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

Abstract

ABSTRACT One of the unique aspects of RNA processing in trypanosomatid protozoa is the presence of a cap 4 structure (m 7 \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathbf{Gpppm}_{2}^{6}\) \end{document} AmpAmpCmpm 3 Um) at the 5′ end of all mRNAs. The cap 4 becomes part of the mRNA through trans -splicing of a 39-nucleotide-long sequence donated by the spliced leader RNA. Although the cap 4 modifications are required for trans -splicing to occur, the underlying mechanism remains to be determined. We now describe an unconventional nuclear cap binding complex (CBC) in Trypanosoma brucei with an apparent molecular mass of 300 kDa and consisting of five protein components: the known CBC subunits CBP20 and importin-α and three novel proteins that are only present in organisms featuring a cap 4 structure and trans -splicing. Competitive binding studies are consistent with a specific interaction between the CBC and the cap 4 structure. Downregulation of several individual components of the T. brucei CBC by RNA interference demonstrated an essential function at an early step in trans -splicing. Thus, our studies are consistent with the CBC providing a mechanistic link between cap 4 modifications and trans -splicing.