the pre-mrna splicing machinery of trypanosomes: complex ... · cis splicing which, in...

EUKARYOTIC CELL, Aug. 2010, p. 1159–1170 Vol. 9, No. 81535-9778/10/$12.00 doi:10.1128/EC.00113-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The Pre-mRNA Splicing Machinery of Trypanosomes:Complex or Simplified?�

Arthur Günzl*Department of Genetics and Developmental Biology and Department of Molecular, Microbial and Structural Biology, University of

Connecticut Health Center, 263 Farmington Avenue, Farmington, Connecticut 06030-3301

Trypanosomatids are early-diverged, protistan parasites of which Trypanosoma brucei, Trypanosoma cruzi, andseveral species of Leishmania cause severe, often lethal diseases in humans. To better combat these parasites, theirmolecular biology has been a research focus for more than 3 decades, and the discovery of spliced leader (SL) transsplicing in T. brucei established a key difference between parasites and hosts. In SL trans splicing, the capped5�-terminal region of the small nuclear SL RNA is fused onto the 5� end of each mRNA. This process, in conjunctionwith polyadenylation, generates individual mRNAs from polycistronic precursors and creates functional mRNA byproviding the cap structure. The reaction is a two-step transesterification process analogous to intron removal bycis splicing which, in trypanosomatids, is confined to very few pre-mRNAs. Both types of pre-mRNA splicing arecarried out by the spliceosome, consisting of five U-rich small nuclear RNAs (U snRNAs) and, in humans, up to�170 different proteins. While trypanosomatids possess a full set of spliceosomal U snRNAs, only a few splicingfactors were identified by standard genome annotation because trypanosomatid amino acid sequences are amongthe most divergent in the eukaryotic kingdom. This review focuses on recent progress made in the characterizationof the splicing factor repertoire in T. brucei, achieved by tandem affinity purification of splicing complexes, bysystematic 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 arediscussed.

Trypanosomatids are protistan parasites infecting hosts asdiverse as mammals, insects, and plants. In humans, vector-borne Trypanosoma brucei, Trypanosoma cruzi, and Leishmaniaspp. cause lethal diseases, and the strong impact of theseparasites on global health has spurred investigations of themolecular processes in these organisms from early on. Oneof the first key discoveries in regard to gene expression wasspliced leader (SL) trans splicing, which was eventually foundto be an essential maturation step for all nuclear pre-mRNA intrypanosomatids.

The initial discoveries of SL trans splicing were made in T.brucei, and until now, this organism has remained the pre-ferred trypanosomatid organism for spliceosomal studies. T.brucei is an extracellular parasite which evades the mammalianimmune system by antigenic variation of its variant surfaceglycoprotein (VSG) coat. VSG expression has therefore been aresearch focus, and it was on VSG mRNAs that the 5�-terminalregion was first discovered to contain a leader sequence whichwas not encoded in the VSG gene (10, 87). Further analysisshowed that the 39-nucleotide (nt)-long leader was derived fromthe 5� terminus of a separate, small nuclear RNA, which has beentermed SL RNA or miniexon-derived RNA (12, 34, 56). Thediscovery of a Y structure intermediate which corresponds tothe cis splicing intron-exon-lariat structure (Fig. 1) stronglyindicated that the SL transfer functions analogously to intronremoval, entailing the same two transesterification reactions(58, 79). This notion was confirmed by the demonstration that

the destruction of spliceosomal uridine-rich small nuclearRNAs (U snRNAs) blocked SL transfer (84).

SL trans splicing is not restricted to VSG mRNA but is anessential maturation step for all trypanosomatid mRNAs. Intrypanosomatid genomes, coding genes are tandemly arrangedin large polygenic clusters which are transcribed in a polycis-tronic fashion (7). Trans splicing and polyadenylation lead toprecursor cleavages up- and downstream of a coding region,respectively, and therefore, are mechanistically required to pro-cess individual mRNAs from polycistronic pre-mRNA. More-over, the SL carries a 7-methylguanosine (m7G) cap and thefirst four nucleotides of its sequence are methylated; some ofthese methylations are unique to trypanosomes, and the unusual5�-terminal structure has been termed cap 4 (4). Since cap 4 istransferred onto mRNA 5� ends as part of the SL, trans splicingrepresents a posttranscriptional mode of mRNA capping and,therefore, is essential in the formation of functional mRNA.

Since all trypanosomatid mRNAs are trans spliced and try-panosomatid genes typically do not harbor introns, it was longthought that these organisms use RNA splicing exclusivelyfor SL transfer, and accordingly, trypanosome-specific devi-ations of splicing factors were hypothesized to be trans splic-ing specific. It therefore came as a surprise when the T.brucei PAP gene (TriTryp database [TriTrypDB] accessionno. Tb927.3.3160), encoding poly(A) polymerase, was shownto harbor a single intron that was removed by conventionalcis splicing (48). The search for further introns revealed onlyone more gene in T. brucei (Tb927.8.1510), encoding a pu-tative RNA helicase (7), whose pre-mRNA was shown to becis spliced (30). Interestingly, a recent characterization ofthe T. brucei transcriptome by high-throughput RNA se-quencing strongly indicates that there are no other intronsdisrupting protein-coding genes (75).

* Mailing address: Department of Genetics and Developmental Bi-ology, University of Connecticut Health Center, 263 Farmington Av-enue, Farmington, CT 06030-3301. Phone: (860) 679-8878. Fax: (860)679-8345. E-mail: [email protected].

� Published ahead of print on 25 June 2010.

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SL trans splicing is a more widespread phenomenon in eu-karyotes, and after its initial discovery in trypanosomes, it wasfound to occur in a variety of organisms, including euglenids (80),nematodes (35), trematodes (70), and even lower chordates, suchas the sea squirt (86). However, there is no indication that thisparticular mode of trans splicing occurs in the hosts of trypano-somatid parasites, e.g., vertebrates or arthropods (19), and there-fore, it can be regarded as a parasite-specific process. This spec-ificity and the ubiquitous requirement of SL trans splicing formRNA maturation have made this process an attractive researchfocus. The long-term aims have been to find out how the trypano-some splicing machinery differs from its human counterpart, toidentify factors or factor domains which are specifically requiredfor the trans splicing process, and to analyze whether these fea-tures can be inactivated in a parasite-specific manner. The chal-lenge of this research is that the splicing machinery, termed thespliceosome, is a huge, dynamic complex composed of structuralRNAs and proteins that is difficult to characterize.

The spliceosome consists of the U1, U2, U4, U5, and U6snRNAs and, in the human system, up to 170 spliceosome-associated protein factors (91). Trypanosomatids possess allfive spliceosomal U snRNAs, which are typically somewhatsmaller and deviate in several aspects from their human coun-terparts. In contrast to the well-characterized human system,until recently, our knowledge of spliceosomal protein factors intrypanosomatids was very limited. A main reason for this lackof knowledge comes from the fact that amino acid sequencesof trypanosomatid proteins have diverged dramatically fromtheir human and yeast orthologs, and thus, only a few splic-

ing factors were identified by standard annotation of thecompleted L. major, Trypanosoma brucei, and Trypanosomacruzi (TriTryp) genomes (29). In recent years, however, ma-jor progress has been made in the identification of spliceoso-mal proteins and the characterization of U small nuclear ribo-nucleoprotein particles (U snRNPs) in trypanosomes. Threefactors have contributed to this success: first, the unre-stricted access to the sequenced and annotated TriTryp ge-nome databases (7) at GeneDB (http://www.genedb.org/)and, recently, also at TriTrypDB (http://tritrypdb.org/) (2);second, the systematic analysis of RNA binding proteinsharboring an RNA recognition motif (RRM) (16); andthird, tandem affinity purification (TAP) of splicing com-plexes combined with mass spectrometric identification ofcopurified proteins (46, 63). A current list of identified splic-ing factors is presented in Table 1.

In an excellent previous review on trypanosomatid RNAsplicing, Liang et al. described the discoveries and functionalcharacterizations of the trypanosome spliceosomal U snRNAsand early characterizations of the corresponding snRNPs (39).This review omits a general discussion of the U snRNAs andinstead focuses on proteins involved in splicing.

U snRNPS AND THE SPLICEOSOME INHIGHER EUKARYOTES

Our mechanistic insight into RNA splicing and our biochem-ical and structural knowledge of snRNPs, splicing factors, andthe spliceosome stem predominantly from work in the human

FIG. 1. Schematic of the mammalian cis splicing and the trypanosome SL trans splicing reactions. Upstream exon and spliced leader are drawnas gray rectangles, and downstream exon and trypanosome gene are drawn as black rectangles. 5� and 3� splice sites (SSs) are represented by smallopen boxes, branch points (BPs) by closed circles, polypyrimidine tracts by small striped boxes, and the cap 4 structure of the spliced leader as anoval. Conserved sequences are provided below the drawing with invariant residues underlined. While in mammalian systems, 5�SSs, BPs, and 3�SSsexhibit 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 unlessa compensatory AG dinucleotide is present within the 5� untranslated region (76). It appears that the importance of the polypyrimidine tractbecomes more important when consensus sequences are lacking. Yeast has highly conserved splice site and BP sequences, and some yeast intronsfunction without a polypyrimidine tract (not shown). The partly conserved sequences in mammals require a small polypyrimidine tract in the rangeof 10 to 12 residues (Y10-12), whereas in trypanosomes, the polypyrimidine tract is large (Y�20), is an essential sequence determinant for efficientsplicing, and typically starts just downstream of the BP (43, 76). After the first transesterification reaction, cis splicing results in a lariat intronstructure, whereas a Y structure intermediate is formed in the SL trans splicing process. After the second transesterification, these intronicstructures are debranched (not shown) and rapidly degraded.

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TABLE 1. Spliceosomal proteins of Trypanosoma bruceig

Annotationa Accession no.b Mr (103) TAPc DescriptionReference(s)

or source E valued

Sm/LSm proteinsSmB Tb927.2.4540 12.3 1, 2, 3, 4 65SmD1 Tb927.7.3120 11.7 1, 2 65SmD2 Tb927.2.5850 12.5 1, 2 65SmD3 Tb927.4.890 12.4 1, 2, 3, 4 65SmE Tb927.6.2700 9.6 1, 2 65SmF Tb09.211.1695 8.4 1, 2 65SmG Tb11.01.5915 8.9 1.2 65SSm2-1/Sm15K Tb927.6.4340 12.8 1, 2 83, 92SSm2-2/Sm16.5K Tb927.10.4950 14.7 1, 2 83, 92SSm4 Tb927.7.6380 23.2 1, 2 83LSm2 Tb927.8.5180 13.2 1, 2 46, 82LSm3 Tb927.7.7380 10.1 41LSm4 Tb11.01.5535 14.2 1, 2 41LSm5 Not assignede 12.0 82LSm6 Tb09.160.2150 9.1 41LSm7 Tb927.5.4030 10.2 1, 2 41LSm8 Tb927.3.1780 14.0 1, 2 41

SMN/Gemin2 and associated proteinsSMN Tb11.01.6640 17.0 1, 2, 3, 4 63Gemin2 Tb927.10.5640 55.4 1, 2, 3, 4 63Coatomer � Tb927.4.450 132.0 3, 4 47Coatomer � Tb927.1.2570 110.0 3, 4 47Coatomer �� Tb927.2.6050 93.9 3, 4 47Coatomer � Tb11.01.3740 97.5 3 47Coatomer � Tb927.8.5250 57.3 3, 4 47Coatomer ε Tb11.01.6530 34.8 3, 4 47Coatomer � Tb927.10.4270 20.5 3 47

U1 proteinsU1-70K Tb927.8.4830 31.7 1, 2 66U1A Tb927.10.8280/8300 18.0 1 46U1-24K Tb927.3.1090 24.2 1, 2 66U1C Tb927.10.2120 21.7 1, 2 66

U2 proteinsU2A� (U2-40K) Tb927.10.2120 36.5 1, 2 15U2B� Tb927.3.3480 13.6 1, 2 69SF3a60 Tb927.6.3160 61.5 TriTrypDB 1e13

SF3b(SAP)155 Tb11.01.3690 122.0 3, 50SF3b(SAP)145 Tb927.6.2000 52.5 50SF3b(SAP)130 Tb927.7.6980 195.0 1 50SF3b(SAP)49 Tb927.3.5280 29.8 50SF3b(SAP)14b Tb927.10.7390 12.7 50 6e12

SF3b(SAP)10 Tb09.211.2205 10.4 SF3b10 domain Pfam DB; 50 4e7

SF3b14 (p14) Tb927.10.7470 13.3 3, 50

U4 proteinsPRP3 Tb09.160.2900 63.2 1, 2 PRP3 domain Pfam DB 2e42

PRP4 Tb927.10.960 65.5 1, 2 46Snu13f Tb09.160.3670 13.6 5e34

U5 proteinsPRP8 Tb09.211.2420 277.0 1, 2, 4 44U5-200Kf Tb927.5.2290 249.3 1 0U5-102K Tb11.01.7330 111.0 1 1e5

U5-116K Tb11.01.7080 105.5 1, 2 8e100

U5-40K Tb11.01.2940 35.0 1, 2 46U5-15K Tb927.8.2560 17.7 1 4e26

U5-Cwc21 Tb09.160.2110 16.2 1, 2 46

PRP19 complexPRP19 Tb927.2.5240 54.3 1, 3, 4 3e42

CDC5f Tb927.5.2060 80.1 1e30

CRN/SYF3 Tb927.10.9660 87.7 1 7e41

SYF1 Tb927.5.1340 92.2 1 7e23

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and yeast systems. Unfortunately, there is no uniform nomen-clature for the protein factors in the two systems, and typically,there are two distinct names for orthologous factors (listed inreference 31). In this review, the default is the denotation fromthe human system.

The main building blocks of the spliceosome are the UsnRNPs, whose biogenesis requires several distinct assemblysteps. First, all spliceosomal U snRNAs, except U6, are ex-ported to the cytoplasm where they bind a set of seven com-mon proteins, known as the Sm proteins B, D1, D2, D3, E, F,and G. These proteins form a heteromeric ring around a con-served Sm binding site that resides in a single-stranded regionin the 3�-terminal domain of the U snRNA. This RNA-proteininteraction is typically very stable, and thus, the U snRNA/Smcomplex is referred to as the core snRNP. Core snRNP assembly

takes place in the cytoplasm and is linked to U snRNA caphypermethylation, which in turn codetermines the reimport of thecore snRNP into the nucleus. The U6 snRNA does not have acytoplasmic phase and, in the nucleus, binds a differentcomplex of seven Sm-like (LSm) proteins termed LSm2 to -8(LSm2-8). Back in the nucleus, the core snRNPs bind vari-ous snRNP-specific proteins, and overall, there are approx-imately 45 different proteins in the human system that in-teract directly with the spliceosomal U snRNAs (91). Asfollows, most of the spliceosome-associated proteins areconsidered to be non-snRNP proteins.

The RNA sequence determinants for the splicing reactioncomprise the 5� splice site (5�SS) and the 3�SS and a branchpoint (BP) sequence upstream of the 3�SS. In addition, a poly-pyrimidine tract is typically present between BP and the 3�SS

TABLE 1—Continued

Annotationa Accession no.b Mr (103) TAPc DescriptionReference(s)

or source E valued

ISY1 Tb927.8.1930 31.7 1 ISY1 domain Pfam DB 6e7

KIAA1604/Cwc22 Tb11.01.2520 66.82 2 2e49

Unannotated proteins that copurifiedin spliceosomal complexesh

Conserved hypothetical Tb927.8.6280 27.1 1, 2 Putative cyclophilin 63Conserved hypothetical Tb927.8.2090 21.6 2 Putative cyclophilin 63Conserved hypothetical Tb927.10.11950 22.4 2 Putative Cwc15 63Conserved hypothetical Tb927.8.4790 26.0 1 NovelConserved hypothetical Tb927.2.3400 42.0 1 NovelConserved hypothetical Tb927.7.1890 31.0 1 NovelConserved hypothetical Tb927.5.2910 20.0 1 NovelConserved hypothetical Tb11.02.0465 12.1 1 Novel

Annotated proteins without knownsplicing function that copurifiedin spliceosomal complexes

eEF-1� Tb927.10.2100 49.1 2HSP70 Tb11.01.3110 75.4 1, 2, 3Importin � Tb927.6.2640 58.0 2La protein Tb927.10.2370 37.7 1, 2, 3NORF1 Tb927.5.2140 93.3 1PABP1 Tb09.211.2150 62.1 1, 2TRYP1 Tb09.160.4250/80 22.4 1

Miscellaneous splicing factorsU2AF35 Tb927.10.3200 29.1 89U2AF65 Tb927.10.3500 96.6 90SF1 Tb927.10.9400 31.6 90PRP17 Tb927.3.1930 52.8 2 4e59

PRP31 Tb927.10.10700 39.7 40PRP43 Tb927.5.1150 82.9 40PTB1 Tb09.211.0560 37.0 78PTB2 Tb11.01.5690 54.7 78TSR1 Tb927.8.900 37.5 SR-like protein 28RRM1 Tb927.2.4710 50.0 SR-like protein 51SR protein Tb09.160.5020 17.6 TriTrypDB 3e06

a Annotation is according to the human system. Conserved hypotheticals are proteins which are conserved among trypanosomatids but dissimilar to proteins of othereukaryotes.

b Accession numbers are from TriTrypDB (http://www.tritrypdb.org/).c Proteins were cotandem affinity purified with SmD1 (1), SmB (2), SMN (3), or Gemin2 (4).d Protein sequences of identifications without experimental support were compared to the human genome and their E values determined by NCBI BLAST.e The gene of LSm5 has not yet been recognized as a protein-coding gene.f These genes were annotated in this study.g Proteins shown in boldface are trypanosome specific. The accession numbers of eight putative spliceosomal DExD/H-box helicases are Tb927.6.4600, Tb927.6.4600,

Tb927.10.5280, Tb927.10.7280, Tb927.10.9130, Tb11.02.3460, Tb927.7.7300, and Tb11.02.1930. More than 20 candidate putative spliceosomal peptidyl-prolyl cis/transisomerases are not shown.

h E values lower than 1e05 were considered not significant.



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(Fig. 1). Importantly, the spliceosome is assembled step-by-step on the pre-mRNA, and before and during splicing, itundergoes highly dynamic changes in which both the RNA andprotein composition are altered (reference 91 and referencestherein). In brief, the U1 snRNP first recognizes the 5�SS, theprotein factor SF1 the branch point, and the heterodimeric U2auxiliary factor (U2AF) both the branch point and 3�SS. Sub-sequently, the U2 snRNP is recruited and the U2 snRNAforms base pairs with the BP sequence, displacing SF1, a pro-cess which is mediated by the U2-associated protein complexesSF3a and SF3b. At this stage, the factor assembly is calledthe prespliceosome or complex A. Subsequently, the U4/U6snRNP and the U5 snRNP enter the spliceosome in the formof the U4/U6.U5 tri-snRNP, which results in the precatalyticcomplex B. Although in this complex, all snRNPs are on board,the spliceosome undergoes major rearrangements for activa-tion (complex B�), including the discard of U1 and U4 snRNPs.After the first transesterification, the spliceosome is trans-formed into complex C, and following the second splicing step,it is disassembled. The snRNPs are then recycled for newrounds of splicing. The different spliceosomal complexes havebeen purified and biochemically characterized in the yeast andhuman systems. Besides the above-described snRNP changes,

these complexes are associated with distinct sets of proteins(Fig. 2) (reviewed in references 31 and 91).

Is the spliceosome different for SL trans splicing? Interest-ingly, as first shown for the nematode Caenorhabditis elegans,the SL RNA splicing substrate itself is assembled into a coresnRNP binding the Sm proteins (11). This finding led to thehypothesis that the SL RNP activates its own splice site andthat trans splicing does not require the U1 snRNP (81, 88).Indeed, in vitro studies of the parasitic nematode Ascaris lum-bricoides showed that the destruction of U1 snRNA affectedonly cis and not trans splicing (27). Moreover, in the samesystem, two specific SL RNP proteins were identified andtermed SL175 and SL30 according to their molecular masses(17). The results of protein-protein interaction experimentssuggested that these proteins bridge the SL RNP and, thus, the5�SS of the SL RNA, via SF1 to the BP, a function which in cissplicing is mediated by the U1-specific FBP11/Prp40p (human/yeast nomenclature) subunit (17). SL175 and SL30 are indis-pensable for SL trans splicing, but they have no function in cissplicing or in an SL-independent mode of trans splicing (17)which has also been described in the human system (reviewedin reference 22). While these factors and their interactions arepotential antiparasitic targets, the amino acid sequences of

FIG. 2. Comparison of known spliceosomal factors of humans and trypanosomes. Schematic drawing of spliceosomal complexes during asplicing reaction as described in the mammalian and yeast systems. For each complex, proteins are listed that enter the spliceosome at the outlinedstage (slightly modified human protein repertoire is according to reference 91). Please note that only incoming proteins are listed and proteinsleaving the spliceosome in the transitions are not recognized. Bold blue lettering indicates proteins for which orthologs have been found intrypanosomes, whereas red lettering specifies trypanosome-specific factors. 1, Highly divergent, putative cyclophilin orthologs have been copurifiedwith 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-boxhelicases (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 splicingor metabolism remains unclear.



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these proteins are not conserved, and putative orthologs havenot been identified outside nematodes.

TRYPANOSOME Sm AND LSm PROTEINS AND SmCORE VARIATION IN U2 AND U4 snRNPs

Sm and LSm proteins are small proteins with a molecularmass typically of �10 to 20 kDa that share a highly conservedbipartite Sm motif. The corresponding Sm fold characteristi-cally consists of an N-terminal helix and a strongly bent, anti-parallel beta-sheet of five strands. While antibodies directedagainst the Sm domain of human proteins recognize Sm pro-teins in a wide range of organisms, they did not cross-react withtrypanosome proteins (55, 61, 62). Hence, it required affinitypurification of U snRNPs and protein analysis to show thattrypanosome U snRNAs and the SL RNA bind a set of com-mon proteins (62). The identity of five of these proteins wasrevealed in the classic way: U snRNPs were affinity purified,amino acid sequence information from common proteins wasobtained by protein microsequencing, and the respective geneswere cloned with the help of degenerate primers and PCR(65). In the same study, the missing SmB and SmD3 orthologs,however, could already be identified by mining the growing T.brucei genome database (65). Later, this was the exclusiveroute to identify the orthologs of LSm2-8 (41). However, whilethe Sm motifs were readily identifiable in all these proteins, theremaining amino acid sequences exhibited limited similarity totheir putative orthologs in other eukaryotes and, therefore,needed functional verification. In the case of the Sm proteins,SmG was shown to complement an SmG-deficient yeast strain(65), whereas the others exhibited protein-protein interactionswhich were consistent with the known arrangement in the ringstructure (63, 65). For the LSm proteins, only LSm8 and LSm3were functionally analyzed at first, and all others were identi-fied by sequence similarity alone (41). This approach backfiredbecause LSm2 and LSm5 turned out to be very interesting Smproteins (see below) but not LSm proteins. Eventually, a sec-ond study clarified the trypanosome LSm repertoire, identifiednew LSm2 and LSm5 orthologs, and provided strong evidencethrough functional studies that the correct set of LSm proteinswas identified (82). The formation of core snRNPs stabilizesthe U snRNAs, and expression silencing of a single Sm or LSmprotein leads to a loss of the cognate U snRNA. Accordingly,conditional RNA interference (RNAi) experiments targetingeach of the seven LSm proteins resulted in a specific loss of U6snRNA, confirming the new identifications (41, 46, 82).

In initial studies of trypanosome snRNPs, it was found thatthe trypanosome U2 core snRNP, in contrast to its humancounterpart and other trypanosome U snRNPs, disassembledcompletely in a cesium chloride density gradient (14) and inhigh-salt buffers (25). While this instability with exposure tosalt was originally attributed to a deviating U2 Sm binding site,which in T. brucei contains an unusual central guanosine resi-due, it was found only recently that the different core includesthe Sm complex as well. U2 snRNP purification revealed twoU2-specific proteins with apparent sizes of 15 and 16.5 kDathat contained the bipartite Sm motif (92). This was odd be-cause the whole Sm repertoire had already been characterizedand, moreover, Sm15K had at that time been identified asLSm5. However, a comprehensive tagging and coimmunopre-

cipitation analysis clarified the issue and showed that Sm15Kand Sm16.5K are paralogs of SmB and SmD3, respectively;they replace these proteins specifically in the U2 Sm core anddo not bind other U snRNAs. Furthermore, snRNP reconsti-tution assays with recombinant Sm proteins and syntheticRNAs demonstrated that the guanosine residue of the U2 Smbinding site is the recognition determinant of the U2-specificSm core complex (92). In an independent study, the identifi-cation of the two U2-specific Sm paralogs was confirmed andthe previously misannotated LSm2 was shown to be a secondSmD3 paralog that replaces SmD3 in the U4 snRNP (83).Importantly, the study by Tkacz et al. (83) provided an in vivoanalysis demonstrating that RNAi-mediated expression silenc-ing of the specific Sm paralogs reduced the abundance of onlythe corresponding U snRNA. The U4-specific Sm core wassubsequently characterized at the biochemical level, verifyingthe U4 association of the SmD3 paralog (30). Unfortunately,the studies on Sm core variation established different nomen-clatures, and Sm15K is also referred to as specific spliceosomalSm2-1 protein (SSm2-1), Sm16.5K as SSm2-2, and the U4-specific protein as SSm4.

What is the significance of Sm core variation? It has beenspeculated that it assists U2 and U4 snRNP assembly (83, 92),and indeed, this has recently been demonstrated for the U2snRNP. Earlier it was found that stable protein binding to the3�-terminal region of U2 snRNA, which included the Sm bind-ing site, was dependent on residues in the 3�-terminal loop IVsequence. This suggested that the U2 core snRNP was notformed by Sm protein binding alone but required cooperativebinding of Sm and loop IV-binding proteins (25). This modelwas recently verified by the demonstration that the U2-specificSm15K/Sm16.5K doublet interacts with the U2 snRNP proteinU2A� which in turn interacts with the loop IV-binding proteinU2B� (69). Only this ternary complex efficiently and specificallyinteracts with the 3�-terminal U2 snRNA region. In the humansystem, U2A� is separated from the Sm core by stem-loop III.Since this structure is completely missing in trypanosome U2snRNA, it is likely that the Sm15K/Sm16.5K-U2A� interactionoccurs through an essential, parasite-specific protein-proteininterface that compensates for the lack of this RNA structure.

Furthermore, it was suggested that Sm core variation mayfacilitate snRNP function in the splicing process (92). For exam-ple, human and yeast U2 snRNAs share a conserved motifwhich is complementary to the BP sequence. Conversely, intrypanosomes, there is no conserved BP sequence and, typi-cally, no complementarity between BP and U2 snRNA se-quences (43). Possibly the U2-specific Sm paralogs undergospecific protein-protein interactions that position the U2snRNP at the BP in the absence of sequence complementarity.A third speculation stated that the different Sm cores may beconnected to different U snRNA cap structures (83). In verte-brate and yeast systems, the U6 snRNA carries a �-mono-methyl phosphate cap, whereas U1, U2, U4, and U5 snRNAsobtain cotranscriptionally an m7G cap which is further meth-ylated to a 2,7,7 trimethylguanosine (m3G) cap after the for-mation of the core snRNP. It was shown that the binding of theSm proteins to the U snRNAs is a prerequisite for the recruit-ment of the enzyme trimethylguanosine synthase 1 (54, 67). Intrypanosomes, U1, U2, U4, and U6 snRNAs have the samecaps as their yeast and human orthologs (18, 57, 64), whereas



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SL RNA has cap 4 (4) and U5 snRNA lacks a cap (20, 95).Theoretically, Sm core variation could facilitate the recruit-ment of different cap-modifying enzymes into the core RNP,but there is no correlation between the type of cap and the typeof Sm core. For example, U1, U2, and U4 snRNAs binddifferent Sm complexes but share the same m3G cap. More-over, it was demonstrated experimentally for the T. bruceiU2 snRNA that cap trimethylation does not depend on thepresence of the Sm binding site or on formation of the coreRNP, thus excluding the possibility that the U2-specific Smcore is involved in cap hypermethylation (24).

SMN-MEDIATED ASSEMBLY OF CANONICALSm CORES

When Wang et al. (92) reconstituted core snRNPs withrecombinantly expressed Sm proteins, they detected specificbinding of the canonical and U2-specific Sm cores to theircognate Sm binding sites only with short RNA fragments,whereas full-length U snRNAs did not discriminate betweenthe two Sm complexes. This suggested that other activities inthe cell confer specificity of Sm core binding. A candidate forsuch an activity was the SMN (survival motor neuron) complex,which in the human system was shown to act as a catalyst forcore snRNP formation (reviewed in references 33 and 59). Thehuman SMN complex consists of the SMN protein and sevenadditional subunits, termed Gemin2 to -8, and it binds theSmD1/SmD2-SmE/SmF/SmG and SmD3/SmB subcomplexesin an open ring formation (13). This interaction then leads toU snRNA binding, ring closure, and dissociation of the SMNcomplex. While standard annotation of the Tritryp genomesdid not identify SMN or Gemin homologs, tandem affinitypurification of the T. brucei SmB protein (see below) led to theidentification of highly divergent orthologs of SMN andGemin2 (63). No other Gemin orthologs were found, and it ispossible that they do not exist in trypanosomes, because lowereukaryotes in general have a strongly reduced Gemin reper-toire and, in Drosophila melanogaster, the SMN/Gemin2 com-plex was sufficient to mediate core RNP assembly in vitro (36).In trypanosomes, in vitro core snRNP assembly experimentsfunctioned efficiently in the absence of SMN, but when thefactor was added it exhibited a striking discriminatory role: inits presence the canonical Sm core was efficiently loaded ontoits cognate U5 snRNA but not onto U4 and U2 snRNAs or aU5 snRNA with a mutated Sm site (63). In contrast, the SMNcomplex had no effect on the binding of the U2-specific Smcore. These findings suggested that the SMN complex specif-ically bound the canonical SmD3/SmB subcomplex and directlyinteracted with SmB, because this protein, in contrast toSmD3, is replaced in both the U2- and U4-specific Sm cores.This was indeed the case. SMN purification coisolated only theSmB and SmD3 proteins and not their paralogs, and pulldownassays with recombinant, tagged SMN proteins identified adirect interaction with SmB and the N-terminal part of SMN(63). The latter finding, again, is highly significant because itidentified an important, potentially parasite-specific pro-tein-protein interaction: human SMN utilizes an internalTudor domain and C-terminal regions to interact with di-methylated arginines in the RG-rich C-termini of Sm pro-teins (73), whereas in trypanosomes, neither Tudor domain

nor RG-rich domains are present in SMN and Sm proteins,respectively (63). Another striking difference from the humansystem was found in regard to SMN localization. In the humansystem, core snRNP assembly takes place in the cytoplasm and,accordingly, human SMN is primarily localized in this com-partment. Conversely, trypanosome SMN was found almostexclusively in the nucleus, suggesting that U snRNP assemblyin this organism is nuclear, a finding which is consistent withlocalizations of SL RNA and U2 snRNA by fluorescence in situhybridization (8, 83). In summary, it appears that despite itssmall size, the trypanosome SMN complex is mechanisticallycomplex, entailing both chaperone and specificity functions incore snRNP assembly.

If the SMN complex only chaperones the assembly of thecanonical Sm core, how, then, are the U2- and U4-specific Smcores put together? One possibility is that they require a dif-ferent, yet-to-be-determined assembly chaperone. Alterna-tively, the specific interactions of the U2 Sm paralogs SmK15/SmK16.5 with U2A�/U2B� may facilitate correct assembly ofthe U2-specific Sm complex onto the U2 Sm binding site. Onthe other hand, U4 core snRNP formation appears to be in-dependent of snRNP-specific proteins, because in core RNPreconstitution assays, SSm4 alone determined efficient andspecific assembly of the U4-specific Sm complex onto the U4snRNA (30).

TANDEM AFFINITY PURIFICATION OF SPLICINGCOMPLEXES IN T. BRUCEI

Until recently, only two snRNP-specific proteins had beenstudied in trypanosomes, namely, the orthologs of humanU2A� (originally termed U2-40K) (15) and the U5-specificPRP8 (44). While the latter was identified by sequence homol-ogy, U2A� was copurified with the U2 snRNA in high-strin-gency U snRNP purifications which typically left only the corestructures intact (62). For a more comprehensive biochemicalcharacterization of U snRNPs and/or of the spliceosome, it wastherefore essential to purify the RNA-protein complexes underconditions of lower stringency. A method well-suited for thispurpose is tandem affinity purification (TAP), which is basedon expressing a known protein factor fused to a compositeTAP tag. TAP comprises two consecutive high-affinity chro-matography steps which are carried out under nearly physio-logical conditions. Since the advent of this technology (71), theTAP tag and the TAP method have been modified in variousways to accommodate different systems, extracts, or proteincomplexes (26). For the purification of nuclear protein com-plexes in trypanosomes, the PTP (protein C epitope-TEVprotease cleavage site-protein A domains) modification ofTAP has proven to be very useful (26, 72). One of the firstapplications of the PTP tag was the purification of the try-panosome U1 snRNP. A first characterization of this snRNPhad revealed a protein with sequence homology to the humanU1-70K protein (64). And indeed, PTP tagging and purifica-tion of T. brucei U1-70K resulted in the specific copurificationof the U1 snRNA (66). The protein profile of the purificationcomprised the Sm proteins, the tagged protein, two additionalproteins of major abundance, and several proteins of minorabundance. The proteins of the two major bands were identi-fied by mass spectrometry and found to be annotated as “con-



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served hypotheticals,” meaning that they were conservedamong trypanosomatids but had no obvious similarity to pro-teins of other eukaryotes. However, when kinetoplastid se-quences of one of the new proteins were compared to those ofknown U1-specific proteins of model organisms, the proteinwas identified as the ortholog of human U1C (66). In contrast,the second protein, termed U1-24K, could not be meaningfullyaligned to known U1 proteins and, therefore, probably repre-sents a trypanosome-specific U1 snRNP subunit.

Since this initial tandem affinity purification of a snRNPwas successful, more comprehensive proteomic analyses oftrypanosomal splicing complexes were carried out by PTP tag-ging and purification of the common proteins SmD1 (46) andSmB (63). Overall, mass spectrometry identified 53 proteins inthese purifications, and the majority of the proteins copurifiedin both studies (Table 1). Moreover, with the exception ofthree LSm proteins and two non-snRNP proteins, all knowntrypanosomal snRNP proteins were identified in these pro-teomic analyses, confirming the high significance of the pro-teomic data sets. Consequently, bioinformatic analyses of theamino acid sequences of unannotated proteins revealed sev-eral new orthologs of known splicing factors (Table 1), and forLSm2 (U6), U1A, PRP4 (U4), and U5-40K, the bioinformaticidentifications were confirmed by coimmunoprecipitation ex-periments which showed that these proteins were bound totheir predicted snRNAs (46).

While these proteomic analyses increased the number ofspliceosomal protein orthologs in trypanosomes considerablyand identified potentially novel splicing factors (see below), thenumber of proteins that copurified with SmD1 or SmB is �3-fold lower than the protein count in human spliceosomes.Proteomics of yeast spliceosomal complexes revealed about 90proteins (21), which is lower than the count in the humansystem but still about 2-fold higher than the identified trypano-some repertoire. One possible interpretation of this finding isthat the splicing machinery of trypanosomatids that divergedearly is simplified. However, this is unlikely because the vastmajority of newly identified proteins are snRNP proteins, andnon-snRNP proteins are strongly underrepresented (Fig. 2). Infact, trypanosome orthologs have been identified for nearly allknown bona fide snRNP proteins, indicating that a trypano-some spliceosome comprises additional non-snRNP proteins,possibly in numbers comparable to those in yeast and humans.Hence, the question arises of why only a few non-snRNP pro-teins copurified with SmB and SmD1. Both proteomics studieswere carried out according to the standard PTP protocol, in-cluding the extract preparation procedure (37, 72). Since theseextracts contain an estimated overall salt concentration of 250to 300 mM, it is likely that the spliceosome did not withstandthe extract preparation procedure. Accordingly, a sucrose gra-dient sedimentation analysis of SmD1-PTP-purified materialshowed that the U snRNPs did not cosediment as part of alarger complex, and complexes with Svedberg values greaterthan 20 were not detected (46). In contrast, spliceosomal 45Scomplexes were characterized by a combination of glycerolgradient sedimentation and native gel electrophoresis in ex-tracts of lower salt concentration (40). It is therefore likely thatmodifying the extract preparation procedure will result in theformation of higher-order spliceosomal complexes which pos-

sibly can be isolated by tandem affinity purification and char-acterized by mass spectrometry in the future.

As discussed above, in both proteomic studies, the highlydivergent SMN and Gemin2 orthologs copurified. To betterunderstand the trypanosome SMN complex, Palfi et al. (63)PTP tagged and tandem affinity purified both proteins andidentified copurified proteins by mass spectrometry. While noother Gemin proteins were detected, which supports the ideathat an SMN/Gemin2 complex is sufficient for chaperone func-tion, surprisingly, all coatomer subunits copurified. While thecoatomer complex functions in vesicular transport betweenGolgi apparatus and endoplasmic reticulum (47), the signifi-cance of the coatomer-SMN/Gemin2 interaction is not under-stood. Possibly, the trypanosome SMN/Gemin2 complex has acytoplasmic function independent of snRNP core assembly(63), or there is a cytoplasmic component of the core snRNPassembly process which is vesicular and has not yet been de-tected.

ANALYSIS OF PROTEINS CARRYING AN RRM

Besides by tandem affinity purification, trypanosome splicingfactors have been identified through a focus on RRM-contain-ing proteins. Since trypanosomatid protein coding genes aretypically arranged in long tandem gene arrays which are tran-scribed polycistronically, differential gene expression is typi-cally regulated posttranscriptionally, for example, at the levelof RNA stability. Many proteins which affect RNA stabilitybind to mRNAs directly by virtue of an RRM motif andthus, RRM-containing proteins have become a research fo-cus in gene expression studies of both T. brucei and Trypano-soma cruzi (16). Since the spliceosome comprises severalRRM proteins, their identification came as a benefit fromthe attempt to determine the role of RRMs in the regulationof gene expression.

One RRM protein that was identified as a splicing factor wasa subunit of the U2-associated SF3b complex. SF3a and SF3bare two essential multisubunit splicing factors that interact withthe U2 snRNP after its recognition of the BP. The trypano-some protein was identified as the ortholog of human SF3b49,and accordingly, expression silencing of the correspondinggene was lethal and affected RNA splicing in T. brucei (50).Moreover, TAP tagging and purification of the protein, usingthe original TAP method, led to the complete characterizationof the trypanosome SF3b complex. Orthologs of all seven hu-man SF3b subunits were identified, including the RRM proteinSF3b14, often referred to as p14 (50). The SF3a complexappears to also be present in trypanosomatids, because a pu-tative homolog of the SF3a60 subunit was annotated in thegenome database (Table 1).

Other RRM proteins that were found to be splicing factorscomprise the snRNP protein U1A (46) and the U2 auxiliaryfactor components U2AF65 and U2AF35 (89, 90). In addition,RRM-containing serine-arginine-rich (SR) proteins have beenidentified. SR proteins comprise a phylogenetically conservedprotein family and, as has been shown in other systems, playsignificant roles in constitutive and alternative splicing of pre-mRNA (reviewed in reference 42). SR proteins contain one ortwo N-terminal RRMs and a C-terminal RS domain, rich inarginine-serine dipeptides. The first such protein discovered in



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trypanosomes was termed RRM1 (51). While RRM1 wasshown to be encoded by an essential gene and located in thenucleus, its specific function has not yet been determined (52).A second SR protein, termed trypanosomal SR-rich protein 1(TSR1), was localized to the nucleus and shown to bind to theheterologous human U2AF complex, and in a yeast three-hybrid system, it appeared to interact with the SL RNA (28).While these findings led to the speculation that TSR1 mayfacilitate recognition of the SL RNA by the trans spliceosome(28), a functional characterization of TSR1 strongly indicatedthat the factor has an essential role in cis splicing but not in SLtrans splicing (Christian Tschudi, Yale University, personalcommunication). This result is in accordance with the results ofa previous study of the T. cruzi ortholog TcSR which showedthat TcSR was functional in cis splicing in a heterologoussystem (68). Finally, RRM protein analysis in trypanosomesrevealed two homologs (PTB1 and PTB2) of the mammalianpolypyrimidine tract binding protein. While mammalian PTBdid not copurify with spliceosomal complexes and has severalnonsplicing functions, it negatively affects the splicing process,presumably by binding to the polypyrimidine tract near the3�SS, thereby interfering with U2AF65 function (77). Func-tional characterization of trypanosome PTB1 and PTB2 didnot reveal a repressor function of these proteins in splicing. Incontrast, a detailed study provided very strong evidence thatboth proteins are essential for trans splicing of pre-mRNAsthat contain C-rich polypyrimidine tracts (78). In addition,expression silencing of PTB1 but not of PTB2 affected cissplicing, indicating that both proteins have distinct activatingfunctions in trypanosome RNA splicing (78).

BIOINFORMATIC IDENTIFICATION OFTRYPANOSOME SPLICING FACTORS

A third route to identify RNA splicing factors has been datamining. Some of the splicing factors in trypanosomes are con-served enough to be identified by in silico analysis alone. Forexample, it was straightforward to identify the missing or-thologs of the human snRNP proteins Snu13 and U5-200K forthis study (Table 1). Similarly, the important CDC5 subunit ofthe PRP19 complex, which is an essential component of theactive spliceosome, was readily identifiable in the trypanosomegenome database (Table 1). Two splicing factors which hadpreviously been identified bioinformatically are PRP43 andPRP31 (40). PRP43 is a conserved spliceosomal helicase withessential functions in intron lariat release from the spliceo-some (53) and in spliceosome disassembly (1), whereas PRP31is a factor of the U4/U6.U5 tri-snRNP that is important fortri-snRNP formation and assembly into the spliceosome (49).The trypanosome PRP31 and PRP43 appear to be functionallyequivalent, because both proteins were shown to be essentialfor both cis and SL trans splicing, and PRP31 was specificallyassociated with the trypanosome U4/U6.U5 tri-snRNP (40).Nevertheless, the bioinformatics route of identifying trypano-some splicing proteins has not been exploited extensively andit is very likely that a systematic approach will reveal additional(putative) orthologs of non-snRNP proteins.

Overall, our knowledge of the spliceosomal protein reper-toire of trypanosomes has greatly increased in the past years.While the set of snRNP proteins appears to be nearly com-

plete, most of the non-snRNP factors have probably not beenidentified yet. However, the identification of individual com-ponents of spliceosomal protein complexes, such as PRP19 andSF3a (Fig. 2), indicate that these complexes are present andthat they can be further analyzed. For example, in yeast, morethan 20 splicing proteins were identified by tandem affinitypurification of the CDC5 ortholog Cef1p (reviewed in refer-ence 31). The identification of CDC5 in this study will enablea comparable analysis in trypanosomes.

Another important aspect of the newly identified trypano-some splicing factors is that they strongly indicate that trypano-somes form a spliceosome that possesses the same basic com-ponents and undergoes the same dynamic rearrangements asits human and yeast counterparts. It should be kept in mindthat, with the exception of a 45S spliceosome detection bynative gel electrophoresis (40), there is so far no biochemicalevidence that trypanosomes do form complexes that corre-spond to the well-characterized spliceosome E, A, B, or Ccomplexes in the yeast and human systems. On the other hand,a comparison of human proteins that enter the spliceosome atthese defined stages and of the known trypanosome repertoireshows that for each spliceosome transition, characteristic try-panosome orthologs have been identified (Fig. 2).

TRYPANOSOME-SPECIFIC ASPECTS OFTHE SPLICEOSOME

Despite the rapidly increasing number of identified trypano-somal RNA splicing factors, only a few functional proteincharacterizations have been published thus far. Nevertheless,several trypanosome-specific characteristics of the splicing ma-chinery have already been identified. As discussed above, Smcore variation, including the trypanosome-specific interactionbetween SmB and SMN, as well as the particular architectureof the U2 RNP core, involving potentially unique interactionsbetween Sm15K/Sm16.5K and U2A�, are trypanosome-specificU snRNP features. Other notable differences, shown in the T.cruzi system, include the demonstration that the U2AF sub-units U2AF35 and U2AF65 exhibit weak or no interaction(90), that instead, U2AF65 forms a stable complex with the BPbinding protein SF1 (90), and that, within the U2-related SF3bcomplex, the protein interface between the SF3b155 andSF3b14 subunits appears to be larger and more complex thanin the human system (3).

Another interesting trypanosome splicing factor is U5-Cwc21. This protein shares a highly conserved N terminus withthe human SRm300/SRRM2 protein and yeast Cwc21p(complexed with Cef1p protein 21). Coimmunoprecipitationanalysis showed that the trypanosome protein is predomi-nantly associated with U5 snRNA, and expression silencingof U5-Cwc21 was lethal and affected both cis and transsplicing (46). In contrast, yeast Cwc21p and human SRm300have redundant, nonessential roles in RNA splicing, becauseCWC21 is a nonessential gene and SRm300 can be immunode-pleted from extract without affecting splicing efficiency in vitro(9). Moreover, while yeast Cwc21p does interact with the U5-protein PRP8 (23), it is predominantly associated with U2snRNA and not with U5 snRNA (32). These findings thereforestrongly indicate that trypanosome U5-Cwc21 has an essentialfunction in RNA splicing that is unique to trypanosomes.



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A further peculiarity of trypanosome splicing factors is theexpression level of U1 snRNP components. In the nematodesystem, the U1 snRNP functions exclusively in cis splicing andnot in SL trans splicing (27). If the trypanosome U1 snRNPfunctions analogously, it would be required only for the re-moval of a single intron from two different pre-mRNAs. How-ever, the U1-specific proteins U1-70K, U1-24K, and U1C areamong the most abundant proteins that copurified with SmD1(46). This discrepancy between intron number and U1 snRNPexpression level suggests that the trypanosome U1 snRNP hasfunctions beyond intron removal. There is evidence that thetrypanosome 45S spliceosome contains both SL and U1snRNA, and it was suggested that there may only be one kindof spliceosome for both cis and trans splicing (40). If this istrue, the U1 snRNP may be essential for spliceosome integrityor it may have a yet-undetected, trypanosome-specific functionin trans splicing. Alternatively, the trypanosome U1 snRNP,like its human counterpart, may function beyond intron re-moval in transcription initiation and/or elongation (reviewed inreference 5).

Finally, there seems to be a difference in SL RNP recruit-ment to the BP in nematode and trypanosome systems. Whilethe nematode SL RNP apparently docks on SF1 via a proteinbridge (17), immunoprecipitation of trypanosome SF1 underlow-stringency conditions did not coprecipitate SL RNA (D. L.Ambrósio and A. Günzl, unpublished results). It is thereforelikely that other proteins and protein-protein interactions thanin the nematode system mediate the recruitment of the try-panosome SL RNP. Possibly, U5 and U6 snRNPs play a role inthis process, because U5 and U6 snRNAs were convincinglyshown to interact with the 5�SS of the SL RNA (93, 96). If SLRNP recruitment to pre-mRNA requires trans splicing-specificfactors, as in the nematode system, potential candidates forsuch proteins are listed in Table 1; there are currently eightproteins which copurified with trypanosomal splicing com-plexes but could not be annotated convincingly. Three of theseproteins may be the orthologs of cyclophylins and of thePRP19-related factor Cwc15, although the sequence similari-ties are very weak. The remaining five proteins are novel insequence because they do not exhibit any sequence similarityto nontrypanosomatid proteins.

PERSPECTIVES

The spliceosome is one of the most complex molecular ma-chineries in the cell, and it is a great challenge to functionallycharacterize this dynamic RNP-protein machinery. In recentyears, major progress has been made in the biochemical andstructural analysis of the human spliceosome (45, 91). If cor-responding studies can be carried out in trypanosomes, it willbe possible to determine in detail essential differences betweentrypanosome and human spliceosomes. While such differencesmay be the consequence of evolutionary divergence or mayrepresent SL trans splicing-specific requirements, they are po-tential antiparasitic drug targets. This notion is not remotesince the spliceosome has been validated as a drug target, forexample, for anticancer treatment (reviewed in reference 85).Although the undertaking of comprehensively analyzing thetrypanosome spliceosome appears overwhelming, the prospectsare nevertheless good because all necessary tools are in place. As

shown in Fig. 2, there are now several new spliceosomal proteinswhich can serve as bait in tandem affinity purification to broadlycharacterize the trypanosome splicing factor repertoire. The con-ditional RNAi-based expression-silencing system in T. brucei (94),in combination with established reverse transcription-PCR andprimer extension assays for the analysis of trans and cis splicingdefects, provides an in vivo platform for determining the splic-ing functions of individual proteins. Moreover, a homologousin vitro trans splicing system was recently established in T.brucei which will allow the functional dissection of importantsplicing factors (74). Finally, the recent demonstration that thetandem affinity-purified trypanosome transcription factor com-plex TFIIH was sufficiently intact and pure to determine itsmolecular structure by macromolecular electron microscopy(38) strongly indicates that similar structures can be obtainedfrom tandem affinity-purified splicing complexes.

And there is another potentially exciting perspective. Whileintrons and alternative splicing greatly enhance the proteinrepertoire in higher eukaryotes (recently reviewed in reference60), the functional role of introns in lower eukaryotes is notwell understood. Trypanosomes appear to have reduced theirintron repertoire to only two (75). Why did they not eliminatethese two introns as well? The fact that the insertion site of thePAP intron is conserved in trypanosomatids argues that theintron has a specific and essential function which was re-tained throughout trypanosomatid evolution. Since it shouldbe straightforward to test the outcome of [conditionally] de-leting these intron sequences in the trypanosome genome, itmay be possible to determine the specific function of theseintrons and understand the functional significance of cis splic-ing in these early-diverged organisms.

ACKNOWLEDGMENTS

I thank Christian Tschudi (Yale University) for communicating un-published data and Tu N. Nguyen and Daniela L. Ambrósio for criticalreading of the manuscript.

This work was supported by National Institutes of Health R01 grantsAI059377 and AI073300 to A.G.

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