Characterization of protein machinery required for synthesis and function of spliced leader RNA in Trypanosoma brucei

Date of Completion

January 2009

Keywords

Health Sciences, Pathology|Biophysics, General

Degree

Ph.D.

Abstract

The phylogenetically early-diverged trypanosomatids are unicellular, flagellated eukaryotes and include the lethal human parasites Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. While each of the latter has developed unique parasite-host interactions, they share a unique gene expression mechanism which does not exist in their hosts: they employ polycistronic transcription of protein coding genes and a unique post-transcriptional step, termed spliced leader (SL) trans splicing, to mature all nuclear pre-mRNA. Therefore, a high supply of SL RNA for trans splicing is crucial for trypanosomatid gene expression and viability, and interference with SL RNA synthesis or function are promising anti-parasitic strategies. ^ Our previous studies showed that SL RNA gene transcription in T. brucei depends on the general transcription factor (GTF) TFIIB and a complex which included the GTF TFIIA. The discovery of these GTFs was surprising because GTFs were not found in annotations of completed trypanosomatid genomes and only orthologues of the multifunctional factors TRF4 and TFIIH were unambiguously identified. The new identifications, however, suggested that trypanosomatids do form a highly divergent transcription pre-initiation complex (PIC) which in other eukaryotic systems comprises six distinct GTFs. We therefore characterized the PIC in T. brucei and found that TFIIH and a novel TFIIH-associated factor (HATF) are essential for SL RNA gene transcription. Characterization of parasite TFIIH demonstrated that the composition of this multisubunit factor differs from that of its human counterpart. Moreover, we showed that HATF is a complex of at least nine subunits, essential for TFIIH recruitment into the PIC, and required for PIC stabilization in vivo. ^ RNA splicing is carried out by the spliceosome which is composed of five U-rich snRNAs and up to 170 proteins in humans. Although SL trans splicing is a parasite-specific way to utilize the spliceosome, only a few spliceosomal proteins were known in trypanosomatids. We have therefore tandem affinity-purified the T. brucei spliceosome and identified a set of 47 proteins which included nearly all of the known splicing factors as well as 21 un-annotated proteins. Our functional analysis revealed five new spliceosomal proteins and demonstrated that two of these are essential for both cis and trans splicing. ^

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