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...

Small nucleolar RNAs (snoRNAs) constitute newly discovered noncoding small RNAs, most of which function in guiding modifications such as 2-O-ribose methylation and pseudouridylation on rRNAs and snRNAs. To investigate the genome organization of Trypanosoma brucei snoRNAs and the pattern of rRNA modifications, we used a whole-genome approach to identify the repertoire of these guide RNAs. Twenty-one clusters encoding for 57 C/D snoRNAs and 34 H/ACA-like RNAs, which have the potential to direct 84 methylations and 32 pseudouridines, respectively, were identified. The number of 2-O-methyls (Nms) identified on rRNA represent 80% of the expected modifications. The modifications guided by these RNAs suggest that trypanosomes contain many modifications and guide RNAs relative to their genome size. Interestingly, ∼40% of the Nms are species-specific modifications that do not exist in yeast, humans, or plants, and 40% of the species-specific predicted modifications are located in unique positions outside the highly conserved domains. Although most of the guide RNAs were found in reiterated clusters, a few single-copy genes were identified. The large repertoire of modifications and guide RNAs in trypanosomes suggests that these modifications possibly play a central role in these parasites.

Most eukaryotic C/D small nucleolar RNAs (snoRNAs) guide 2-O methylation (Nm) on rRNA and are also involved in rRNA processing. The four core proteins that bind C/D snoRNA in Trypanosoma brucei are fibrillarin (NOP1), NOP56, NOP58, and SNU13. Silencing of NOP1 by RNA interference identified rRNAprocessing and modification defects that caused lethality. Systematic mapping of 2-O-methyls on rRNA revealed the existence of hypermethylation at certain positions of the rRNA in the bloodstream form of the parasites, suggesting that this modification may assist the parasites in coping with the major temperature changes during cycling between their insect and mammalian hosts. The rRNA-processing defects of NOP1-depleted cells suggest the involvement of C/D snoRNA in trypanosome-specific rRNA-processing events to generate the small rRNA fragments. MRP RNA, which is involved in rRNA processing, was identified in this study in one of the snoRNA gene clusters, suggesting that trypanosomes utilize a combination of unique C/D snoRNAs and conserved snoRNAs for rRNA processing.

SUMMARYThe loss of rooting capability following the transition from the juvenile to the mature phase is a known phenomenon in woody plant development. Eucalyptus grandis was used here as a model system to study the differences in gene expression between juvenile and mature cuttings. RNA was prepared from the base of the two types of cuttings before root induction and hybridized to a DNA microarray of E. grandis. In juvenile cuttings, 363 transcripts were specifically upregulated, enriched in enzymes of oxidation/reduction processes. In mature cuttings, 245 transcripts were specifically upregulated, enriched in transcription factors involved in the regulation of secondary metabolites. A gene encoding for nitrate reductase (NIA), which is involved in nitric oxide (NO) production, was among the genes that were upregulated in juvenile cuttings. Concomitantly, a transient burst of NO was observed upon excision, which was higher in juvenile cuttings than in mature ones. Treatment with an NO donor improved rooting of both juvenile and mature cuttings. A single NIA gene was found in the newly released E. grandis genome sequence, the cDNA of which was isolated, overexpressed in Arabidopsis plants and shown to increase NO production in intact plants. Therefore, higher levels of NIA in E. grandis juvenile cuttings might lead to increased ability to produce NO and to form adventitious roots. Arabidopsis transgenic plants constantly expressing EgNIA did not exhibit a significantly higher lateral or adventitious root formation, suggesting that spatial and temporal rather than a constitutive increase in NO is favorable for root differentiation.

A pair of proteins is defined to be related by a circular permutation if the N-terminal region of one protein has significant sequence similarity to the C-terminal of the other and vice versa. To detect pairs of proteins that might be related by circular permutation, we implemented a procedure based on a combination of a fast screening algorithm that we had designed and manual verification of candidate pairs. The screening algorithm is a variation of a dynamic programming string matching algorithm, in which one of the sequences is doubled. This algorithm, although not guaranteed to identify all cases of circular permutation, is a good first indicator of protein pairs related by permutation events. The candidate pairs were further validated first by application of an exhaustive string matching algorithm and then by manual inspection using the dotplot visual tool. Screening the whole Swissprot database, a total of 25 independent protein pairs were identified. These cases are presented here, divided into three categories depending on the level of functional similarity of the related proteins. To validate our approach and to confirm further the small number of circularly permuted protein pairs, a systematic search for cases of circular permutation was carried out in the Pfam database of protein domains. Even with this more inclusive definition of a circular permutation, only seven additional candidates were found. None of these fitted our original definition of circular permutations. The small number of cases of circular permutation suggests that there is no mechanism of local genetic manipulation that can induce circular permutations; most examples observed seem to result from fusion of functional units.