Type 1 interferons (IFNs) are cytokines with critical roles in innate immunity that can act on a large number of cell types. They induce changes on different cellular levels, such as transcriptional, translational and metabolic. This study began with our discovery of a novel aspect of global post-transcriptional changes, shortening of 3’ untranslated regions (UTRs) of mature mRNAs.
An increase of transcripts with shorter 3’-UTRs was detected in IFNb-treated human and murine macrophages using Poly-A-Tail-sequencing. Genes expressed with shorter 3’-UTRs are not typical IFN-regulated genes (IRGs) and are not differentially expressed on gene level. They make up a new class of IRGs regulated through alternative polyadenylation rather than changes to overall expression.Initial investigations using a NanoString array have shown that 3’-UTR shortening is mediated by IRGs. A flow cytometry-based RNA detection method was adapted to measure 3’-UTR isoforms and used as a read-out for a genome-wide CRISPR/Cas9 knock out screen. This allowed further identification of IRGs and other components, such as RNA processing factors, as putative mediators of IFNb-induced 3’-UTR shortening.
A change in 3’-UTR length generally doesn’t alter the protein coding sequence of a transcript or affect the abundance of the corresponding protein. Instead, recent studies have described a scaffold-like role for 3’-UTRs. They can facilitate the formation of different protein complexes depending on 3’-UTR length, which can affect the encoded proteins localization and function. Here, sets of overexpression constructs for selected candidates were used for a co-IP followed by mass spectrometry to identify 3’-UTR length-dependent protein-protein interactors. Network and ontology analyses of the identified interactors suggest differences in function and localization of candidate RNA and protein derived from different 3’-UTR isoforms, independent of protein coding sequence. This discovery expands our current understanding of IRGs and their functions and highlights the importance of including non-coding sequences in future research.
This study shows that global shortening of 3’-UTRs is a novel aspect of macrophage activation by IFNb. Genes regulated through alternative polyadenylation make up a new class of IRG that may have diverse, 3’-UTR length-dependent functions. Further investigations will show how the expression of distinct isoforms shapes innate immune responses.