On the role of exon and intron sequences in trans-splicing utilization and cap 4 modification of the trypanosomatid Leptomonas collosoma SL RNA

In trypanosomatid protozoa the biogenesis of mature mRNA involves addition of the spliced leader (SL) sequence from the SL RNA to polycistronic pre-mRNA via trans-splicing. Here we present a mutational analysis of the trypanosomatid Leptomonas collosoma SL RNA to further our understanding of its functional domains important for trans-splicing utilization. Mutant SL RNAs were analyzed for defects in modification of the hypermethylated cap structure (cap 4) characteristic of trypanosomatid SL RNAs, for defects in the first step of the reaction and overall utilization in trans-splicing. Single substitution of the cap 4 nucleotides led to undermethylation of the cap 4 structure, and these mutants were all impaired in their utilization in trans-splicing. Abrogation of the sequence of the Sm-like site and sequences downstream to it also showed cap modification and trans-splicing defects, thus providing further support for a functional linkage between cap modifications and trans-splicing. Further, we report that in L. collosoma both the exon and intron of the SL RNA contribute information for efficient function of the SL RNA in trans-splicing. This study, however, did not provide support for the putative SL RNA-U6 small nuclear RNA (snRNA) interaction at the Sm site like in the nematodes, suggesting differences in the bridging role of U6 in the two trans-splicing systems.     

Trypanosoma brucei encodes a bifunctional capping enzyme essential for cap 4 formation on the spliced leader RNA

The 5' end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m7GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA.     

Mass spectrometry of mRNA cap 4 from trypanosomatids reveals two novel nucleosides.

Synthesis of mRNA in kinetoplastid protozoa involves the process of trans-splicing, in which an identical 39-41-nucleotide (depending on the species) mini-exon is placed at the 5' end of mature mRNAs. The mini-exon sequence is highly conserved among all members of the Kinetoplastida, nucleotides 1-6 being identical in the four genera so far examined. Prior to trans-splicing, the mini-exon donor RNA is capped by the addition of a (5'-5') triphosphate-linked 7-methylguanosine, followed by modification of the first four transcribed nucleotides. Partial structures have been previously deduced for this cap 4 moiety from Trypanosoma brucei and Leptomonas collosoma. We have purified enough cap 4 from T. brucei and Crithidia fasciculata to allow definitive structural analysis by combined liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry. The results, together with the known mini-exon sequence, show that cap 4 in both species has the structure m7G(5')ppp(5')m6(2)AmpAmpCmpm3Ump. The presence of N6,N6,2'-O-trimethyladenosine and 3,2'-O-dimethyluridine, nucleosides previously unknown in nature, were confirmed by rigorous comparison with synthetic standards. The conservation of cap 4 between these divergent genera suggests that this structure may be common to most if not all Kinetoplastida.     

A protein related to the vaccinia virus cap-specific methyltransferase VP39 is involved in cap 4 modification in Trypanosoma brucei

The spliced-leader (SL) RNA plays a key role in the biogenesis of mRNA in trypanosomes by providing the m(7)G-capped SL sequence to the 5' end of every mRNA. The cap structure of the SL RNA is unique in eukaryotes with 4 nucleotides after the cap carrying a total of seven methyl groups and by convention is referred to as "cap 4". Although the enzymatic machinery for cap addition has been characterized in several organisms, including Trypanosoma brucei, the identification of methyltransferases dedicated to the generation of higher order cap structures has lagged behind, except in viruses. Here we describe T. brucei MT57 (TbMT57), a primarily nuclear polypeptide with structural and functional similarities to vaccinia virus VP39, a bifunctional protein acting at the mRNA 5' end as a cap-specific 2'-O-methyltransferase. Down-regulation by RNAi or genetic ablation of TbMT57 resulted in the accumulation of SL RNA missing 2'-O-methyl groups at positions +3 and +4 and thus bearing a cap 2 rather than a cap 4. Furthermore, competitive binding studies indicated that modifications at the +3 and +4 positions are important for binding to the nuclear cap-binding complex. Genetic ablation of MT57 resulted in viable cells with no apparent defect in SL RNA trans-splicing, suggesting that MT57 is not essential or that trypanosomes have developed alternate mechanisms to counteract the absence of this protein. Interestingly, MT57 homologs are only found in trypanosomatid protozoa that have a cap 4 structure and in poxviruses, of which vaccinia virus is a prototype.     

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome.     

Down-regulation of the trypanosomatid signal recognition particle affects the biogenesis of polytopic membrane proteins but not of signal peptide-containing proteins

Protein translocation across the endoplasmic reticulum is mediated by the signal recognition particle (SRP). In this study, the SRP pathway in trypanosomatids was down-regulated by two approaches: RNA interference (RNAi) silencing of genes encoding SRP proteins in Trypanosoma brucei and overexpression of dominant-negative mutants of 7SL RNA in Leptomonas collosoma. The biogenesis of both signal peptide-containing proteins and polytopic membrane proteins was examined using endogenous and green fluorescent protein-fused proteins. RNAi silencing of SRP54 or SRP68 in T. brucei resulted in reduced levels of polytopic membrane proteins, but no effect on the level of signal peptide-containing proteins was observed. When SRP deficiency was achieved in L. collosoma by overexpression of dominant-negative mutated 7SL RNA, a major effect was observed on polytopic membrane proteins but not on signal peptide-containing proteins. This study included two trypanosomatid species, tested various protein substrates, and induced depletion of the SRP pathway by affecting either the levels of SRP binding proteins or that of SRP RNA. Our results demonstrate that, as in bacteria but in contrast to mammalian cells, the trypanosome SRP is mostly essential for the biogenesis of membrane proteins.     

Discontinuously synthesized mRNA from Trypanosoma brucei contains the highly methylated 5' cap structure, m7GpppA*A*C(2'-O)mU*A

Trypanosoma brucei mRNA is discontinuously synthesized via the 5' addition of a "mini-exon" sequence. The mini-exon-specific cap structure was purified from a complete RNase T2 and phosphatase digest of in vivo 32P-labeled poly(A)+RNA. The purified cap structure was sequenced by a series of partial and complete enzymatic digests by nuclease P1 and venom phosphodiesterase. This approach demonstrated that the T. brucei mini-exon cap structure consists of N7-methylguanosine linked in a conventional 5'-5' triphosphate bond to five nucleotides, in the sequence A*A*C(2'-O)mU*A (asterisks denote modifications that were not fully characterized in this work). 2'-O-methylations and other modifications appear to be present in this novel cap structure, which could have a functional role in the metabolism of the mini-exon.     

Complete cap 4 formation is not required for viability in Trypanosoma brucei

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.     

Spectrin deficient inherited hemolytic anemias in the mouse: Characterization by spectrin synthesis and mRNA activity in reticulocytes

The 35 nucleotide spliced leader (SL) sequence is found on the 5' end of numerous trypanosome mRNAs, yet the tandemly organized reiteration units encoding this leader are not detectably linked to any of these structural genes. Here we report the presence of a class of discrete small SL RNA molecules that are derived from the genomic SL reiteration units of Trypanosoma brucei, Trypanosoma cruzi, and Leptomonas collosoma. These small SL RNAs are 135, 105, and 95 nucleotides, respectively, and contain a 5'-terminal SL or SL-like sequence. S1 nuclease analyses demonstrate that these small SL RNAs are transcribed from continuous sequence within the respective SL reiteration units. With the exception of the SL sequence and a concensus donor splice site immediately following it, these small RNAs are not well conserved. We suggest that the small SL RNAs may function as a donor of the SL sequence in an intermolecular process that places the SL at the 5' terminus of many trypanosomatid mRNAs.     

Changes in 7SL RNA conformation during the signal recognition particle cycle.

The structure of 7SL RNA has been probed by chemical modification followed by primer extension, using four substrates: (i) naked 7SL RNA; (ii) free signal recognition particle (SRP); (iii) polysome bound SRP; and (iv) membrane bound SRP. Decreasing sensitivity to chemical modification between these different substrates suggests regions on 7SL RNA that: bind proteins associated with SRP might interact with ribosomes; and are protected by binding to membranes. Other areas increase in chemical sensitivity, exemplified by a tertiary interaction present in naked 7SL RNA but not in free SRP. Such changes suggest that 7SL RNA changes its conformation during the SRP cycle. These conformational changes could be a necessary component to move through the SRP cycle from one stage to the next.     

Evidence for the presence of a small U5-like RNA in active trans-spliceosomes of Trypanosoma brucei.

The existence of the Trypanosoma brucei 5' splice site on a small RNA of uniform sequence (the spliced leader or SL RNA) has allowed us to characterize the RNAs with which it interacts in vivo by psoralen crosslinking treatment. Analysis of the most abundant crosslinks formed by the SL RNA allowed us previously to identify the spliced leader-associated (SLA) RNA. The role of this RNA in trans-splicing, as well as the possible existence of an analogous RNA interaction in cis-splicing, is unknown. We show here that the 5' splice site region of the SL RNA is also crosslinked in vivo to a second small RNA. Although it is very small and lacks a 5' trimethylguanosine (TMG) cap, the SLA2RNA possesses counterparts of the conserved U5 snRNA stem-loop 1 and internal loop 1 sequence elements, as well as a potential trypanosome snRNA core protein binding site; these combined features meet the phylogenetic definition of U5 snRNA. Like U5, the SLA2 RNA forms an RNP complex with the U4 and U6 RNAs, and interacts with the 5' splice site region via its putative loop 1 sequence. In a final analogy with U5, the SLA2 RNA is found crosslinked to a molecule identical to the free 5' exon splicing intermediate. These data present a compelling case for the SLA2 RNA not only as an active trans-spliceosomal component, but also for its identification as the trypanosome U5 structural homolog. The presence of a U5-like RNA in this ancient eukaryote establishes the universality of the spliceosomal RNA core components.     

Structure-function analysis of the trypanosomatid spliced leader RNA.

In trypanosomes, all mRNAs possess a spliced leader (SL) at their 5' end. SL is added to pre-mRNA via trans -splicing from a small RNA, the SL RNA. To examine structure-function aspects of the trypanosomatid SL RNA, an in vivo system was developed in the monogenetic trypanosomatid Leptomonas collosoma to analyze the function of chimeric and site-directed SL RNA mutants in trans -splicing. Stable cell lines expressing chimeric and mutated SL RNA from the authentic SL RNA regulatory unit were obtained. The chimeric RNA was expressed and assembled into an SL RNP particle, but could not serve as a substrate in splicing. Mutations in loop II and III of L.collosoma SL RNA formed the Y structure intermediate. In addition, a double SL RNA mutant in loop II, and positions 7 and 8 of the intron, also formed the Y structure intermediate, suggesting that these intron positions, although proposed to participate in the interaction of SL RNA with U5, may not be crucial for the first step of the trans -splicing reaction. A mutation in the exon located in loop I was not utilized in splicing, suggesting the importance of exon sequences for trans -splicing in trypanosomes. However, a double SL RNA mutant in loop II and exon position 31 was utilized in both steps of splicing; the mutant thus provides a model molecule for further analysis of positions essential for the function of the SL RNA.     

Structural analyses of the 7SK ribonucleoprotein (RNP), the most abundant human small RNP of unknown function.

The human 7SK ribonucleoprotein (RNP) has been analyzed to determine its RNA secondary structure and protein constituents. HeLa cell 7SK RNA alone and within its RNP have been probed by chemical modification and enzymatic cleavage, and sites of modification or cleavage have been mapped by primer extension. The resulting secondary structure suggests that structural determinants necessary for capping (a 5' stem followed by the sequence AUPuUPuC) and nuclear migration (the sequence AUPuUPuC) of 7SK RNA may be similar to those for U6 small nuclear RNA (snRNA). It also supports existence of a 3' stem structure which could serve to self-prime cDNA synthesis during pseudogene formation. Oligonucleotide-directed RNase H digestion indicated regions of 7SK RNA capable of base pairing with other nucleic acids. Antisense 2'-O-methyl RNA oligonucleotides were used to affinity select the 7SK RNP from an in vivo 35S-labeled cell sonic extract and identify eight associated proteins of 83, 48, 45, 43, 42, 21, 18, and 13 kDa. 7SK RNA has extensive sequence complementarity to U4 snRNA, within the U4/U6 base pairing domain, and also to U11 snRNA. The possibility that the 7SK RNP is an unrecognized component of the pre-mRNA processing machinery is discussed.     

Silencing of Sm proteins in Trypanosoma brucei by RNA interference captured a novel cytoplasmic intermediate in spliced leader RNA biogenesis

In Trypanosoma brucei the small nuclear (sn) RNAs U1, U2, U4, and U5, as well as the spliced leader (SL) RNA, bind the seven Sm canonical proteins carrying the consensus Sm motif. To determine the function of these proteins in snRNA and SL RNA biogenesis, two of the Sm core proteins, SmE and SmD1, were silenced by RNAi. Surprisingly, whereas the level of all snRNAs, including U1, U2, U4, and U5 was reduced during silencing, the level of SL RNA was dramatically elevated, but the levels of U6 and spliced leader-associated RNA (SLA1) remained unchanged. The SL RNA that had accumulated in silenced cells lacked modification at the cap4 nucleotide but harbored modifications at the cap1 and cap2 nucleotides and carried the characteristic psi. This SL RNA possessed a longer tail and had accumulated in the cytoplasm in 10 and 50 S particles that were found by in situ hybridization to be present in "speckles." We propose a model for SL RNA biogenesis involving a cytoplasmic phase and suggest that the trypanosome-specific "cap4" nucleotides function as a signal for export and import of SL RNA out and into the nucleus. The SL RNA biogenesis pathway differs from that of U sn ribonucleoproteins (RNPs) in that it is the only RNA that binds Sm proteins that were stabilized under Sm depletion in a novel RNP, which we termed SL RNP-C.