AUF1 Is Expressed in the Developing Brain, Binds to AT-rich Double-stranded DNA, and Regulates Enkephalin Gene Expression

Abstract

During our search for transcriptional regulators that control the developmentally regulated expression of the enkephalin (ENK) gene, we identified AUF1. ENK, a peptide neurotransmitter, displays precise cell-specific expression in the adult brain. AUF1 (also known as heterogeneous nuclear ribonucleoprotein D) has been known to regulate gene expression through altering the stability of AU-rich mRNAs. We show here that in the developing brain AUF1 proteins are expressed in a spatiotemporally defined manner, and p37 and p40/42 isoforms bind to an AT-rich double-stranded (ds) DNA element of the rat ENK (rENK) gene. This AT-rich dsDNA sequence acts as a cis-regulatory DNA element and is involved in regulating the cell-specific expression of the ENK gene in primary neuronal cultures. The AT-rich dsDNA elements are present at ϳ2.5 kb 5 upstream of the rat, human, and mouse ENK genes. AUF1 proteins are shown here to provide direct interaction between these upstream AT-rich DNA sequences and the TATA region of the rENK gene. Double immunohistochemistry demonstrated that in the developing brain AUF1 proteins are expressed by proliferating neural progenitors and by differentiating neurons populating brain regions, which will not express the ENK gene in the adult, suggesting a repressor role for AUF1 proteins during enkephalinergic differentiation. Their subnuclear distribution and interactions with AT-rich DNA suggest that in the developing brain they can be involved in complex nuclear regulatory mechanisms controlling the development-and cell-specific expression of the ENK gene.The expression of the ENK 3 gene in the adult brain is highly cell-specific and spatially restricted. Many enkephalinergic neurons (i.e. express the ENK gene and use enkephalin peptides as neurotransmitters) are in the striatum (caudate putamen), and most of the other brain structures, such as cerebral cortex, are devoid of enkephalinergic neurons (1). This contrasting distribution is the result of the developmental process.ENK is a single copy gene encoding for preproenkephalin polypeptide, which is proteolytically cleaved, yielding four copies of Met-enkephalin and one copy of Leu-enkephalin peptides, which are endogenous ligands of opiate receptors (2). Enkephalins in the basal forebrain mediate social behavior, aggression, and reward and are implicated in mediating euphoric properties of drugs.The identity of neurons is established during neurodevelopment when multipotent progenitors differentiate into their various neurotransmitter phenotypes (3). Enkephalinergic differentiation, the cell-specific expression of the ENK gene, i.e. in spiny projection neurons in the striatum, takes place between embryonic day 14 (E14) and postnatal day 2 (P2) in the rat (4). It is during this period when transcription factors that directly regulate the developmental expression of neurotransmitter genes, such as ENK, are specifically expressed. They bind to cis-regulatory DNA elements and regulate the expression of their target genes, such as ENK (5), by restrict...

Multiple and Specific mRNA Processing Targets for the Major Human hnRNP Proteins

“…Given that hnRNP D and DL are mostly known as cytoplasmic proteins involved in controlling mRNA stability, it is possible that the changes that we observed are caused by differences in the stability of the specific mRNA splice isoforms. Nonetheless, nuclear functions for hnRNP D have also been proposed (16,30) and a direct impact on alternative splicing remains possible but will need to be confirmed in future experiments.…”

Similar Regulation of Human Inducible Nitric-oxide Synthase Expression by Different Isoforms of the RNA-binding Protein AUF1

“…Activity-Besides its post-transcriptional effects, AUF1 has been described to regulate the promoter activity of genes like ␣-fetoprotein or enkephalin by binding to AT-rich DNA sequences (28,29,40). Therefore, we investigated if the AUF1 isoforms mediate their effect on iNOS expression by modulation of human iNOS promoter activity.…”

Upf1/Upf2 Regulation of 3′ Untranslated Region Splice Variants of AUF1 Links Nonsense-Mediated and A+U-Rich Element-Mediated mRNA Decay

“…Small interfering RNA (siRNA)-mediated depletion of AUF1 stabilizes ARE-containing mRNAs, including those for GADD45␣, cyclin D1, and the cell cycle inhibitors p21 and p16 INK4a , and a reporter mRNA containing the interleukin-3 ARE (33,34,49). In addition to its role in AMD, AUF1 participates in other cellular processes, including telomere maintenance and transcriptional activation/repression (12,14,16,18,20,26).…”

“…For example, as an AMD factor, AUF1 promotes the decay of mRNAs encoding proteins involved in cell growth, apoptosis, and signaling (34,35). Moreover, the AUF1 protein isoforms are multifunctional and are known to regulate the transcription of several genes (12,14,20,26,53) as well as to contribute to telomere maintenance through interaction with telomere repeats (18). Interestingly, a link between NMD and telomere maintenance via regulation of RNA-binding proteins has already been reported for simple eukaryotes.…”