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