Characterisation and regulation of the alternative cap binding complex consisting of NCBP1 and -3

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The flow of genomic information from DNA to protein requires polymerase-II transcripts, which are characterized by the presence of a 5 primes cap structure. The cap-binding complex (CBC), consisting of the nuclear cap binding protein (NCBP) 2 and its adaptor NCBP1, is believed to bind all capped RNA and to be necessary for processing and intracellular localization. However, NCBP1 but not NCBP2 is required for cell viability and mRNA export suggesting the existence of additional factors involved in mRNA biology. We recently identified an alternative cap-binding complex consisting of the highly conserved protein C17orf85 (now officially named NCBP3 for Nuclear cap-binding protein 3) and NCBP1. Only simultaneous depletion of NCBP2 and -3 diminishes association of NCBP1 to cap-RNA, impairs cell growth and nuclear mRNA export. mRNA binds either NCBP2 or -3 whereas small RNA species such as snRNA preferentially associates to NCBP2. Thus, besides the canonical CBC consisting of NCBP1/2, an alternative CBC consisting of NCBP1/3 is operating in most eukaryotic cells. It is currently unclear why our organism evolved two CBCs that under steady state conditions share large parts of their activity. Intriguingly, under cellular stress conditions such as virus infection NCBP3 becomes pivotal to regulate a proper antiviral immune response. This suggests that mRNA maturation and translocation may be differentially regulated depending on environmental stimuli. Here, we aim to functionally characterize the alternative CBC under steady state and stimulated conditions. Therefore, the interactions between NCBP1 and -3 as well as association of RNA processing factors to the NCBP1/3 complex should be studied in detail. A further aim is to study why mRNA but not other types of capped RNAs (e.g. snRNA, lincRNA,...) are associated with the alternative CBC. Functionally, the alternative CBC appears to be required during the antiviral immune response suggesting influence of innate pathogen sensing on the mRNA export pathway. In preliminary experiments we could identify unique phosphorylation sites on NCBP1 and -3 that are dynamically changing during virus infection. The function of these phosphorylation sites will be evaluated in the context of the antiviral immune response. A recently developed NCBP3 deficient mouse will be of critical help to gain functional insights on an organismal level. In sum this proposal aims to gain further insights in a fundamental cellular process, namely the regulation of information transfer from DNA to protein. Preliminary data let us hypothesize that this process is controlled through integrating signals elicited by environmental stimuli.