Non-coding RNA (ncRNA)-directed regulation of epigenetic processes has recently emerged as an important feature of mammalian differentiation and development. Long non-coding RNAs (lncRNAs) are non-protein coding transcripts longer than 200 nucleotides RNAs. Although they are highly expressed within the mammalian brain, their function in behaviour remains equivocal. Deregulation of the lncRNA regulatory systems in the brain may contribute to the development of neuropsychiatric disorders.
This project aimed to identify the role of lncRNAs in association with complex mammalian behaviours.
In this thesis, sequencing technologies were used to identify ncRNAs that are dynamically regulated in fear-related learning in mice. These technologies included nuclear enriched RNA-capture sequencing, whole-genome RNA sequencing (RNA-Seq) and small RNA-Seq. The use of complementary sequencing approaches was expected to cover the expression of all possible sizes of ncRNAs. RNA-capture sequencing was used to identify newly described ncRNAs transcripts within the brain-derived neurotrophic factor (Bdnf) locus that were modulated in association with fear conditioning. Whole-genome RNA-Seq analysis showed that several lncRNAs (such as Neat1, Malat1, Mirg, Rmst and Gomafu) were dynamically regulated in response to fear learning. This transcriptome profiling revealed that most of the transcribed lncRNAs were proximal to coding genes, which suggested in cis regulatory activity of these transcripts. Knock down of the antisense lncRNA to Cacng2 altered the level of Cacng2 mRNA expression, which indicated the potential role of lncRNAs in regulating proximal coding genes in response to neural activation and behaviour.
To establish the relevance of lncRNAs in association with behaviour, this investigation focused on Gomafu, which has previously been linked to schizophrenia, drug addiction and brain development. To determine whether Gomafu affects behaviour, chimeric antisense oligonucleotides (ASOs) were designed to knock down this lncRNA in vivo. Infusion of ASO in the mouse mPFC did not affect fear-associated long-term memory but instead appeared to modulate the level of anxiety.
The molecular mechanisms by which Gomafu exerts its function in the development of anxiety-related behaviours were explored further. A possible in cis regulatory function of Gomafu within the schizophrenia locus was investigated by examining the level of expression of proximal genes after Gomafu knockdown. Gomafu knockdown resulted in the up-regulation of Crybb1, gene that is antisense to the Gomafu lncRNA. Previous investigations have suggested that lncRNAs regulate the expression of coding genes in association with Polycomb group repressive complexes (PRCs). Immunoprecipitation assays were performed to investigate the molecular interplay between Gomafu and Crybb1 through PRCs. Gomafu recruited PRC1 to the Crybb1 promoter, which regulated Crybb1 expression levels in response to neuronal stimulation. Experimental knock down of Crybb1 also exposed that its expression plays a role in anxiety. Gomafu knockdown increased anxiety in mice, whereas Crybb1 knockdown consistently attenuated this response. These results suggest that lncRNAs control the expression of proximal genes by recruiting the PRC, and that such epigenetic mechanisms may be associated with the development of fear-induced anxiety.
Lastly, small RNA-Seq analysis of fear-conditioned mice showed the dynamic regulation of micro-RNAs (miRNAs) and small nucleolar RNAs (snoRNAs), which are transcribed from regions associated with lncRNAs such as Gas5, Taf1d, Ipw, Rian, Snhgs and Mirg. The dynamic expression of Piwi-interacting small non-coding RNAs (piRNAs), including piRNA-30, a well-characterised piRNA in the mouse brain, was also detected. A copy variant of the known orphan snoRNAs, snord115, was also found, which suggested that snoRNAs are involved in fear learning, possibly by targeting coding genes and lncRNAs beyond the expected ribosomal genomic fractions.
Overall the results of this thesis endorse the concept that lncRNAs are dynamically regulated in response to neuronal activation and fear learning within the mammalian brain. As proof of principle, this investigation demonstrated that Gomafu expression is necessary for the modulation of anxiety-related behaviours. This thesis also found evidence of the involvement of sncRNAs, transcribed from lncRNAs loci, in altering adaptive behaviours. These results support the idea that non-coding transcription is involved in the development of psychiatric illness.