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.
| Original language | English |
|---|---|
| Pages (from-to) | 36492-36499 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 286 |
| Issue number | 42 |
| DOIs | |
| State | Published - Oct 21 2011 |
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ASJC Scopus subject areas
- Biochemistry
- Cell Biology
- Molecular Biology
Cite this
Translational regulation of acetylcholinesterase by the RNA-binding protein pumilio-2 at the neuromuscular synapse. / Marrero, Emilio; Rossi, Susana G; Darr, Andrew; Tsoulfas, Pantelis; Rotundo, Richard L.
In: Journal of Biological Chemistry, Vol. 286, No. 42, 21.10.2011, p. 36492-36499.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Translational regulation of acetylcholinesterase by the RNA-binding protein pumilio-2 at the neuromuscular synapse
AU - Marrero, Emilio
AU - Rossi, Susana G
AU - Darr, Andrew
AU - Tsoulfas, Pantelis
AU - Rotundo, Richard L
PY - 2011/10/21
Y1 - 2011/10/21
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=80054700059&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=80054700059&partnerID=8YFLogxK
U2 - 10.1074/jbc.M111.285510
DO - 10.1074/jbc.M111.285510
M3 - Article
C2 - 21865157
AN - SCOPUS:80054700059
VL - 286
SP - 36492
EP - 36499
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 42
ER -