The 3'-untranslated region of CaMKII alpha is a cis-acting signal for the localization and translation of mRNA in dendrites - PubMed (original) (raw)

The 3'-untranslated region of CaMKII alpha is a cis-acting signal for the localization and translation of mRNA in dendrites

M Mayford et al. Proc Natl Acad Sci U S A. 1996.

Abstract

Neuronal signaling requires that synaptic proteins be appropriately localized within the cell and regulated there. In mammalian neurons, polyribosomes are found not just in the cell body, but also in dendrites where they are concentrated within or beneath the dendritic spine. The alpha subunit of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII alpha) is one of only five mRNAs known to be present within the dendrites, as well as in the soma of neurons. This targeted subcellular localization of the mRNA for CaMKII alpha provides a possible cell biological mechanism both for controlling the distribution of the cognate protein and for regulating independently the level of protein expression in individual dendritic spines. To characterize the cis-acting elements involved in the localization of dendritic mRNA we have produced two lines of transgenic mice in which the CaMKII alpha promoter is used to drive the expression of a lacZ transcript, which either contains or lacks the 3'-untranslated region of the CaMKII alpha gene. Although both lines of mice show expression in forebrain neurons that parallels the expression of the endogenous CaMKII alpha gene, only the lacZ transcripts bearing the 3'-untranslated region are localized to dendrites. The beta-galactosidase protein shows a variable level of expression along the dendritic shaft and within dendritic spines, which suggests that neurons can control the local biochemistry of the dendrite either through differential localization of the mRNA or variations in the translational efficiency at different sites along the dendrite.

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Figures

Figure 1

Figure 1

Expression of lacZ mRNA in mouse forebrain. (A) Schematic representation of the DNA constructs used for the generation of transgenic mice.lac-CMK: the 8.5-kbp CaMKIIα promoter region, as well as 84 nucleotides of the 5′ non-coding exon, was fused to the E. coli lacZ gene. The entire 3′-UTR of the CaMKIIα mRNA was placed downstream of the lacZ coding region.lac_-A: identical to_lac_-CMK except that the bovine growth hormone polyadenylylation signal was substituted for the CaMKIIα 3′-UTR. nls-lac-CMK, the tet·O promoter (16) was linked to a modified lacZ gene with an in-phase fusion to the green fluorescent protein (GFP) and a nuclear localization sequence. (B) Northern blot analysis of poly(A)+ RNA isolated from the forebrain of the lac-CMK and_lac-A mice.

Figure 2

Figure 2

β-gal histochemistry.

Figure 3

Figure 3

In situ localization of_lacZ_ mRNA in hippocampus. (A) In situ hybridization using a _lacZ_-specific oligonucleotide probe. SM (stratum molecular), dendritic layer of the dentate gyrus granule cells; SR (stratum radiatum), dendritic layer of the CA1 pyramidal cells. (B) X-gal staining of hippocampus from 20-μm horizontal sections as described in Fig. 2. (Bar = 300 μm.)

Figure 4

Figure 4

Differential expression of β-gal within dendrites. (A) In situ hybridization against the nls-lac-CMK mouse using a lacZ specific probe. (B) Histochemical detection of β-gal in the nls-lac-CMK mouse hippocampus (Bar = 300 μM.) (C) Immunofluorescent detection of MAP2 and β-gal expression in an nls-lac-CMK neuron in culture. The MAP2 antibody specifically labels microtubules along the dendritic shaft. MAP2 labeling is indicated in red. β-gal labeling is shown in green. Arrows denote β-gal in presumptive dendritic spines. Arrowheads indicate areas of punctate β-gal staining along the dendrite. (D) Expression of β-gal in a distal portion of the dendritic arbor. Arrowheads denote areas of punctate β-gal staining. Open arrow shows a dendrite arising from a neuron, which did not express the nls-lac-CMK transgene (Bar = 10 μM.)

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