Identification and characterization of nuclear non-canonical poly(A) polymerases from Trypanosoma brucei.

Regulation of nuclear genome expression in Trypanosoma brucei is critical for this protozoan parasite's successful transition between its vertebrate and invertebrate host environments. The canonical eukaryotic circuits such as modulation of transcription initiation, mRNA splicing and polyadenylation appear to be nearly non-existent in T. brucei suggesting that the transcriptome is primarily defined by mRNA turnover. Our previous work has highlighted sequence similarities between terminal RNA uridylyl transferases (TUTases) and non-canonical poly(A) polymerases, which are widely implicated in regulating nuclear, cytoplasmic and organellar RNA decay throughout the eukaryotic lineage. Here, we have continued characterization of TUTase-like proteins in T. brucei and identified two nuclear non-canonical poly(A) polymerases (ncPAPs). The 82kDa TbncPAP1 is essential for viability of procyclic and bloodstream forms of T. brucei. Similar to Trf4/5 proteins from budding yeast, TbncPAP1 requires protein cofactor(s) to exert poly(A) polymerase activity in vitro. The recombinant 54kDa TbncPAP2 showed a PAP activity as an individual polypeptide. Proteomic analysis of the TbncPAP1 interactions demonstrated its association with Mtr4 RNA helicase and several RNA binding proteins, including a potential ortholog of Air1p/2p proteins, which indicates the presence of a stable TRAMP-like complex in trypanosomes. Our findings suggest that TbncPAP1 may be a "quality control" nuclear PAP involved in targeting aberrant or anti-sense transcripts for degradation by the 3'-exosome. Such mechanisms are likely to play a major role in alleviating promiscuity of the transcriptional machinery.