mRNA maturation in trypanosomes differs from the process in most eukaryotes mainly because protein-coding genes are transcribed into polycistronic RNAs in this organism (78). Studies from the ongoing genome project suggest that the entire chromosome may be transcribed as large transcripts, but thus far there is no evidence to support the existence of conventional polymerase II promoters (62). The process of trans splicing was discovered 20 years ago when it was found that the different variant surface glycoprotein mRNAs in Trypanosoma brucei carry a common 39-nucleotide (nt) sequence, namely, the spliced leader (SL) sequence (9). It was later established that all trypanosome mRNAs undergo trans splicing (2). The source of the SL sequence was found to be a small capped RNA, the SL RNA (14, 58). Thus, the SL addition serves two purposes: it functions together with polyadenylation in dissecting the polycistronic transcripts, and it provides the cap to mRNAs (2). trans splicing proceeds through a two-step transesterification reaction, analogous to cis splicing but forming a Y structure instead of a lariat intermediate (illustrated in Fig. 1A) (61,88). Although first discovered in trypanosomes, the process was later found in nematodes (41), euglenoids (91), trematodes (73), and recently in chordates (97). Surprisingly, after almost a decade of searching for cis splicing, a single gene carrying a cis-spliced intron was discovered, suggesting that these two splicing processes coexist in trypanosomes, as in all other organisms capable of trans splicing (51).In the last decade, studies have focused on elucidating the mechanism and machinery of pre-mRNA processing in these organisms. The major findings included (i) the identification of the first cis-spliced intron and U1 snRNA that may function exclusively in this process, (ii) the finding and unraveling the function of U5 and SLA1, and (iii) the existence of coupling between trans splicing and polyadenylation. In this review we summarize studies performed mainly in vivo to elucidate structure-function aspects of the SL RNA and the snRNAs with which it interacts. The unique modifications on the SL RNA, including capping and pseudouridylation and their role in SL RNA function and biogenesis, are described. The regulation of splicing and its linkage to polyadenylation is discussed, and data are provided for the existence of splicing factors whose function is well characterized in other eukaryotes. STRUCTURE-FUNCTION ANALYSIS OF THE SL RNAIn the absence of an in vitro system for trans splicing, most of the structure-function studies were performed in vivo in Leptomonas seymouri, Leptomonas collosoma, and Leishmania tarentolae by using tagged SL RNAs (49,53,85,86,108). For some unknown reason, this approach does not work for T. brucei (Elisabetta Ullu, unpublished data). The SL RNA secondary structure of all organisms carrying out trans splicing is similar; it is composed of three stem-loops. In trypanosomatids, The SL sequence is 39 to 41 nt, followed by an intron of varia...