BackgroundThe spliced leader (SL) RNA provides the 5' m7G cap and first 39 nt for all nuclear mRNAs in kinetoplastids. This small nuclear RNA is transcribed by RNA polymerase II from individual promoters. In Leishmania tarentolae the SL RNA genes reside in two multi-copy tandem arrays designated MINA and MINB. The transcript accumulation from the SL promoter on the drug-selected, episomal SL RNA gene cassette pX-tSL is ~10% that of the genomic array in uncloned L. tarentolae transfectants. This disparity is neither sequence- nor copy-number related, and thus may be due to interference of SL promoter function by epigenetic factors. To explore these possibilities we examined the nucleoplasmic localization of the SL RNA genes as well as their nucleosomal architecture.ResultsThe genomic SL RNA genes and the episome did not co-localize within the nucleus. Each genomic repeat contains one nucleosome regularly positioned within the non-transcribed intergenic region. The 363-bp MINA array was resistant to micrococcal nuclease digestion between the -258 and -72 positions relative to the transcription start point due to nucleosome association, leaving the promoter elements and the entire transcribed region exposed for protein interactions. A pattern of ~164-bp protected segments was observed, corresponding to the amount of DNA typically bound by a nucleosome. By contrast, nucleosomes on the pX-tSL episome were randomly distributed over the episomal SL cassette, reducing transcription factor access to the episomal promoter by approximately 74%. Cloning of the episome transfectants revealed a range of transcriptional activities, implicating a mechanism of epigenetic heredity.ConclusionThe disorganized nucleosomes on the pX episome are in a permissive conformation for transcription of the SL RNA cassette approximately 25% of the time within a given parasite. Nucleosome interference is likely the major factor in the apparent transcriptional repression of the SL RNA gene cassette. Coupled with the requirement for run-around transcription that drives expression of the selectable drug marker, transcription of the episomal SL may be reduced even further due to sub-optimal nucleoplasmic localization and initiation complex disruption.

In all trypanosomatids, trans splicing of the spliced leader (SL) RNA is a required step in the maturation of all nucleus-derived mRNAs. The SL RNA is transcribed with an oligo-U 3 extension that is removed prior to trans splicing. Here we report the identification and characterization of a nonexosomal, 335 exonuclease required for SL RNA 3-end formation in Trypanosoma brucei. We named this enzyme SNIP (for snRNA incomplete 3 processing). The central 158-amino-acid domain of SNIP is related to the exonuclease III (ExoIII) domain of the 335 proofreading subunit of Escherichia coli DNA polymerase III holoenzyme. SNIP had a preference for oligo(U) 3 extensions in vitro. RNA interference-mediated knockdown of SNIP resulted in a growth defect and correlated with the accumulation of one-to two-nucleotide 3 extensions of SL RNA, U2 and U4 snRNAs, a five-nucleotide extension of 5S rRNA, and the destabilization of U3 snoRNA and U2 snRNA. SNIP-green fluorescent protein localized to the nucleoplasm, and substrate SL RNA derived from SNIP knockdown cells showed wild-type cap 4 modification, indicating that SNIP acts on SL RNA after cytosolic trafficking. Since the primary SL RNA transcript was not the accumulating species in SNIP knockdown cells, SL RNA 3-end formation is a multistep process in which SNIP provides the ultimate 3-end polishing. We speculate that SNIP is part of an organized nucleoplasmic machinery responsible for processing of SL RNA.The kinetoplastid protozoa are deep-branching eukaryotes that have evolved unusual mechanisms of nuclear gene expression. Transcription initiation of protein-coding genes in these organisms is regulated minimally, and all nucleus-derived premRNAs are transcribed polycistronically. To resolve these polycistronic pre-mRNAs into mature mRNAs, kinetoplastids use the trans-splicing reaction. Through trans splicing, the 5Ј 39-nucleotide (nt) spliced leader (SL) is transferred from the substrate SL RNA to the polycistronic pre-mRNA, yielding transspliced mRNAs. In addition to substrate SL RNA, trans splicing also requires the coordinated functions of mature U2, U4, U5, and U6 small nuclear RNAs (snRNAs) and associated proteins.Although there are differences in the details of individual snRNA gene transcription among eukaryotes, all snRNAs share at least one common feature: nascent snRNAs contain 3Ј extensions as a consequence of transcription termination. These 3Ј extensions are of variable sequence and length and are removed to yield the mature 3Ј end of each snRNA (10). In metazoans 3Ј-extension removal of nascent U1 snRNA was shown to require (i) intracellular trafficking of U1 snRNA, (ii) the activities of at least two nucleases, and (iii) a cis-acting element downstream of the mature 3Ј end of U1 snRNA (5). The first nuclease acting on nascent U1 snRNA was cytosolic and left a 2-nt extension, whereas the final 3Ј-end trimming activity was nuclear (20). 3Ј-Extension removal of human precursor U2 snRNA also required intracellular trafficking, a 3Ј cis-acting sequence element, and at...

Post-transcriptional 3' end formation is an essential step in the maturation of most small RNAs. Knowledge of the precise 3' ends of mature RNAs is essential for defining 3'-processing activities. We have mapped the mature 3' ends of Spliced Leader RNA, the U1, U2, U4, U5, and U6 small nuclear RNAs, the U3 small nucleolar RNA, and the 5S and 5.8S ribosomal RNAs by ligation-mediated PCR in Trypanosoma brucei. With the exception of U5, two classes of 3' ends were observed: flush with the base of a stem-loop structure, and 1-2 nt extended from a stem-loop. Multiple mechanisms and structural features are likely to influence 3' end maturation of RNAs.

Post-transcriptional 3' end formation is an essential step in the maturation of most small RNAs. Knowledge of the precise 3' ends of mature RNAs is essential for defining 3'-processing activities. We have mapped the mature 3' ends of Spliced Leader RNA, the U1, U2, U4, U5, and U6 small nuclear RNAs, the U3 small nucleolar RNA, and the 5S and 5.8S ribosomal RNAs by ligation-mediated PCR in Trypanosoma brucei. With the exception of U5, two classes of 3' ends were observed: flush with the base of a stem-loop structure, and 1-2 nt extended from a stem-loop. Multiple mechanisms and structural features are likely to influence 3' end maturation of RNAs.