Novel and Essential Subunits in the 300-Kilodalton Nuclear Cap Binding Complex of Trypanosoma brucei

Flagellate protozoa of the family Trypanosomatidae include a large number of diverse organisms, many of which are parasites of great economical and medical importance. The study of the parasitic life style of these organisms has resulted in the original discovery of phenomena of fundamental biological significance, including among others antigenic variation of surface glycoproteins (5), glycosylphosphatidylinositol anchors of membrane proteins (11), and mitochondrial RNA editing (35). The examination of antigenic variation at the gene expression level revealed yet another complexity of eukaryotic biology, namely, polycistronic transcription and trans-splicing of precursor RNAs (20, 30, 43). In particular, transcription of protein coding genes by RNA polymerase II (Pol II) generates polycistronic pre-mRNAs, which are then processed by the coupled action of trans-splicing and polyadenylation to yield monocistronic mature mRNAs (24). trans-splicing transfers the 39-nucleotide (nt)-long capped spliced leader (SL) from the SL RNA to the 5′ end of mRNAs, and this process is mechanistically similar to the removal of intervening sequences (24). At present, there is no evidence for regulation of gene expression at the transcriptional level and, thus, it is very likely that posttranscriptional events play a major role in fine-tuning the output of gene products (7). In this scenario, pre-mRNA and SL RNA cis-acting signals for RNA processing, by virtue of their interaction(s) with components of RNA processing machineries, could be major determinants for regulating gene expression in trypanosomes. Studies performed mainly in yeast and human cells have shown that the m7GpppN cap structure of nascent Pol II transcripts has key functions in various aspects of RNA processing and that these effects are mediated by two distinct cap binding complexes (CBCs) (17, 22, 39). The predominantly nuclear CBC is comprised of two subunits, termed cap binding proteins 20 (CBP20) (16) and 80 (CBP80) (17, 31). CBP20, in contrast to CBP80, is highly conserved from yeast to humans and contains an RNA binding motif (the RNP domain). Of note is that binding to capped RNA requires an association of the two subunits, with CBP20 directly contacting the cap and CBP80 ensuring high-affinity cap binding (6, 17, 28). Although the CBC subunits are not essential for viability in Saccharomyces cerevisiae (8), they play an active role in both splicing and RNA export (17, 19). CBC augments pre-mRNA processing by increasing the splicing efficiency of cap-proximal 5′ introns. Furthermore, CBC positively affects the efficiency of 3′-end formation (12). In metazoa, where U-snRNP assembly has a cytoplasmic phase, CBC, in cooperation with an adaptor protein named PHAX (for phosphorylated adaptor for RNA export), is required for the nuclear export of U-snRNAs (32). In contrast, export of mRNA does not appear to require CBC, although it accompanies mRNA to the cytoplasm (15, 22, 47), where it is exchanged for eukaryotic initiation factor 4E, the cytoplasmic cap binding protein of eukaryotic translation initiation factor 4F (13). The mRNA cap in T. brucei has the unusual feature of containing, in addition to 7-methylguanosine, four modified nucleotides making it by definition a cap 4 structure (m7AmpAmpCmpm3Um) (3). Whereas this highly modified cap appears to be conserved throughout the evolution of the family Trypanosomatidae (34), there is at present no evidence that a similar cap structure exists outside this group of organisms. Although permeable cell studies showed that the hypermodified cap is essential for utilization of the SL RNA in trans-splicing (46) and genetic analysis in Leptomonas collosoma established that each of the four nucleotides of the cap 4 is essential for SL RNA function in trans-splicing (27), a specific role for the modified nucleotides has not been elucidated. Nevertheless, these observations reinforce the notion that cap structures have key functions in RNA metabolism. In this paper we have characterized the T. brucei nuclear CBC, which consists of five subunits and is essential for cell viability.