Abstract
Acetylcholinesterase (AChE) is highly expressed at sites of nerve-muscle contact where it is regulated at both the transcriptional and post-transcriptional levels. Our understanding of the molecular mechanisms underlying its regulation is incomplete, but they appear to involve both translational and post-translational events as well. Here, we show that Pumilio-2 (PUM2), an RNA binding translational repressor, is highly localized at the neuromuscular junction where AChE mRNA concentrates. Immunoprecipitation of muscle cell extracts with a PUM2 specific antibody precipitated AChE mRNA, suggesting that PUM2 binds to the AChE transcripts in a complex. Gel shift assays using a bacterially expressed PUM2 RNA binding domain showed specific binding using wild type AChE 3'-UTR RNA segment that was abrogated by mutation of the consensus recognition site. Transfecting skeletal muscle cells with shRNAs specific for PUM2 up-regulated AChE expression, whereas overexpression of PUM2 decreased AChE activity. We conclude that PUM2 binds to AChE mRNA and regulates AChE expression translationally at the neuromuscular synapse. Finally, we found that PUM2 is regulated by the motor nerve suggesting a trans-synaptic mechanism for locally regulating translation of specific proteins involved in modulating synaptic transmission, analogous to CNS synapses.
Highlights
Acetylcholinesterase RNA and protein are concentrated at the neuromuscular synapse but how its translation is regulated is not known
We show that the AChE mRNA 3Ј-untranslated region (3Ј-UTR) contains one consensus Pumilio-binding element (PBE) that is highly conserved in mammals and that PUM2 is highly localized at the mammalian neuromuscular junction (NMJ)
PUM2 Is Localized at NMJ—Cloning and sequencing of the full-length mammalian AChE cDNAs by several laboratories (26 –28) showed that they were highly conserved in both their coding and non-coding sequences
Summary
Acetylcholinesterase RNA and protein are concentrated at the neuromuscular synapse but how its translation is regulated is not known. We show that Pumilio-2 (PUM2), an RNA binding translational repressor, is highly localized at the neuromuscular junction where AChE mRNA concentrates. The PUF proteins are characterized by a highly conserved C-terminal RNA-binding domain, the homology domain (HD) [14, 15], composed of eight tandem repeats This domain binds to a specific eight nucleotide sequence in the 3Ј-UTR of target mRNAs known as the Nanos response element [16] or Pumilio-binding element (PBE) [12]. PUM2 binds to the AChE transcript 3Ј-UTR where it regulates its translation in tissue-cultured skeletal muscle These studies provide the first evidence for translational regulation of a synaptic component, AChE, by an identified RNA-binding protein PUM2 at the neuromuscular synapse and provide a model system for studying translational controls at both PNS and CNS synapses. In vivo studies suggest that PUM2 itself is under regulation by the motor nerve
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