Characterization of multiple exon 1 variants in mammalian HuD mRNA and neuron-specific transcriptional control via neurogenin 2.
The RBP (RNA-binding protein) and Hu/ELAV family member HuD regulates mRNA metabolism of genes directly or indirectly involved in neuronal differentiation, learning and memory, and several neurological diseases. Given the important functions of HuD in a variety of processes, we set out to determine the mechanisms that promote HuD mRNA expression in neurons using a mouse model. Through several complementary approaches, we determined that the abundance of HuD mRNA is predominantly under transcriptional control in developing neurons. Bioinformatic and 5'RACE (rapid amplification of cDNA ends) analyses of the 5' genomic flanking region identified eight conserved HuD leader exons (E1s), two of which are novel. Expression of all E1 variants was determined in mouse embryonic (E14.5) and adult brains. Sequential deletion of the 5' regulatory region upstream of the predominantly expressed E1c variant revealed a well conserved 400 bp DNA region that contains five E-boxes and is capable of directing HuD expression specifically in neurons. Using EMSA (electrophoretic mobility shift assay), ChIP (chromatin immunoprecipitation), and 5' regulatory region deletion and mutation analysis, we found that two of these E-boxes are targets of Neurogenin 2 (Ngn2) and that this mechanism is important for HuD mRNA induction. Together, our findings reveal that transcriptional regulation of HuD involves the use of alternate leader exons and Ngn2 mediates neuron-specific mRNA expression. To our knowledge, this is the first study to identify molecular events that positively regulate HuD mRNA expression.
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5, Canada.
Lucas M
LM
Bronicki
Guy
G
Bélanger
Bernard J
BJ
Jasmin
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D001921:Brain / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D002417:Cattle; D002461:Cell Line, Transformed; D002466:Cell Nucleolus / Q000235:genetics / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D019295:Computational Biology; D003609:Dactinomycin / Q000494:pharmacology; D024721:E-Box Elements / Q000187:drug effects / Q000235:genetics; D051959:ELAV Proteins / Q000235:genetics* / Q000378:metabolism; D000067779:ELAV-Like Protein 4; D024202:Electrophoretic Mobility Shift Assay; D004622:Embryo, Mammalian; D005091:Exons / Q000235:genetics*; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D016415:Sequence Alignment
D051792:Basic Helix-Loop-Helix Proteins; D003609:Dactinomycin; D051959:ELAV Proteins; D000067779:ELAV-Like Protein 4; D009419:Nerve Tissue Proteins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; C498011:Elavl4 protein, mouse; C102938:Neurog2 protein, mouse