A Potential Regulatory Role for Intronic microRNA-338-3p for Its Host Gene Encoding Apoptosis-Associated Tyrosine Kinase

MicroRNAs (miRNAs) are important gene regulators that are abundantly expressed in both the developing and adult mammalian brain. These non-coding gene transcripts are involved in post-transcriptional regulatory processes by binding to specific target mRNAs. Approximately one third of known miRNA genes are located within intronic regions of protein coding and non-coding regions, and previous studies have suggested a role for intronic miRNAs as negative feedback regulators of their host genes. In the present study, we monitored the dynamic gene expression changes of the intronic miR-338-3p and miR-338-5p and their host gene Apoptosis-associated Tyrosine Kinase (AATK) during the maturation of rat hippocampal neurons. This revealed an uncorrelated expression pattern of mature miR-338 strands with their host gene. Sequence analysis of the 3′ untranslated region (UTR) of rat AATK mRNA revealed the presence of two putative binding sites for miR-338-3p. Thus, miR-338-3p may have the capacity to modulate AATK mRNA levels in neurons. Transfection of miR-338-3p mimics into rat B35 neuroblastoma cells resulted in a significant decrease of AATK mRNA levels, while the transfection of synthetic miR-338-5p mimics did not alter AATK levels. Our results point to a possible molecular mechanism by which miR-338-3p participates in the regulation of its host gene by modulating the levels of AATK mRNA, a kinase which plays a role during differentiation, apoptosis and possibly in neuronal degeneration

MiR-338 is encoded within the AATK gene and is expressed during maturation of hippocampal neurons.

<p>(A) A schematic overview of rat miR-338 encoded within the seventh intron (depicted in blue) of the AATK gene located on chromosome 11, with the exons shown in red. The depicted genes are <i>Rattus norvegicus</i> AATK (rno-AATK) and miR-338 (rno-miR-338). (B) qPCR assay was used to assess levels of pre-miR-338, mature miR-338-3p and miR-338-5p, and AATK mRNA in cultured rat hippocampal neurons (DIV 0–21). The data represents relative fold change in AATK and miR-338 expression levels to DIV 0.</p

MiR-338 targets AATK.

<p>(A) Pre-miR-338, and miR-338-3p levels in B35 cells (transfected with pmiR-338 or pmiR-null plasmids) were quantified following PCR. Pre-miR-338 levels are visualized on 4% agarose gels containing ethidium bromide using UV absorption (254 nm wavelength), and pre-miR-338 band intensities are expressed relative to U6 snRNA. Furthermore qRT-PCR assessment of miR-338-3p levels, expressed relative to U6 snRNA following pmiR-338 overexression versus the null condition. (B) Quantification of AATK mRNA levels in B35 cells transfected with pmiR-338 or pmiR-null vectors, as determined 72 hrs following transfection using qRT-PCR. (C) Relative firefly luciferase activity in B35 cells measured in light units. Cells were co-transfected with luciferase encoding the 3′UTR of rat AATK (indicated as 3′AATK) and either with the pmiR-null control vector, or the pmiR-338 overexpression vector. Luciferase activity was normalized to Renilla luciferase activity. Error bars represent the SEM for n = 3 independent experiments, * is <i>p</i><0.05 with pmiR-null vs. pmiR-338 (Student's t test).</p

An overview of <i>in silico</i> identified putative miR-338 target sites within the AATK 3′UTR.

<p>Three AATK genes are depicted namely <i>Homo sapiens</i> AATK (hsa-AATK), <i>Mus musculus</i> AATK (mmu-AATK) and <i>Rattus norvegicus</i> AATK (rno-AATK). The miR-338 seed sequence is indicated in red.</p

MicroRNA-326 acts as a molecular switch in the regulation of midbrain urocortin 1 expression

Contains fulltext : 167946.pdf (publisher's version ) (Closed access)BACKGROUND: Altered levels of urocortin 1 (Ucn1) in the centrally projecting Edinger-Westphal nucleus (EWcp) of depressed suicide attempters or completers mediate the brain's response to stress, while the mechanism regulating Ucn1 expression is unknown. We tested the hypothesis that microRNAs (miRNAs), which are vital fine-tuners of gene expression during the brain's response to stress, have the capacity to modulate Ucn1 expression. METHODS: Computational analysis revealed that the Ucn1 3' untranslated region contained a conserved binding site for miR-326. We examined miR-326 and Ucn1 levels in the EWcp of depressed suicide completers. In addition, we evaluated miR-326 and Ucn1 levels in the serum and the EWcp of a chronic variable mild stress (CVMS) rat model of behavioural despair and after recovery from CVMS, respectively. Gain and loss of miR-326 function experiments examined the regulation of Ucn1 by this miRNA in cultured midbrain neurons. RESULTS: We found reduced miR-326 levels concomitant with elevated Ucn1 levels in the EWcp of depressed suicide completers as well as in the EWcp of CVMS rats. In CVMS rats fully recovered from stress, both serum and EWcp miR-326 levels rebounded to nonstressed levels. While downregulation of miR-326 levels in primary midbrain neurons enhanced Ucn1 expression levels, miR-326 overexpression selectively reduced the levels of this neuropeptide. LIMITATIONS: This study lacked experiments showing that in vivo alteration of miR-326 levels alleviate depression-like behaviours. We show only correlative data for miR-325 and cocaine- and amphetamine-regulated transcript levels in the EWcp. CONCLUSION: We identified miR-326 dysregulation in depressed suicide completers and characterized this miRNA as an upstream regulator of the Ucn1 neuropeptide expression in midbrain neurons