Exportin 1 Mediates Nuclear Export of the Kinetoplastid Spliced Leader RNA

Abstract

The kinetoplastid protozoan spliced leader (SL) RNA is the common substrate pre-mRNA utilized in all trans-splicing reactions. Here we show by fluorescence in situ hybridization that the SL RNA is present in the cytoplasm of Leishmania tarentolae and Trypanosoma brucei. Treatment with the karyopherin-specific inhibitor leptomycin B was toxic to T. brucei and eliminated the cytoplasmic SL RNA, suggesting that cytoplasmic SL RNA was dependent on the nuclear exporter exportin 1 (XPO1). Ectopic expression of xpo1 with a C506S mutation in T. brucei conferred resistance to leptomycin B. A reduction in SL RNA 3 extension removal and 5 methylation of nucleotide U 4 was observed in wild-type T. brucei treated with leptomycin B, suggesting that the cytoplasmic stage is necessary for SL RNA biogenesis. This study demonstrates spatial and mechanistic similarities between the posttranscriptional trafficking of the kinetoplastid protozoan SL RNA and the metazoan cis-spliceosomal small nuclear RNAs.The kinetoplastid protozoa use the trans-splicing reaction to resolve individual mRNAs from polycistronically transcribed pre-mRNAs. Transcription initiation for pre-mRNA in these early branching organisms is regulated minimally, if at all, and steady-state mRNA levels are determined posttranscriptionally at the level of trans splicing (6) or transcript stability (11). In trans splicing a 39-nt exon, the spliced leader (SL) is transferred from the SL RNA to the 5Ј end of each protein-coding pre-mRNA (2). Mechanistically, cis and trans splicing are related in that they share common machinery, such as U2, U4, and U6 small nuclear RNAs (snRNAs) (46) and associated core proteins (17,39).SL RNA is the best-characterized molecule unique to the machinery of trans splicing. All kinetoplastid and nematode SL RNAs studied to date fold into a conserved, three-stem-loop structure (7). An unusual feature of the kinetoplastid SL RNA is the 5Ј cap 4 structure that has been characterized for Trypanosoma brucei and Crithidia fasciculata (3,15,40). Kinetoplastid cap 4 is comprised of 2Ј-O-methylation of the sugar groups for the first four nucleotides (AACU), with additional methylations on the bases of first (m 6,6 A) and fourth (m 3 U) nucleotides.A posttranscriptional processing model for trypanosomatid SL RNA maturation was proposed based on mutagenesis studies with Leishmania tarentolae. SL RNA genes are transcribed from individual promoters by RNA polymerase II (8). Transcription termination occurs in a T-tract-producing 3Ј-extended SL RNA. The Sm protein binding site and downstream stem-loop III structure are required for cytoplasmic 3Ј-nucleolytic processing of the primary SL RNA transcripts (45). Formation of the mature 3Ј end is linked to cap 4 methylation; however, a mature 3Ј end is not the sole determinant of cap 4 formation, since some correctly 3Ј-processed SL RNA mutants do not receive cap 4 (43).While SL RNA exon content is largely tolerant to mutagenesis in L. tarentolae, intron sequence and structural changes reduce or eliminate tra...

Identification of novel snRNA-specific Sm proteins that bind selectively to U2 and U4 snRNAs in Trypanosoma brucei

“…It was initially proposed that since cap-4 formation is cotranscriptional, SL RNA assembly takes place in the nucleus (Mair et al 2000). Later it was suggested that XPO1, an export factor, may mediate the transport of the SL RNA from the nucleus to the cytoplasm, suggesting that at least part of the assembly process may occur in the cytoplasm (Zeiner et al 2003). The observation of large cytoplasmic SL RNA speckles (SL RNP-C) during Sm depletion supports the cytoplasmic assembly of SL RNA.…”

“…Most recently two distinct methyltransferases that carry out the 29-O9-methylations at positions +3 and +4 and +2 of the cap-4 were identified and localized to the nucleus (Arhin et al 2006a,b) . Despite the fact that many factors involved in SL RNA biogenesis are found within the nucleus, studies from recent years suggested that SL RNA biogenesis, nevertheless, involves a cytoplasmic phase (Mandelboim et al 2003;Zeiner et al 2003). It was suggested that export of SL RNA to the cytoplasm is mediated by XPO1 (Zeiner et al 2003).…”

“…Despite the fact that many factors involved in SL RNA biogenesis are found within the nucleus, studies from recent years suggested that SL RNA biogenesis, nevertheless, involves a cytoplasmic phase (Mandelboim et al 2003;Zeiner et al 2003). It was suggested that export of SL RNA to the cytoplasm is mediated by XPO1 (Zeiner et al 2003). The finding that SL RNA accumulated in the cytoplasm during Sm depletion, and that snRNP assembly occurs in the cytoplasm in metazoa, led to the proposal that SL RNA assembly with Sm proteins takes place in the cytoplasm in trypanosomes (Mandelboim et al 2003).…”

Chemical synthesis and binding activity of the trypanosomatid cap-4 structure

“…Cap formation on the SL RNA proceeds in a 5Ј-3Ј direction, as indicated by experiments performed with permeable trypanosomes (Ullu and Tschudi 1995). It appears that at least part of the modifications occur cotranscriptionally, as shown in studies using permeable trypanosomes (Mair et al 2000) and that sequences within the SL RNA coding gene are required for obtaining the complete cap-4 structure (Ullu and Tschudi 1995;Zeiner et al 2003). However, it was recently shown that the cytoplasmic stage is necessary for SL RNA biogenesis (Zeiner et al 2003).…”

“…It appears that at least part of the modifications occur cotranscriptionally, as shown in studies using permeable trypanosomes (Mair et al 2000) and that sequences within the SL RNA coding gene are required for obtaining the complete cap-4 structure (Ullu and Tschudi 1995;Zeiner et al 2003). However, it was recently shown that the cytoplasmic stage is necessary for SL RNA biogenesis (Zeiner et al 2003). It was further suggested that U4 modification of the SL RNA most probably serves as a signal for transport of snRNPs between the nucleus and the cytoplasm (Mandelboim et al 2003).…”