title: Functional genomic analysis and neuroanatomical localization of miR-2954, a song-responsive sex-linked microRNA in the zebra finch.
abstract: Natural experience can cause complex changes in gene expression in brain centers for cognition and perception, but the mechanisms that link perceptual experience and neurogenomic regulation are not understood. MicroRNAs (miRNAs or miRs) have the potential to regulate large gene expression networks, and a previous study showed that a natural perceptual stimulus (hearing the sound of birdsong in zebra finches) triggers rapid changes in expression of several miRs in the auditory forebrain. Here we evaluate the functional potential of one of these, miR-2954, which has been found so far only in birds and is encoded on the Z sex chromosome. Using fluorescence in situ hybridization and immunohistochemistry, we show that miR-2954 is present in subsets of cells in the sexually dimorphic brain regions involved in song production and perception, with notable enrichment in cell nuclei. We then probe its regulatory function by inhibiting its expression in a zebra finch cell line (G266) and measuring effects on endogenous gene expression using Illumina RNA sequencing (RNA-seq). Approximately 1000 different mRNAs change in expression by 1.5-fold or more (adjusted p < 0.01), with increases in some but not all of the targets that had been predicted by Targetscan. The population of RNAs that increase after miR-2954 inhibition is notably enriched for ones involved in the MAP Kinase (MAPK) pathway, whereas the decreasing population is dominated by genes involved in ribosomes and mitochondrial function. Since song stimulation itself triggers a decrease in miR-2954 expression followed by a delayed decrease in genes encoding ribosomal and mitochondrial functions, we suggest that miR-2954 may mediate some of the neurogenomic effects of song habituation.
authors.affiliation: Genomics of Neural and Behavioral Plasticity Theme, Institute for Genomic Biology, University of Illinois Urbana-Champaign, IL, USA ; Department of Cell and Developmental Biology, University of Illinois Urbana-Champaign, IL, USA.
authors.forename: Ya-Chi
authors.initials: YC
authors.lastname: Lin
authors.affiliation: Department of Biology and Center for Biodiversity, East Carolina University Greenville, NC, USA.
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Balakrishnan
authors.affiliation: Genomics of Neural and Behavioral Plasticity Theme, Institute for Genomic Biology, University of Illinois Urbana-Champaign, IL, USA ; Department of Cell and Developmental Biology, University of Illinois Urbana-Champaign, IL, USA ; Department of Biological and Experimental Psychology, School of Biological and Chemical Sciences, Queen Mary University of London London, UK.
authors.forename: David F
authors.initials: DF
authors.lastname: Clayton
keywords: In situ hybridization; MAPK and ERK signaling; NR4A3; RNAseq analysis; cell line; fluorescence; microRNA; zebra finch