Characterization of Multiple Exon 1 Variants in Mammalian HuD mRNA and Neuron-Specific Transcriptional Control via Neurogenin 2

Abstract

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 5ЈRACE (rapid amplification of cDNA ends) analyses of the 5Ј 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 5Ј 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 5Ј 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.

Positive feedback between RNA-binding protein HuD and transcription factor SATB1 promotes neurogenesis

“…In WT NSCs, HuD mRNA expression levels increased during neuronal differentiation (SI Appendix, Fig. S5 A and B), consistent with previous findings in cell lines (15,23). We then found that HuD KO NSCs differentiated into fewer Tuj1 + neurons ( Fig.…”

“…5D). This result is consistent with the earlier finding that a ∼400-bp sequence between −1002 and −606 is responsible for transcriptional activation of the HuD E1c variant during neuronal differentiation (15).…”

“…Although an extensive literature focuses on HuD regulation of its downstream targets, much less is known about how HuD expression is regulated. Recent evidence suggests that the levels of HuD mRNAs in neurons are determined predominantly by transcription (2,3,15). In our study, both HuD and Satb1 mRNA levels increased during NSC neuronal differentiation, suggesting that a positive regulatory loop may exist between these two proteins.…”

“…We next determined whether SATB1 activates HuD transcription through the HuD promoter. The mammalian HuD gene is known to contain eight conserved leader exons (E1a to E1c), and each of them, except for E1c 1 , is individually spliced into a common exon 2 (E2), which results in HuD transcript variants with alternative 5′ ends (15). We first examined the expression levels of all eight E1 mRNA variants in NSCs using variant-specific forward primers together with a reverse primer derived from the common E2 (Fig.…”

“…Thyroid hormone is known to repress HuD transcription (13,14). Recently, Ngn2 was identified as the first transcriptional activator of HuD during neuronal differentiation of P19 neuroblastoma cells (15). Therefore, the spatial and temporal induction of HuD is regulated at least in part via transcriptional regulation.…”