HuD binds to three AU-rich sequences in the 3'-UTR of neuroserpin mRNA and promotes the accumulation of neuroserpin mRNA and protein.
Neuroserpin is an axonally secreted serine protease inhibitor expressed in the nervous system that protects neurons from ischemia-induced apoptosis. Mutant neuroserpin forms have been found polymerized in inclusion bodies in a familial autosomal encephalopathy causing dementia, or associated with epilepsy. Regulation of neuroserpin expression is mostly unknown. Here we demonstrate that neuroserpin mRNA and the RNA-binding protein HuD are co-expressed in the rat central nervous system, and that HuD binds neuroserpin mRNA in vitro with high affinity. Gel-shift, supershift and T1 RNase assays revealed three HuD-binding sequences in the 3'-untranslated region (3'-UTR) of neuroserpin mRNA. They are AU-rich and 20, 51 and 19 nt in length. HuD binding to neuroserpin mRNA was also demonstrated in extracts of PC12 pheochromocytoma cells. Additionally, ectopic expression of increasing amounts of HuD in these cells results in the accumulation of neuroserpin 3'-UTR mRNA. Furthermore, stably transfected PC12 cells over-expressing HuD contain increased levels of both neuroserpin mRNAs (3.0 and 1.6 kb) and protein. Our results indicate that HuD stabilizes neuroserpin mRNA by binding to specific AU-rich sequences in its 3'-UTR, which prolongs the mRNA lifetime and increases protein level.
Cuadrado
Ana
A
Instituto de Investigaciones Biomédicas 'Alberto Sols', Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier 4, E-28029 Madrid, Spain.
Navarro-Yubero
Cristina
C
Furneaux
Henry
H
Kinter
Jochen
J
Sonderegger
Peter
P
Muñoz
Alberto
A
D020413:3' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism; D051959:ELAV Proteins; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D017403:In Situ Hybridization; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D000097522:Neuroserpin
D020413:3' Untranslated Regions; D051959:ELAV Proteins; D005982:Glutathione Transferase; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015843:Serpins; D000097522:Neuroserpin