Dissecting the mechanistic connection between inositol pyrophosphate signaling and mRNA polyadenylation in different eukaryotes.

Inositol pyrophoshates (PP-InsPs) are fully phosphorylated myo-inositols carrying at least one diphosphate group. In recent years these enigmatic signalling molecules have been implicated in regulating growth and development, cellular bioenergetics and tumor progression in animals. Very little is known about the molecular functions of PP-InsPs in other eukaryotes. PP-InsPs may signal by promoting or blocking protein-protein interaction sites, and/or inducing conformational changes in their protein targets. The detailed molecular mechanisms and the full spectrum of PP-InsP-mediated signalling pathways are presently unknown.

The Hothorn lab at UNIGE previously discovered that SPX domains act as conserved high-affinity sensors for PP-InsP in all eukaryotes (Wild et al., Science, 2016). To identify the PP-InsP interactome in plants and to explore PP-InsP-governed signalling pathways, the Hothorn lab has recently performed a systems-scale proteomics study, based on a non-hydrolysable and matrix-adsorbed PP-InsP analogue. This yielded a comprehensive data-set of the Arabidopsis PP-InsP-interacting proteins, comprising more that 600 candidates. Remarkably, a large fraction of novel PP-InsP-interactors identified in this screen include components of the highly conserved mRNA 3’ end polyadenylation machinery, which is composed of four multiprotein complexes.

3’ terminal polyadenylation of mRNAs occurs following transcription termination on specific sites that are determined within the transcript sequence. However, alternative polyadenlyation (APA) may occur in response to external stimuli, or in a cell/tissue-specific manner, making it a major mechanism for regulation of gene expression. Recent work in the Jinek lab, UZH (Clerici et al., eLife 2017; Nat Struct Mol Biol 2018) has provided detailed structural insights into the molecular architecture and mechanism of transcript recognition by the core human polyadenylation complex CPSF. The exact mechanisms that determine the choice of (alternative) polyadenylation sites are not well understood, but it has been hypothesised that differences within the composition of the polyadenylation machinery, or recruitment of additional factors, may play a critical role.

To date, no connection between PP-InPs and mRNA polyadenylation has been reported. Our finding that polyadenylation factors are PP-InsP-interacting proteins opens the door to novel regulatory mechanisms mediated by PP-InsP that could influence complex formation, RNA recruitment and/or enzymatic activities. We now propose a joint effort of the Hothorn and Jinek labs to: (i) confirm and describe in detail the interactions between PP-InsPs and the polyadenylation machinery in vitro; (ii) investigate how PP-InsPs influence the assembly of the polyadenlation machinery; (iii) test whether PP-InsPs have a direct regulatory role over polyadenylation using in vitro polyadenylation assays; and finally (iv) investigate the biological meaning of our findings. We envision that the powerful combination of structural biology and biochemistry with plant genetics will enable us to define a novel signalling paradigm connecting cell nutrition with mRNA processing in different higher organisms.

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