Internal initiation of translation of five dendritically localized neuronal mRNAs - PubMed (original) (raw)
Internal initiation of translation of five dendritically localized neuronal mRNAs
J K Pinkstaff et al. Proc Natl Acad Sci U S A. 2001.
Abstract
In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5' leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the alpha subunit of calcium-calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5' untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and alpha-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5' untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5' leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.
Figures
Figure 1
Analysis of monocistronic mRNAs containing the transcribed leader sequences of dendritically localized mRNAs. (A) Schematic illustration of monocistronic reporter mRNAs containing the transcribed leader sequences (TLS) 5′ of the Photinus luciferase gene. (B) Northern blot analysis of monocistronic mRNAs using equivalent amounts of total RNA from B104 cells transfected with the monocistronic constructs indicated and probed with a radiolabeled cRNA to the Photinus luciferase gene. (C) Photinus luciferase activity generated by monocistronic reporter mRNAs. Luciferase activity was normalized to that generated by the parent vector_P_M. (D) Monocistronic constructs were cotransfected with either a plasmid expressing hypophosphorylated 4E-BP1 or control plasmid and assayed for luciferase activity. The activities obtained in cells cotransfected with the mutated 4E-BP1 is represented as a percentage of the activity obtained in cells cotransfected with the control plasmid.
Figure 2
Analysis of dicistronic mRNAs containing the transcribed leader sequences of dendritically localized mRNAs. (A) Schematic representation of the dicistronic luciferase mRNAs. IRES activity generated by each construct after transfection into neural (B) and nonneural (C) cell lines is shown as the ratio of Photinus to Renilla luciferase activities (P:R) and normalized to the activity generated by the parent construct_RP_. (D) Total RNA harvested from B104 cells transfected with the dicistronic constructs was hybridized with a cRNA probe specific for the ORF of the Photinus luciferase gene. The migration of the 28S and 18S rRNAs is indicated. (E) Analysis of luciferase activity generated from each dicistronic construct in the presence of the hairpin structure. Luciferase activity is shown as the P:R ratio and is normalized to the activity of the hairpin control construct, _RP_h.
Figure 3
Analysis of the RC3 IRES in the intronless _RP_i construct. (A) Northern blot analysis of total RNA extracted from B104 cells transfected with the RC3/RP_i construct containing the RC3 leader sequence and hybridized with a cRNA probe to the Photinus luciferase gene. (B) P:R ratios produced from dicistronic luciferase mRNAs containing the β-globin, RC3, and EMCV leader sequences and normalized to the_P:R ratio generated by_RP_i. (C)P:R ratios in the presence of a 5′ hairpin structure after transfection into B104 cells.
Figure 4
Analysis of the RC3 3′ UTR as a dendritic targeting sequence and demonstration of translation in dendrites. Cultured hippocampal neurons were transfected with a construct encoding EYFP with a nuclear-localization sequence containing either the RC3 3′ UTR (A) or a simian virus 40 polyadenylation sequence alone (B). In situ hybridization of neurons transfected with the EYFP/NLS constructs with (C) or without (D) the RC3 3′ UTR using a digoxygenin-labeled cRNA probe to the EYFP coding region. Arrows in A and_C_ indicate the localization of the EYFP protein and exogenous mRNA, respectively, in neuronal processes of transfected neurons. Arrows in B and D identify the nucleus of a transfected neuron.
Figure 5
In situ analysis of IRES activity in hippocampal neurons. Dicistronic constructs were created containing ECFP and EYFP as the first and second cistron, respectively. The 5′ leader sequence from β-globin or RC3 mRNAs was inserted into the intercistronic region. The constructs also contained the RC3 3′ UTR to direct transport of the mRNA into the dendrites. The dicistronic constructs were transfected into mature primary hippocampal neurons that were cultured in vitro for 3 weeks. Fluorescent photomicrographs of dendrites transfected with the dicistronic construct containing the β-globin (A) or RC3 (B) leader sequence in the intercistronic region under both ECFP and EYFP filters are shown. Cap-dependent translation is seen as blue; IRES-dependent translation is seen as yellow. Where both cap- and IRES-dependent translation occur, the blue and yellow fluorescence combine to exhibit turquoise. (C) Fluorescent emission was measured from each cistron in the cell body and in the dendrites. IRES activity is quantified as a ratio of the fluorescence emitted from the second cistron (EYFP) to the fluorescence emitted from the first cistron (ECFP).
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