Regulation of mRNA availability and translation is critical in neuronal differentiation; moreover, in mature neurons the response to activity relies on tight temporospatial availability of appropriate transcripts. We hypothesise that as regulators of mRNA availability, miRNA will be functionally compartmentalised in neurons. Although miRNA function is typically associated with the cytoplasm, a growing body of evidence supports the activity of mature miRNA in the nucleus in a variety of roles beyond canonical gene silencing.
To investigate miRNA expression and localisation in differentiating neuronal cell cultures, nuclear fractions were prepared by hypo-osmotic lysis and differential centrifugation of SH-SY5Y human neuroblastoma differentiated with retinoic acid and matured with BDNF. Gene and miRNA expression was analysed by microarray and transcripts of interest validated by qRT-PCR; to investigate the impact of normalisation on data derived from divergent compartments, 3 normalisation methods were compared by validation of putative nuclear-enriched molecules derived from each method, with un-normalised data demonstrating superior consistency in this context. Compartmental miRNA and mRNA expression were paired in IPA software to identify and characterise putative regulatory networks.
We found mature miRNAs in the nucleus of SH-SY5Y, including 35 demonstrating preferential nuclear localisation whose nuclear component comprised >50% of total expression, while overall nuclear composition was significantly altered by neuronal differentiation. Excitingly, analysis of nucleus-enriched and –depleted miRNA identified a 4-nucleotide motif significantly associated with nuclear enrichment. Integration of miRNA and mRNA datasets found a putative network with considerable co-regulation significantly enriched for terms associated with mRNA availability, including “nucleic acid metabolism” (p=0.01) and “RNA processing” (p=0.002).
These findings support a role for miRNA in the nucleus in the gene-silencing context, and further elaboration of this mechanism is being investigated by small RNAseq analysis of differential Argonaute-associated transcripts via co-immunoprecipitation of Ago1 and Ago2 from these cells.