The innate immune response of an organism is the primary response to challenges including inflammatory stimuli, infectious agents or the presence of cancer cells. The important protective role of IFN activation and signalling is a key component of this response. While IFN signalling is essential for host survival, fundamental to vaccine responses, can be activated therapeutically and is often the target for pathogen evasion strategies uncontrolled or aberrant responses can be toxic and result in disease. It is therefore critical to balance signaling pathways to attain benefit while minimizing toxicity. While there is considerable knowledge of transcriptional regulation and role of protein coding transcripts in the IFN response equivalent knowledge of non-coding responses and their regulatory mechanism remains an active research area. In this context, an understanding of the role of small non-coding RNA, particularly miRNA, is rapidly being gained with important insights in many areas of biology.
High throughput sequencing technologies have unlocked the ability to measure the complete transcriptional landscape of a cell ultimately revolutionizing our ability to understand these complex systems. Combined with HITS-CLIP sequencing, a recently developed experimental sequencing based approach capable of detecting microRNA regulatory interaction with target genes can provide important regulatory insights into this system. We present the results of parallel IFNβ time course stimulation of small, ribosomal depleted total RNA and HITS-CLIP sequencing complemented with miRNA expression and target network identification and experimental validation in murine bone marrow derived macrophages. Analysis of this data has resulted in comprehensive network level understanding of the complete transcriptional response to IFNβ including the identification of almost 100, IFNβ inducible putative novel miRNA and associated characterization of potential targets. Novel computational algorithms and analysis approaches within our custom designed RNA-eXpress framework have been developed and applied resulting in the generation of predictive target networks. Results of experimental validation of these related miRNA pathways will also be presented.
This systems level interpretation of IFN mediated signaling has the potential to provide a basis for novel insights into both host-pathogen interactions and host response critical in the efficient development of effectively targeted therapeutics.