Trimethylguanosine Cap Research Articles

Trimethylguanosine cap-fluorescent molecular rotor (TMG-FMR) conjugates are potent, specific snurportin1 ligands enabling visualization in living cells.

The trimethylguanosine (TMG) cap is a motif present inter alia at the 5' end of small nuclear RNAs, which are involved in RNA splicing. The TMG cap plays a crucial role in RNA processing and stability as it protects the RNA molecule from degradation by exonucleases and facilitates its export from the nucleus. Additionally, the TMG cap plays a role in the recognition of snRNA by snurportin, a protein that facilitates nuclear import. TMG cap analogs are used in biochemical experiments as molecular tools to substitute the natural TMG cap. To expand the range of available TMG-based tools, here we conjugated the TMG cap to Fluorescent Molecular Rotors (FMRs) to open the possibility of detecting protein-ligand interactions in vitro and, potentially, in vivo, particularly visualizing interactions with snurportin. Consequently, we report the synthesis of 34 differently modified TMG cap-FMR conjugates and their evaluation as molecular probes for snurportin. As FMRs we selected three GFP-like chromophores (derived from green fluorescent protein) and one julolidine derivative. The evaluation of binding affinities for snurportin showed unexpectedly a strong stabilizing effect for TMGpppG-derived dinucleotides containing the FMR at the 2'-O-position of guanosine. These newly discovered compounds are potent snurportin ligands with nanomolar KD (dissociation constant) values, which are two orders of magnitude lower than that of natural TMGpppG. The effect is diminished by ∼50-fold for the corresponding 3'-regioisomers. To deepen the understanding of the structure-activity relationship, we synthesized and tested FMR conjugates lacking the TMG cap moiety. These studies, supported by molecular docking, suggested that the enhanced affinity arises from additional hydrophobic contacts provided by the FMR moiety. The strongest snurportin ligand, which also gave the greatest fluorescence enhancement (Fm/F0) when saturated with the protein, were tested in living cells to detect interactions and visualize complexes by fluorescence lifetime monitoring. This approach has potential applications in the study of RNA processing and RNA-protein interactions.

TGS1 impacts snRNA 3'-end processing, ameliorates survival motor neuron-dependent neurological phenotypes in vivo and prevents neurodegeneration.

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5'-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3' tails that are often uridylated. snRNAs with defective 3' terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

Trimethylguanosine synthase 1 (Tgs1) is involved in Swi6/HP1-independent siRNA production and establishment of heterochromatin in fission yeast.

In fission yeast, siRNA generated by RNA interference (RNAi) factors plays critical roles in establishment and maintenance of heterochromatin. To achieve efficient siRNA synthesis, RNAi factors assemble on heterochromatin via association with Swi6, a homologue of heterochromatin protein 1 (HP1), and heterochromatic noncoding RNA (hncRNA) retained on chromatin. In addition, spliceosomes formed on hncRNA introns recruit RNAi factors to hncRNA and heterochromatin. Small nuclear RNAs, components of the spliceosome, have a trimethylguanosine (TMG) cap that is generated by Tgs1-dependent hypermethylation of the normal m7G cap; this cap is required for efficient splicing of some mRNAs in budding yeast and Drosophila. In this study, we found that loss of Tgs1 in fission yeast destabilizes centromeric heterochromatin. Tgs1 was required for Swi6-independent siRNA synthesis, as well as for the establishment of centromeric heterochromatin. Loss of Tgs1 affected the splicing efficiency of hncRNA introns in the absence of Swi6. Furthermore, some hncRNAs have a TMG cap, and we found that loss of Tgs1 diminished the chromatin binding of these hncRNAs. Together, these results suggest that the Tgs1-dependent TMG cap plays critical roles in establishment of heterochromatin by ensuring spliceosome-dependent recruitment of RNAi factors and regulating the binding between chromatin and hncRNA.

Loss of the RNA trimethylguanosine cap is compatible with nuclear accumulation of spliceosomal snRNAs but not pre-mRNA splicing or snRNA processing during animal development.

The 2,2,7-trimethylguanosine (TMG) cap is one of the first identified modifications on eukaryotic RNAs. TMG, synthesized by the conserved Tgs1 enzyme, is abundantly present on snRNAs essential for pre-mRNA splicing. Results from ex vivo experiments in vertebrate cells suggested that TMG ensures nuclear localization of snRNAs. Functional studies of TMG using tgs1 mutations in unicellular organisms yield results inconsistent with TMG being indispensable for either nuclear import or splicing. Utilizing a hypomorphic tgs1 mutation in Drosophila, we show that TMG reduction impairs germline development by disrupting the processing, particularly of introns with smaller sizes and weaker splice sites. Unexpectedly, loss of TMG does not disrupt snRNAs localization to the nucleus, disputing an essential role of TMG in snRNA transport. Tgs1 loss also leads to defective 3’ processing of snRNAs. Remarkably, stronger tgs1 mutations cause lethality without severely disrupting splicing, likely due to the preponderance of TMG-capped snRNPs. Tgs1, a predominantly nucleolar protein in Drosophila, likely carries out splicing-independent functions indispensable for animal development. Taken together, our results suggest that nuclear import is not a conserved function of TMG. As a distinctive structure on RNA, particularly non-coding RNA, we suggest that TMG prevents spurious interactions detrimental to the function of RNAs that it modifies.

Intimate functional interactions betweenTGS1 and the Smncomplex revealed by an analysis of the Drosophila eye development.

Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to understand the molecular mechanisms underlying SMA pathogenesis and devise new therapies for this genetic disease.

Loss of Human TGS1 Hypermethylase Promotes Increased Telomerase RNA and Telomere Elongation

SUMMARYBiogenesis of the human telomerase RNA (hTR) involves a complex series of posttranscriptional modifications, including hypermethylation of the 5′ mono-methylguanosine cap to a tri-methylguanosine cap (TMG). How the TMG cap affects hTR maturation is unknown. Here, we show that depletion of trimethylguanosine synthase 1 (TGS1), the enzyme responsible for cap hypermethylation, increases levels of hTR and telomerase. Diminished trimethylation increases hTR association with the cap-binding complex (CBC) and with Sm chaperone proteins. Loss of TGS1 causes an increase in accumulation of mature hTR in both the nucleus and the cytoplasm compared with controls. In TGS1 mutant cells, increased hTR assembles with telomerase reverse transcriptase (TERT) protein to yield elevated active telomerase complexes and increased telomerase activity, resulting in telomere elongation in cultured human cells. Our results show that TGS1-mediated hypermethylation of the hTR cap inhibits hTR accumulation, restrains levels of assembled telomerase, and limits telomere elongation.

Functional characterization of the translation initiation factor eIF4E of Echinococcus granulosus.

The eukaryotic initiation factor 4E (eIF4E) specifically recognizes the 5' mRNA cap, a rate-limiting step in the translation initiation process. Although the 7-methylguanosine cap (MMGcap) is the most common 5' cap structure in eukaryotes, the trans-splicing process that occurs in several organism groups, including nematodes and flatworms, leads to the addition of a trimethylguanosine cap (TMGcap) to some RNA transcripts. In some helminths, eIF4E can have a dual capacity to bind both MMGcap and TMGcap. In the present work, we evaluated the distribution of eIF4E protein sequences in platyhelminths and we showed that only one gene coding for eIF4E is present in most parasitic flatworms. Based on this result, we cloned the Echinococcus granulosus cDNA sequence encoding eIF4E in Escherichia coli, expressed the recombinant eIF4E as a fusion protein to GST, and tested its ability to capture mRNAs through the 5' cap using pull-down assay and qPCR. Our results indicate that the recombinant eIF4E was able to bind preferentially 5'-capped mRNAs compared with rRNAs from total RNA preparations of E. granulosus. By qPCR, we observed an enrichment in MMG-capped mRNA compared with TMG-capped mRNAs among Eg-eIF4E-GST pull-down RNAs. Eg-eIF4E structural model using the Schistosoma mansoni eIF4E as template showed to be well preserved with only a few differences between chemically similar amino acid residues at the binding sites. These data showed that E. granulosus eIF4E can be used as a potential tool to study full-length 5'-capped mRNA, besides being a potential drug target against parasitic flatworms.

Clickable trimethylguanosine cap analogs modified within the triphosphate bridge: synthesis, conjugation to RNA and susceptibility to degradation

Phosphate-modified m3G cap analogs were synthesized, conjugated to RNA using “click chemistry”, and studied for susceptibility to hNUDT16 enzyme.

Two Routes to Genetic Suppression of RNA Trimethylguanosine Cap Deficiency via C-Terminal Truncation of U1 snRNP Subunit Snp1 or Overexpression of RNA Polymerase Subunit Rpo26.

The trimethylguanosine (TMG) caps of small nuclear (sn) RNAs are synthesized by the enzyme Tgs1 via sequential methyl additions to the N2 atom of the m7G cap. Whereas TMG caps are inessential for Saccharomyces cerevisiae vegetative growth at 25° to 37°, tgs1∆ cells that lack TMG caps fail to thrive at 18°. The cold-sensitive defect correlates with ectopic stoichiometric association of nuclear cap-binding complex (CBC) with the residual m7G cap of the U1 snRNA and is suppressed fully by Cbc2 mutations that weaken cap binding. Here, we show that normal growth of tgs1∆ cells at 18° is also restored by a C-terminal deletion of 77 amino acids from the Snp1 subunit of yeast U1 snRNP. These results underscore the U1 snRNP as a focal point for TMG cap function in vivo. Casting a broader net, we conducted a dosage suppressor screen for genes that allowed survival of tgs1∆ cells at 18°. We thereby recovered RPO26 (encoding a shared subunit of all three nuclear RNA polymerases) and RPO31 (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of tgs1∆ cold sensitivity, respectively. A structure-guided mutagenesis of Rpo26, using rpo26∆ complementation and tgs1∆ suppression as activity readouts, defined Rpo26-(78-155) as a minimized functional domain. Alanine scanning identified Glu89, Glu124, Arg135, and Arg136 as essential for rpo26∆ complementation. The E124A and R135A alleles retained tgs1∆ suppressor activity, thereby establishing a separation-of-function. These results illuminate the structure activity profile of an essential RNA polymerase component.

Towards novel efficient and stable nuclear import signals: synthesis and properties of trimethylguanosine cap analogs modified within the 5',5'-triphosphate bridge.

A trimethylguanosine (TMG) cap is present at the 5' end of several small nuclear and nucleolar RNAs. Recently, it has been reported that the TMG cap is a potential nuclear import signal for nucleus-targeting therapeutic nucleic acids and proteins. The import is mediated by recognition of the TMG cap by the snRNA transporting protein, snurportin1. This work describes the synthesis and properties of a series of dinucleotide TMG cap (m3(2,2,7)GpppG) analogs modified in the 5',5'-triphosphate bridge as tools to study TMG cap-dependent biological processes. The bridge was altered at different positions by introducing either bridging (imidodiphosphate, O to NH and methylenebisphosphonate, O to CH2) or non-bridging (phosphorothioate, O to S and boranophosphate, O to BH3) modifications, or by elongation to tetraphosphate. The stability of novel analogs in blood serum was studied to reveal that the α,β-bridging O to NH substitution (m3(2,2,7)GppNHpG) confers the highest resistance. Short RNAs capped with analogs containing α,β-bridging (m3(2,2,7)GppNHpG) or β-non-bridging (m3(2,2,7)GppSpG D2) modifications were resistant to decapping pyrophosphatase, hNudt16. Preliminary studies on binding by human snurportin1 revealed that both O to NH and O to S substitutions support this binding. Due to favorable properties in all three assays, m3(2,2,7)GppNHpG was selected as a promising candidate for further studies on the efficiency of the TMG cap as a nuclear import signal.

Identification and characterization ofDrosophilaSnurportin reveals a role for the import receptor Moleskin/importin-7 in snRNP biogenesis

Nuclear import is an essential step in small nuclear ribonucleoprotein (snRNP) biogenesis. Snurportin1 (SPN1), the import adaptor, binds to trimethylguanosine (TMG) caps on spliceosomal small nuclear RNAs. Previous studies indicated that vertebrate snRNP import requires importin-β, the transport receptor that binds directly to SPN1. We identify CG42303/snup as the Drosophila orthologue of human snurportin1 (SNUPN). Of interest, the importin-β binding (IBB) domain of SPN1, which is essential for TMG cap-mediated snRNP import in humans, is not well conserved in flies. Consistent with its lack of an IBB domain, we find that Drosophila SNUP (dSNUP) does not interact with Ketel/importin-β. Fruit fly snRNPs also fail to bind Ketel; however, the importin-7 orthologue Moleskin (Msk) physically associates with both dSNUP and spliceosomal snRNPs and localizes to nuclear Cajal bodies. Strikingly, we find that msk-null mutants are depleted of the snRNP assembly factor, survival motor neuron, and the Cajal body marker, coilin. Consistent with a loss of snRNP import function, long-lived msk larvae show an accumulation of TMG cap signal in the cytoplasm. These data indicate that Ketel/importin-β does not play a significant role in Drosophila snRNP import and demonstrate a crucial function for Msk in snRNP biogenesis.

Structure-function analysis of the 5′ end of yeast U1 snRNA highlights genetic interactions with the Msl5•Mud2 branchpoint-binding complex and other spliceosome assembly factors

Yeast pre-mRNA splicing initiates via formation of a complex comprising U1 snRNP bound at the 5′ splice site (5′SS) and the Msl5•Mud2 heterodimer engaged at the branchpoint (BP). Here, we present a mutational analysis of the U1 snRNA, which shows that although enlarging the 5′ leader between the TMG cap and the 3ACUUAC8 motif that anneals to the 5′SS is tolerated, there are tight constraints on the downstream spacer between 3ACUUAC8 and helix 1 of the U1 fold. We exploit U1 alleles with 5′ extensions, variations in the 3ACUUAC8 motif, downstream mutations and a longer helix 1 to discover new intra-snRNP synergies with U1 subunits Nam8 and Mud1 and the trimethylguanosine (TMG) cap. We describe novel mutations in U1 snRNA that bypass the essentiality of the DEAD-box protein Prp28. Structure-guided mutagenesis of Msl5 distinguished four essential amino acids that contact the BP sequence from nine other BP-binding residues that are inessential. We report new synthetic genetic interactions of the U1 snRNP with Msl5 and Mud2 and with the nuclear cap-binding subunit Cbc2. Our results fortify the idea that spliceosome assembly can occur via distinct genetically buffered microscopic pathways involving cross-intron-bridging interactions of the U1 snRNP•5′SS complex with the Mud2•Msl5•BP complex.

Genetic interactions of hypomorphic mutations in the m7G cap-binding pocket of yeast nuclear cap binding complex: an essential role for Cbc2 in meiosis via splicing of MER3 pre-mRNA.

Nuclear cap binding protein complex (CBC) is a heterodimer of a small subunit (Cbc2 in yeast) that binds the m(7)G cap and a large subunit (Sto1 in yeast) that interacts with karyopherins. In order to probe the role of cap recognition in yeast CBC function, we introduced alanine mutations (Y24A, F91A, D120A, D122A, R129A, and R133A) and N-terminal deletions (NΔ21 and NΔ42) in the cap-binding pocket of Cbc2. These lesions had no effect on vegetative growth, but they ameliorated the cold-sensitivity of tgs1Δ cells that lack trimethylguanosine caps (a phenotype attributed to ectopic association of CBC with the m(7)G cap of the normally TMG-capped U1 snRNA), thereby attesting to their impact on cap binding in vivo. Further studies of the Cbc2-Y24A variant revealed synthetic lethality or sickness with null mutations of proteins involved in early steps of spliceosome assembly (Nam8, Mud1, Swt21, Mud2, Ist3, and Brr1) and with otherwise benign mutations of Msl5, the essential branchpoint binding protein. Whereas the effects of weakening CBC-cap interactions are buffered by other actors in the splicing pathway during mitotic growth, the NΔ42 allele causes a severe impediment to yeast sporulation and meiosis. RNA analysis revealed a selective defect in the splicing of MER3 and SAE3 transcripts in cbc2-NΔ42 diploids during attempted sporulation. An intronless MER3 cDNA fully restored sporulation and spore viability in the cbc2-NΔ42 strain, signifying that MER3 splicing is a limiting transaction. These studies reveal a new level of splicing control during meiosis that is governed by nuclear CBC.

Synthesis of 13C- and 14C-labeled dinucleotide mRNA cap analogues for structural and biochemical studies

Herein we describe the first simple and short method for specific labeling of mono- and trimethylated dinucleotide mRNA cap analogues with 13C and 14C isotopes. The labels were introduced within the cap structures either at the N7 for monomethylguanosine cap or N7 and N2 position for trimethylguanosine cap. The compounds designed for structural and biochemical studies will be useful tools for better understanding the role of the mRNA cap structures in pre-mRNA splicing, nucleocytoplasmic transport, translation initiation and mRNA degradation.

Defining the Mer1 and Nam8 meiotic splicing regulons by cDNA rescue

Meiosis-specific pre-mRNA splicing in budding yeast embraces multiple pre-mRNA targets grouped into regulons defined by their genetic requirements for vegetatively optional splicing factors (e.g., splicing enhancer Mer1 and the U1 snRNP subunit Nam8) or snRNA modifications (trimethylguanosine caps). Here, we genetically demarcate a complete meiotic splicing regulon by the criterion of cDNA bypass of the requirement for the governing splicing regulators to execute sporulation. We thereby show that the Mer1 and Nam8 regulons embrace four essential pre-mRNAs: MER2, MER3, SPO22, and AMA1. Whereas Nam8 also regulates PCH2 splicing, PCH2 cDNA is not needed for sporulation by nam8Δ diploids. Our results show that there are no essential intron-containing RNAs missing from the known roster of Mer1 and Nam8 targets. Nam8 is composed of three RRM domains, flanked by N-terminal leader and C-terminal tail segments. We find that the RRM2 and RRM3 domains, and their putative RNA-binding sites, are essential for yeast sporulation, whereas the leader, tail, and RRM1 modules are not.

Composition of yeast snRNPs and snoRNPs in the absence of trimethylguanosine caps reveals nuclear cap binding protein as a gained U1 component implicated in the cold-sensitivity of tgs1Δ cells

Small nuclear and nucleolar RNAs that program pre-mRNA splicing and rRNA processing have a signature 5′-trimethylguanosine (TMG) cap. Whereas the mechanism of TMG synthesis by Tgs1 methyltransferase has been elucidated, we know little about whether or how RNP biogenesis, structure and function are perturbed when TMG caps are missing. Here, we analyzed RNPs isolated by tandem-affinity purification from TGS1 and tgs1Δ yeast strains. The protein and U-RNA contents of total SmB-containing RNPs were similar. Finer analysis revealed stoichiometric association of the nuclear cap-binding protein (CBP) subunits Sto1 and Cbc2 with otherwise intact Mud1- and Nam8-containing U1 snRNPs from tgs1Δ cells. CBP was not comparably enriched in Lea1-containing U2 snRNPs from tgs1Δ cells. Moreover, CBP was not associated with mature Nop58-containing C/D snoRNPs or mature Cbf5- and Gar1-containing H/ACA snoRNPs from tgs1Δ cells. The protein composition and association of C/D snoRNPs with the small subunit (SSU) processosome were not grossly affected by absence of TMG caps, nor was the composition of H/ACA snoRNPs. The cold-sensitive (cs) growth defect of tgs1Δ yeast cells could be suppressed by mutating the cap-binding pocket of Cbc2, suggesting that ectopic CBP binding to the exposed U1 m7G cap in tgs1Δ cells (not lack of TMG caps per se) underlies the cs phenotype.

Nuclear activity of sperm cells during Hyacinthus orientalis L. in vitro pollen tube growth

In this study, the transcriptional state and distribution of RNA polymerase II, pre-mRNA splicing machinery elements, and rRNA transcripts were investigated in the sperm cells of Hyacinthus orientalis L. during in vitro pollen tube growth. During the second pollen mitosis, no nascent transcripts were observed in the area of the dividing generative cell, whereas the splicing factors were present and their pools were divided between newly formed sperm cells. Just after their origin, the sperm cells were shown to synthesize new RNA, although at a markedly lower level than the vegetative nucleus. The occurrence of RNA synthesis was accompanied by the presence of RNA polymerase II and a rich pool of splicing machinery elements. Differences in the spatial pattern of pre-mRNA splicing factors localization reflect different levels of RNA synthesis in the vegetative nucleus and sperm nuclei. In the vegetative nucleus, they were localized homogenously, whereas in the sperm nuclei a mainly speckled pattern of small nuclear RNA with a trimethylguanosine cap (TMG snRNA) and SC35 protein distribution was observed. As pollen tube growth proceeded, inhibition of RNA synthesis in the sperm nuclei was observed, which was accompanied by a gradual elimination of the splicing factors. In addition, analysis of rRNA localization indicated that the sperm nuclei are likely to synthesize some pool of rRNA at the later steps of pollen tube. It is proposed that the described changes in the nuclear activity of H. orientalis sperm cells reflect their maturation process during pollen tube growth, and that mature sperm cells do not carry into the zygote the nascent transcripts or the splicing machinery elements.

The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.

5′ and 3′ end modifications of spliceosomal RNAs in Plasmodium falciparum

5' caps provide recognition sequences for the nuclear import of snRNAs. The 5' and 3' ends of snRNAs were studied in Plasmodium falciparum with a modified adapter ligation method, which showed that 5' ends of U1, U2, U4, U5 and U6 snRNAs are capped. In P. falciparum, the 3' ends of U1, U2, U4 and U5 snRNAs have free hydroxyl groups whereas U6 snRNA has a blocked 3' end. An immunoprecipitation assay for trimethyl guanosine caps shows that the cap structures of parasite U1-U5 snRNAs are hypermethylated while U6 snRNA may be gamma-mono-methylated. Bioinformatics analysis of proteins involved in hypermethylation and trafficking of snRNAs indicates that the methyltransferase TGS1 is present in the P. falciparum genome. PfTGS1 is larger than its orthologs and may have transmembrane domains in the C-terminus. Surprisingly, the snRNA trafficking protein Snurportin is absent from the P. falciparum genome suggesting that reminiscent of yeast, parasite snRNAs may be retained in the nucleus.

In new company: U1 snRNA associates with TAF15

In new company: U1 snRNA associates with TAF15