronic region of a dicistronic luciferase construct and transfected individually into C6 cells. The P:R ratios were normalized to that obtained from the control RP construct.
Figure 7
Figure 7
A model [Mfold (42,43)] of the secondary structure of the 353 nt TrkB 5′ leader identifying regions that promote and inhibit IRES activity. The green and red arrows define the 5′ and 3′ end of the 5′ leader. Parentheses labeled (A–D) demarcate 10 nt, which when serially truncated result in a loss of IRES activity. The area delineated by the red parenthesis identifies 25 nt that when internally deleted result in an increase in IRES activity. The blue parenthesis demarcates 2 nt, which when serially truncated in the absence of the inhibitory region results in a loss of IRES activity.
Figure 8
Figure 8
Internal deletions of the TrkB 5′ leader reveal regions that promote IRES activity. The 10 nt regions that were identified from the serial truncation analysis (Figure 6) were deleted individually or in different combinations within the TrkB 5′ leader. The 5′ leaders were inserted into the intercistronic region of a dicistronic luciferase construct and transfected individually into C6 cells. The P:R ratios were normalized to that obtained from the control RP construct.
Figure 9
Figure 9
IRES activity within a full-length TrkB 5′ leader resides in exon 5. (A) The TrkB 5′ leader containing exon 5, a full-length TrkB 5′ leader consisting of exons 1, 2 and 5, and the full-length TrkB 5′ leader with IRES promoting regions B, C and D deleted were inserted into the intercistronic region of a dicistronic luciferase construct and transfected individually into C6 cells. The P:R ratios were normalized to that obtained from the control RP construct. (B) Monocistronic mRNA containing the β-globin, a full-length TrkB 5′ leader consisting of exons 1, 2 and 5, and the full-length TrkB 5′ leader with IRES promoting regions B, C and D deleted were translated in rabbit reticulocyte lysate in the presence of increasing concentrations of cap analog. The initial level of Photinus luciferase activity from each monocistronic mRNA was normalized to 100.
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