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In the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans -spliced to generate a common 5' exon derived from the spliced leader (SL) RNA. Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA-binding protein TRF4, leading to the shutoff of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structures of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3 F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all of the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments. IMPORTANCE In this study, we found that SLS is induced by depletion of the essential ER-resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi complex-localized quiescin sulfhydryl oxidase (QSOX). Most strikingly, silencing of Rhomboid-like 1 (TIMRHOM1), involved in mitochondrial protein import, also induces SLS. We also report on the autophosphorylation of PK3 during SLS induction. This study has implications for our understanding of how trypanosomes keep the homeostasis between the ER and the mitochondria and suggests that PK3 may participate in the connection between these two organelles. The pathway, when induced, leads to the suicide of these parasites, and its induction offers a potential novel drug target against these parasites.

Spliced-leader-associated RNA (SLA1) guides the pseudouridylation at position -12 (relative to the 5' splice site) of the spliced-leader (SL) RNA in all trypanosomatid species. Nevertheless, the exact role of this RNA is currently unknown. Here, we demonstrate that the absence of pseudouridine on Leptomonas collosoma SL RNA has only a minor effect on the ability of this RNA to function in trans splicing in vivo. To investigate the possible role of SLA1 during SL RNA biogenesis, the structure of the SL RNA was examined in permeable Trypanosoma brucei cells depleted for CBF5, the H/ACA pseudouridine synthase, lacking SLA1. Our results suggest that in the absence of SLA1, the SL RNA secondary structure is changed, as was detected by differential sensitivity to oligonucleotide-directed RNase H cleavage, suggesting that the association of SLA1 maintains the SL RNA in a structural form which is distinct from the structure of the SL RNA in the steady state. In T. brucei cells depleted for the SL RNA core protein SmD1, SL RNA first accumulates in large amounts in the nucleus and then is expelled to the cytoplasm. Here, we demonstrate by in vivo aminomethyltrimethyl UV cross-linking studies that under SmD1 depletion, SLA1 remains bound to SL RNA and escorts the SL RNA to the cytoplasm. In situ hybridization with SLA1 and SL RNA demonstrates colocalization between SLA1 and the SL RNA transcription factor tSNAP42, as well as with Sm proteins, suggesting that SLA1 associates with SL RNA early in its biogenesis. These results demonstrate that SLA1 is a unique chaperonic RNA that functions during the early biogenesis of SL RNA to maintain a structure that is most probably suitable for cap 4 modification.

The fepB gene encodes a periplasmic binding protein that is essential for the uptake of ferric enterobactin by Escherichia coli. Its transcription is regulated in response to iron levels by the Fur repressor. The fepB transcript includes a 217-nucleotide leader sequence with several features suggestive of posttranscriptional regulation. To investigate the fepB leader for its contribution to fepB expression, defined deletions and substitution mutations in the leader were characterized using fepB-phoA translational fusions. The fepB leader was found to be necessary for maximal fepB expression, primarily due to the influence of an AU-rich translational enhancer (TE) located 5' to the Shine-Dalgarno sequence. Deletions or substitutions within the TE sequence decreased fepB-phoA expression fivefold. RNase protection and in vitro transcription-translation assays demonstrated that the TE augmented translational efficiency, as well as RNA levels. Moreover, primer extension inhibition assays showed that the TE increases ribosome binding. In contrast to the enhancing effect of the TE, the natural fepB GUG start codon decreased ribosome binding and reduced fepB expression 2.5-fold compared with the results obtained with leaders bearing an AUG initiation codon. Thus, the TE-GUG organization in fepB results in an intermediate level of expression compared to the level with AUG, with or without the TE. Furthermore, we found that the TE-GUG sequence is conserved among the eight gram-negative strains examined that have fepB genes, suggesting that this organization may provide a selective advantage.

Trypanosomatid parasites share a gene expression mode which differs greatly from that of their human and insect hosts. In these unicellular eukaryotes, protein-coding genes are transcribed polycistronically and individual mRNAs are processed from precursors by spliced leader (SL) trans splicing and polyadenylation. In trans splicing, the SL RNA is consumed through a transfer of its 5'-terminal part to the 5' end of mRNAs. Since all mRNAs are trans spliced, the parasites depend on strong and continuous SL RNA synthesis mediated by RNA polymerase II. As essential factors for SL RNA gene transcription in Trypanosoma brucei, the general transcription factor (GTF) IIB and a complex, consisting of the TATA-binding protein-related protein 4, the small nuclear RNA-activating protein complex, and TFIIA, were recently identified. Although T. brucei TFIIA and TFIIB are extremely divergent to their counterparts in other eukaryotes, their characterization suggested that trypanosomatids do form a class II transcription preinitiation complex at the SL RNA gene promoter and harbor orthologues of other known GTFs. TFIIH is a GTF which functions in transcription initiation, DNA repair, and cell cycle control. Here, we investigated whether a T. brucei TFIIH is important for SL RNA gene transcription and found that silencing the expression of the highly conserved TFIIH subunit XPD in T. brucei affected SL RNA gene synthesis in vivo, and depletion of this protein from extract abolished SL RNA gene transcription in vitro. Since we also identified orthologues of the TFIIH subunits XPB, p52/TFB2, and p44/SSL1 copurifying with TbXPD, we concluded that the parasite harbors a TFIIH which is indispensable for SL RNA gene transcription.

In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5' ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m(7)G cap, ribose 2'-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2'-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A(2) and is not required for subsequent steps; TbMT511 methylates C(3), without which U(4) methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m(7)G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A(2), C(3), and U(4) methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.

Transcription by RNA polymerase II in trypanosomes deviates from the standard eukaryotic paradigm. Genes are transcribed polycistronically and subsequently cleaved into functional mRNAs, requiring trans splicing of a capped 39-nucleotide leader RNA derived from a short transcript, the spliced leader (SL) RNA. The only identified trypanosome RNA polymerase II promoter is that of the SL RNA gene. We have previously shown that transcription of SL RNA requires divergent trypanosome homologs of RNA polymerase II, TATA binding protein, and the small nuclear RNA (snRNA)-activating protein complex. In other eukaryotes, TFIIB is an additional key component of transcription for both mRNAs and polymerase II-dependent snRNAs. We have identified a divergent homolog of the usually highly conserved basal transcription factor, TFIIB, from the pathogenic parasite Trypanosoma brucei. T. brucei TFIIB (TbTFIIB) interacted directly with the trypanosome TATA binding protein and RNA polymerase II, confirming its identity. Functionally, in vitro transcription studies demonstrated that TbTFIIB is indispensable in SL RNA gene transcription. RNA interference (RNAi) studies corroborated the essential nature of TbTFIIB, as depletion of this protein led to growth arrest of parasites. Furthermore, nuclear extracts prepared from parasites depleted of TbTFIIB, after the induction of RNAi, required recombinant TbTFIIB to support spliced leader transcription. The information gleaned from TbTFIIB studies furthers our understanding of SL RNA gene transcription and the elusive overall transcriptional processes in trypanosomes.

RNA polymerase II of trypanosomes, early diverging eukaryotes, transcribes long polycistronic messages, which are not capped but are processed by trans-splicing and polyadenylation to form mature mRNAs. The same RNA polymerase II also transcribes the genes coding for the spliced leader RNA, which are capped, exported to the cytoplasm, processed, and reimported into the nucleus before they are used as splicing donors to form mRNAs from pre-mRNA polycistronic transcripts. As pre-mRNA and spliced leader transcription events appear to be uncoupled, we studied how the RNA polymerase II is distributed in the nucleus of Trypanosoma cruzi. Using specific antibodies to the T. cruzi RNA polymerase II unique carboxy-terminal domain, we demonstrated that large amounts of the enzyme are found concentrated in a domain close to the parasite nucleolus and containing the spliced leader genes. The remaining RNA polymerase II is diffusely distributed in the nucleoplasm. The spliced leader-associated RNA polymerase II localization is dependent on the cell transcriptional state. It disperses when transcription is blocked by alpha-amanitin and actinomycin D. Tubulin genes are excluded from this domain, suggesting that it may exclusively be the transcriptional site of spliced leader genes. Trypomastigote forms of the parasite, which have reduced spliced leader transcription, show less RNA polymerase II labeling, and the spliced leader genes are more dispersed in the nucleoplasm. These results provide strong evidences that transcription of spliced leader RNAs occurs in a particular domain in the T. cruzi nucleus.

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.

While the 5' cis-acting sequence of mouse hepatitis virus (MHV) for genomic RNA replication has been determined in several defective interfering (DI) RNA systems, it remains elusive for subgenomic RNA transcription. Previous studies have shown that the leader RNA in the DI genome significantly enhances the efficiency of DI subgenomic mRNA transcription, indicating that the leader RNA is a cis-acting sequence for mRNA transcription. To further characterize the cis-acting sequence, we made a series of deletion mutants, all but one of which have an additional deletion of the cis-acting signal for replication in the 5' untranslated region. This deletion effectively eliminated the replication of the DI-chloramphenicol acetyltransferase (CAT)-reporter, as demonstrated by the sensitive reverse transcription (RT)-PCR. The ability of these replication-minus mutants to transcribe subgenomic mRNAs was then assessed using the DI RNA-CAT reporter system. Results from both CAT activity and mRNA transcripts detected by RT-PCR showed that a 5'-proximal sequence of 35 nucleotides (nt) at nt 25 to 59 is a cis-acting sequence required for subgenomic RNA transcription, while the consensus repeat sequence of the leader RNA does not have such effect. Analyses of the secondary structure indicate that this 35-nt sequence forms two stem-loops conserved among MHVs. Deletion of this sequence abrogated transcriptional activity and disrupted the predicted stem-loops and overall RNA secondary structure at the 5' untranslated region, suggesting that the secondary structure formed by this 35-nt sequence may facilitate the downstream consensus sequence accessible for the discontinuous RNA transcription. This may provide a mechanism by which the 5' cis-acting sequence regulates subgenomic RNA transcription. The 5'-most 24 nt are not essential for transcription, while the 9 nt immediately downstream of the leader enhances RNA transcription. The sequence between nt 86 and 135 had little effect on transcription. This study thus defines the cis-acting transcription signal at the 5' end of the DI genome.