Characterization of the nuclear cap-binding complex in Trypanosoma brucei: Novel and essential subunits play a role in pre-mRNA splicing

African trypanosomes are protozoan parasites, which are the etiological agents of devastating diseases both in man and in cattle. In the last years, trypanosomatid protozoa have attained the status of model systems for unicellular pathogens, as underscored by the ongoing genome sequencing projects for a number of trypanosomatids, including Trypanosoma brucei. Furthermore, the study of RNA metabolism in these organisms has been instrumental in the discovery of new concepts in eukaryotic RNA biology, like trans-splicing and mitochondrial RNA editing.; In T. brucei protein coding genes are transcribed as polycistronic pre-mRNAs, which are then processed by trans-splicing and cleavage/polyadenylation into monocistronic mature mRNAs. Trans-splicing transfers the 39-nt long spliced leader (SL), including the cap, from the SL RNA to the 5' end of every mRNA. One distinctive characteristics of this process is that the involved cap is uniquely modified (m7 Gpppm62 AmpAmpCmpm3Um, termed "cap 4"). A properly modified cap 4 structure is essential for trans-splicing and hence, for mRNA biogenesis. However, it remains to be determined how this hypermodified cap functions in trans-splicing.; The focus of my dissertation research was to investigate the link between the cap 4 structure and trans-splicing. I describe the characterization of an unconventional nuclear RNA cap binding complex (CBC) in T. brucei with an apparent molecular weight of 300 kDa. In addition to two bona fide CBC components, namely CBP20 and importin-alpha, three novel proteins (TbCBP30, TbCBP66 and TbCBP110) have been identified as subunits of this complex. Interestingly, these novel proteins are only present in organisms featuring a cap 4 structure and trans-splicing. Downregulation by RNA interference of several subunits of the T. brucei CBC demonstrated an essential function in an early step of trans-splicing. Furthermore, competitive binding studies revealed that the T. brucei CBC binds with a 15-fold higher affinity to cap 4-containing RNA than to m7G-capped RNA. Taken together, the CBC offers an attractive link between the cap 4 structure and trans-splicing. My results lay the foundation for future functional and structural studies intended to understand how the CBC interacts with the cap 4 structure, as well as the trans-splicing machinery.