The polyadenylation complex of <i>Trypanosoma brucei:</i> Characterization of the functional poly(A) polymerase

The generation of mature mRNA in the protozoan parasite <i>Trypanosoma brucei</i> requires coupled polyadenylation and <i>trans</i> splicing. In contrast to other eukaryotes, we still know very little on components, mechanisms, and dynamics of the 3′ end-processing machinery in trypanosomes. To characterize the catalytic core of the polyadenylation complex in <i>T. brucei</i>, we first identified the poly(A) polymerase [Tb927.7.3780] as the major functional, nuclear-localized enzyme in trypanosomes. In contrast, another poly(A) polymerase, encoded by an intron-containing gene [Tb927.3.3160], localizes mainly in the cytoplasm and appears not to be functional in general 3′ end processing of mRNAs. Based on tandem-affinity purification with tagged CPSF160 and mass spectrometry, we identified ten associated components of the trypanosome polyadenylation complex, including homologues to all four CPSF subunits, Fip1, CstF50/64, and Symplekin, as well as two hypothetical proteins. RNAi-mediated knockdown revealed that most of these factors are essential for growth and required for both <i>in vivo</i> polyadenylation and <i>trans</i> splicing, arguing for a general coupling of these two mRNA-processing reactions.