The pre-mRNA splicing machinery of trypanosomes: complex or simplified?

Abstract

Trypanosomatids are early-diverged, protistan parasites of which Trypanosoma brucei, Trypanosoma cruzi, and several species of Leishmania cause severe, often lethal diseases in humans. To better combat these parasites, their molecular biology has been a research focus for more than 3 decades, and the discovery of spliced leader (SL) trans splicing in T. brucei established a key difference between parasites and hosts. In SL trans splicing, the capped 5'-terminal region of the small nuclear SL RNA is fused onto the 5' end of each mRNA. This process, in conjunction with polyadenylation, generates individual mRNAs from polycistronic precursors and creates functional mRNA by providing the cap structure. The reaction is a two-step transesterification process analogous to intron removal by cis splicing which, in trypanosomatids, is confined to very few pre-mRNAs. Both types of pre-mRNA splicing are carried out by the spliceosome, consisting of five U-rich small nuclear RNAs (U snRNAs) and, in humans, up to approximately 170 different proteins. While trypanosomatids possess a full set of spliceosomal U snRNAs, only a few splicing factors were identified by standard genome annotation because trypanosomatid amino acid sequences are among the most divergent in the eukaryotic kingdom. This review focuses on recent progress made in the characterization of the splicing factor repertoire in T. brucei, achieved by tandem affinity purification of splicing complexes, by systematic analysis of proteins containing RNA recognition motifs, and by mining the genome database. In addition, recent findings about functional differences between trypanosome and human pre-mRNA splicing factors are discussed.

Fig. 1.

Schematic of the mammalian cis splicing and the trypanosome SL trans splicing reactions. Upstream exon and spliced leader are drawn as gray rectangles, and downstream exon and trypanosome gene are drawn as black rectangles. 5′ and 3′ splice sites (SSs) are represented by small open boxes, branch points (BPs) by closed circles, polypyrimidine tracts by small striped boxes, and the cap 4 structure of the spliced leader as an oval. Conserved sequences are provided below the drawing with invariant residues underlined. While in mammalian systems, 5′SSs, BPs, and 3′SSs exhibit partly conserved sequences (R, purine; Y, pyrimidine; N, any base), there is no obvious sequence conservation at trypanosome BPs (43) and 3′SSs, although it was shown for the latter that an AC dinucleotide (*) preceding the AG residues drastically reduces splicing efficiency unless a compensatory AG dinucleotide is present within the 5′ untranslated region (76). It appears that the importance of the polypyrimidine tract becomes more important when consensus sequences are lacking. Yeast has highly conserved splice site and BP sequences, and some yeast introns function without a polypyrimidine tract (not shown). The partly conserved sequences in mammals require a small polypyrimidine tract in the range of 10 to 12 residues (Y_10-12), whereas in trypanosomes, the polypyrimidine tract is large (Y_∼20), is an essential sequence determinant for efficient splicing, and typically starts just downstream of the BP (43, 76). After the first transesterification reaction, cis splicing results in a lariat intron structure, whereas a Y structure intermediate is formed in the SL trans splicing process. After the second transesterification, these intronic structures are debranched (not shown) and rapidly degraded.

Fig. 2.

Comparison of known spliceosomal factors of humans and trypanosomes. Schematic drawing of spliceosomal complexes during a splicing reaction as described in the mammalian and yeast systems. For each complex, proteins are listed that enter the spliceosome at the outlined stage (slightly modified human protein repertoire is according to reference 91). Please note that only incoming proteins are listed and proteins leaving the spliceosome in the transitions are not recognized. Bold blue lettering indicates proteins for which orthologs have been found in trypanosomes, whereas red lettering specifies trypanosome-specific factors. ¹, Highly divergent, putative cyclophilin orthologs have been copurified with SmD1 and SmB1 (Table 1); ², U5-100K is a DExD/H-box helicase, and it is unclear whether one of the putative trypanosome DExD/H-box helicases (Table 1) represents a U5-100K ortholog; ³, U5-Cwc21 is possibly the ortholog of human SRM300 but seems to have a trypanosome-specific function (see text); ⁴, the trypanosome exon junction complex has recently been characterized (6), but its specific function in RNA splicing or metabolism remains unclear.