Abstract
Inflammation is a host defense process against infection. Inflammatory mediators include cytokines of the interleukin-1 family, such as IL-1α and IL-1β. Unlike IL-1β, IL-1α carries an N-terminal nuclear localisation sequence (NLS) and is trafficked to the nucleus. The importance of IL-1α nuclear localisation is poorly understood. Here, we used CRISPR/Cas9 to make inactivating mutations to the NLS on the Il1a gene. A colony of NLS mutant mice was successfully generated with precise knock-in mutations to incapacitate NLS function. NLS mutant mice had no gross changes in immunophenotype or inflammatory response but, surprisingly, failed to express IL-1α. We deduced that, in making specific mutations in the Il1a gene, we also mutated a long-noncoding (lnc)RNA in the complementary strand which has cis-regulatory transcriptional control of the Il1a gene itself. The mutations generated in the Il1a gene also result in mutation of the lncRNA sequence and a predicted alteration of its secondary structure, potentially explaining a subsequent failure to function as a transcriptional activator of Il1a expression. Thus, lncRNA secondary structure may regulate IL-1α expression. Our results serve as a cautionary note that CRISPR –mediated genome editing without full knowledge of genomic context can result in unexpected, yet potentially informative observations.
Highlights
Inflammation is generally a protective host response to injury and infection
IL-1α and β initiate an inflammatory response by binding IL-1 receptor 1 (IL-1R1) which, following recruitment of IL-1R accessory protein, triggers signalling pathways leading to further pro-inflammatory gene transcription[2]
The nuclear localisation sequence (NLS) of IL-1α can be successfully mutated in vivo by CRISPR gene editing
Summary
Inflammation is initiated by detection of either ‘pathogen’ or ‘damage’-associated molecular patterns by pattern recognition receptors and subsequent secretion of pro-inflammatory cytokines which amplify the inflammatory response by recruiting immune cells to the site of injury/infection[1]. IL-1α and β initiate an inflammatory response by binding IL-1 receptor 1 (IL-1R1) which, following recruitment of IL-1R accessory protein, triggers signalling pathways leading to further pro-inflammatory gene transcription[2]. The mechanisms of IL-1α regulation are poorly understood Both IL-1α and IL-1β are synthesised as 31 kDa precursor proteins (pro-IL-1β and pro-IL-1α) in response to a pathogen or damage associated signals, and are secreted from cells via unconventional secretory mechanisms after processing to active 17 kDa forms[11]. IL-1α is transcriptionally regulated by a long, noncoding RNA (lncRNA) located on the antisense strand of the Il1a gene.
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