Pre-mRNA processing complex in Trypanosoma brucei: structural assembly and function of core polyA factors

Pre mRNA 3 end processing is unique in trypanosomes as it involves polycistronic transcription, polyadenylation coupled to trans splicing and elusive polyadenylation signals. The mechanism is poorly understood. Here we reported that nine putative polyA factors from Trypanosoma brucei form a stable complex which then subsequently develops into two tight subcomplexes. One contains the proteins tbCPSF160, tbCPSF30, tbFip1 and tbWdr33 with tbCPSF160 as the scaffold; while the other contains tbCPSF100, tbCPSF73, tbSYMK and two trypanosome unique proteins, designated CPF30 and CPF15. No CstF homologs were found. The protein tbCPSF100 seems to coordinate the association of these two subcomplexes by interacting with tbCPSF160. RNAi knockdown of these polyA factors resulted in RNA processing disorders and variable disruption of the complex and RNA processing disorders. Depletion of either factors caused dicistronic transcripts formation; depletion of either tbCPSF30, tbFip1 or tbWdr33 caused increased usage of proximal polyadenylation sites of the transcripts; depletion of tbCPSF30 caused mRNA polyA tail shortening. This indicates that these two polysA factors have a critical role in polyadenylation site selection. Genome-wide UV crosslinking assays showed that tbCPSF30 and tbFip1 have strong RNA-binding activity in this complex. Altogether, these data indicate that the 3 end processing complex of T. brucei is more like the human CPSF homolog. However, it displays trypanosome specific features in composition, structural assembly and function of components, which facilitate a common, yet unique, cleavage polyadenylation reaction. CPSF30 were knockdown or not, poly(A) enriched RNA were extracted, direct RNA sequencing were performed (Minion, sequencing kit SQK-RNA002), poly(A) tail length were evaculated with tailfindr software. Direct sequencing of poly(A) RNA from cells of TRRM2 knockdown or not were used as control.