Defining a function for the archaeal CPSF protein

In eukaryotes the cleavage/polyadenylation specific factor (CPSF) is a multisubunit complex which together with the cleavage stimulation factor (CstF) carries out the essential mRNA 3´ end processing coupled to polyadenylation and histone mRNA 3´ processing. The CPSF subunit CPSF-73 has been identified as the endonuclease responsible for mRNA 3´end cleavage. CPSF-73 belongs to the familiy of metallo-b-lactamases like tRNase Z and RNase J.

Interestingly, homologues to the eukaryal CPSF proteins are present in bacteria and archaea. We identified the CPSF-73 homolog in the halophilic archaeon, Haloferax volcanii. To investigate the function of that protein we employ in vitro and in vivo methods.

Expression of the cpsf gene in E. coli resulted in a recombinant protein, which cleaves the CPSF SVL40 substrate. Affinity purification of a tagged CPSF protein was used to identify protein and RNA interaction partners, which are currently further investigated.

Several approaches to delete the cpsf gene in H. volcanii have not been successful; hence we assume that the gene is essential. We therefore replaced the chromosomal cpsf gene promoter with a regulable promoter enabeling us to shut down cpsf gene expression. Phenotypical analysis and investigation of differences on the transcriptome and proteome level will lead to further insights into the role of CPSF in the metabolism of H. volcanii.