Selective dendrite-targeting of mRNAs of NR1 splice variants without exon 5: identification of a cis-acting sequence and isolation of sequence-binding proteins

Abstract

Both protein and mRNA for the NR1 subunit of N-methyl- d-aspartate receptors are present in neuronal dendrites and undergo changes in distribution following synaptic excitation. However, the expression of all exonic splice variants of NR1 in dendrites has not been determined. In the present study, antibodies against the exon 5 (ex5) peptide sequence labeled proteins mostly in the soma of hippocampus neurons, whereas antibodies against ex21 or ex22 labeled cell bodies and dendrites. Antisense cRNAs for ex5 hybridized with mRNAs in cell bodies, whereas cRNAs for ex21 with mRNAs in both cell bodies and dendrites. Antisense DNA to a 24-base sequence identified as being present only in the 5′-UTR of cDNAs lacking ex5 (ex5 −), hybridized with mRNAs in soma and dendrites and this labeling was coincident, mostly, with RNA granules. Insertion of the 24-base DNA ahead of that for enhanced green fluorescent protein (EGFP) increased the transport of EGFP mRNA and the expression of EGFP in neurites of neurons in culture. Fluorescent sense mRNA that contained the 24-base sequence bound to proteins in dendrites and to two proteins, 60 and 70 kDa, in brain microsomes. Proteins of similar size were also labeled by [ 32P]CTP-mRNA for NR1- 1a , which contains the 24-base 5′-UTR sequence, but not for NR1- 2b , which does not. Biotinylated 24-base sense mRNA was used to purify from brain microsomes two RNA-binding proteins (60 and 70 kDa). We concluded that the 24-base sequence in 5′-UTR of ex5 − mRNA functioned as a cis-acting, dendrite-targeting element recognized selectively by two microsome proteins.