Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS.

Analogs of the mRNA cap are widely employed to study processes involved in mRNA metabolism as well as being useful in biotechnology and medicinal applications. Here we describe synthesis of six dinucleotide cap analogs bearing a single phosphorothioate modification at either the alpha, beta, or gamma position of the 5',5'-triphosphate chain. Three of them were also modified with methyl groups at the 2'-O position of 7-methylguanosine to produce anti-reverse cap analogs (ARCAs). Due to the presence of stereogenic P centers in the phosphorothioate moieties, each analog was obtained as a mixture of two diastereomers, D1 and D2. The mixtures were resolved by RP HPLC, providing 12 different compounds. Fluorescence quenching experiments were employed to determine the association constant (K(AS)) for complexes of the new analogs with eIF4E. We found that phosphorothioate modifications generally stabilized the complex between eIF4E and the cap analog. The most strongly bound phosphorothioate analog (the D1 isomer of the beta-substituted analog m(7)Gpp(S)pG) was characterized by a K(AS) that was more than fourfold higher than that of its unmodified counterpart (m(7)GpppG). All analogs modified in the gamma position were resistant to hydrolysis by the scavenger decapping pyrophosphatase DcpS from both human and Caenorhabditis elegans sources. The absolute configurations of the diastereomers D1 and D2 of analogs modified at the alpha position (i.e., m(7)Gppp(S)G and m(2) (7,2'-O )Gppp(S)G) were established as S(P) and R(P) , respectively, using enzymatic digestion and correlation with the S(P) and R(P) diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.

Assignment of the absolute configuration of P-chiral 5' mRNA cap analogues containing phosphorothioate moiety.

Enzymatic cleavage of the P-chiral diastereoisomers of the 5' mRNA cap analogue bearing phosphorothioate moiety in alfa position of 5',5'-triphosphate bridge (m(7)Gppp(S)G D1 and D2) was performed by human Decapping Scavenger (DcpS) enzyme. Analysis of the degradation products allowed to estimate the absolute configuration at the asymmetric phosphorus atoms in examined compounds via correlation with the R(P) and S(P) diastereoisomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS).

Solid-supported synthesis of 5'-mRNA CAP-4 from Trypanosomatids.

The unique structure of 5' mRNA cap from Trypanosomatids is the most modified cap found in nature. Here we present the synthesis of cap-4 (m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up) on a disulfide-tethered solid support. This approach allows obtaining cap-4 more efficiently then previously described. Moreover such modified resin could be a useful tool for affinity purification of Leishmania proteins interacting with cap-4. For the final step of synthesis, namely coupling of phosphorylated tetranucleotide with activated 7-methylguanosine 5'-diphosphate two systems were compared. Surprisingly, the coupling in water with Mn(2+) as a catalyst, gave better results than usually more effective coupling in DMF with ZnCl(2).

Synthesis of Leishmania cap-4 intermediates, cap-2 and cap-3.

Synthesis of Leishmania mRNA 5'-cap analogs, m(7)Gpppm(2)(6)AmpAm (cap-2), and m(7)Gpppm(2)(6)AmpAmpCm (cap-3) is reported. Binding affinities of those cap analogs for LeishIF4E proteins were determined using fluorescence spectroscopy. Cap-3 showed similar affinity to LeishIF4Es compared to the mature trypanosomatids cap structure (cap-4).

Binding specificities and potential roles of isoforms of eukaryotic initiation factor 4E in Leishmania.

The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.

Synthesis and properties of mRNA cap analogs containing phosphorothioate moiety in 5',5'-triphosphate chain.

Nucleosides and oligonucleotides with an oxygen replaced by sulfur atom are an interesting class of compounds because of their improved stability toward enzymatic cleavage by nucleases. We have synthesized several dinucleotide mRNA cap analogs containing a phosphorothioate moiety in the alpha, beta, or gamma position of 5',5'-triphosphate chain [m7Gp(s)ppG, m7Gpp(s)pG, and m7Gppp(s)G]. These are the first examples of the biologically important 5'mRNA cap analogs containing a phosphorothioate moiety, and these compounds may be useful in a variety of biochemical and biotechnological applications. Incorporation of a sulfur atom in the alpha or gamma position within the dinucleotide cap analog was achieved using PSCl3 in a nucleoside phosphorylation reaction followed by coupling the phosphorothioate of nucleoside with a second nucleotide. Synthesis of cap analogs with the phosphorothioate moiety in beta position was performed using an organic phosphorothioate salt in a coupling reaction with an activated nucleotide. The structures of newly synthesized compounds was confirmed using MS and 1H and 31P NMR spectroscopy. We present here the results of preliminary studies on their interaction with translation initiation factor eIF4E and enzymatic hydrolysis with human and nematode DcpS scavengers.

Cap-binding activity of an eIF4E homolog from Leishmania.

All eukaryotic mRNAs possess a 5'-cap (m(7)GpppN) that is recognized by a family of cap-binding proteins. These participate in various processes, such as RNA transport and stabilization, as well as in assembly of the translation initiation complex. The 5'-cap of trypanosomatids is complex; in addition to 7-methyl guanosine, it includes unique modifications on the first four transcribed nucleotides, and is thus denoted cap-4. Here we analyze a cap-binding protein of Leishmania, in an attempt to understand the structural features that promote its binding to this unusual cap. LeishIF4E-1, a homolog of eIF4E, contains the conserved cap-binding pocket, similar to its mouse counterpart. The mouse eIF4E has a higher K(as) for all cap analogs tested, as compared with LeishIF4E-1. However, whereas the mouse eIF4E shows a fivefold higher affinity for m(7)GTP than for a chemically synthesized cap-4 structure, LeishIF4E-1 shows similar affinities for both ligands. A sequence alignment shows that LeishIF4E-1 lacks the region that parallels the C terminus in the murine eIF4E. Truncation of this region in the mouse protein reduces the difference that is observed between its binding to m(7)GTP and cap-4, prior to this deletion. We hypothesize that variations in the structure of LeishIF4E-1, possibly also the absence of a region that is homologous to the C terminus of the mouse protein, promote its ability to interact with the cap-4 structure. LeishIF4E-1 is distributed in the cytoplasm, but its function is not clear yet, because it cannot substitute the mammalian eIF4E in a rabbit reticulocyte in vitro translation system.

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

Leishmania and other trypanosomatids are early eukaryotes that possess unusual molecular features, including polycistronic transcription and trans-splicing of pre-mRNAs. The spliced leader RNA (SL RNA) is joined to the 5' end of all mRNAs, thus donating a 5' cap that is characterized by complex modifications. In addition to the conserved m7GTP, linked via a 5'-5'-triphosphate bound to the first nucleoside of the mRNA, the trypanosomatid 5' cap includes 2'-O methylations on the first four ribose moieties and unique base methylations on the first adenine and the fourth uracil, resulting in the cap-4 structure, m7Gpppm3(6,6,2')Apm2'Apm2' Cpm2(3,2')U, as reported elsewhere previously. A library of analogs that mimic the cap structure to different degrees has been synthesized. Their differential affinities to the cap binding proteins make them attractive compounds for studying the molecular basis of cap recognition, and in turn, they may have potential therapeutic significance. The interactions between cap analogs and eIF4E, a cap-binding protein that plays a key role in initiation of translation, can be monitored by measuring intrinsic fluorescence quenching of the tryptophan residues. In the present communication we describe the multistep synthesis of the trypanosomatid cap-4 structure. The 5' terminal mRNA tetranucleotide fragment (pm3(6,6,2')Apm2'Apm2'Cpm2(3,2')U) was synthesized by the phosphoramidite solid phase method. After deprotection and purification, the 5'-phosphorylated tetranucleotide was chemically coupled with m7GDP to yield the cap-4 structure. Biological activity of this newly synthesized compound was confirmed in binding studies with eIF4E from Leishmania that we recently cloned (LeishIF4E-1), using the fluorescence time-synchronized titration method.

Binding studies of eukaryotic initiation factor eIF4E with novel mRNA dinucleotide cap analogues.

Studies on the interaction of the murine translation initiation factor 4E with two new-synthesized cap-analogues, modified at C2' of 7-methylguanosine, have been performed by means of the fluorescence titration method. No difference in the binding affinity for eIF4E was observed compared with the "anti reversed" cap analogues, possessing the analogous modifications at C3'. Potential significance of the novel caps as research tools for examination of the nuclear cap binding complex CBC80/20 has been discussed.

Novel "anti-reverse" cap analogs with superior translational properties.

Synthetic analogs of the 5'-terminal caps of eukaryotic mRNAs and snRNAs are used in elucidating such physiological processes as mRNA translation, pre-mRNA splicing, intracellular transport of mRNA and snRNAs, and mRNA turnover. Particularly useful are RNAs capped with synthetic analogs, which are produced by in vitro transcription of a DNA template using a bacteriophage RNA polymerase in the presence of ribonucleoside triphosphates and a cap dinucleotide such as m(7)Gp(3)G. Unfortunately, because of the presence of a 3'-OH on both the m(7)Guo and Guo moieties, up to half of the mRNAs contain caps incorporated in the reverse orientation. Previously we designed and synthesized two "anti-reverse" cap analogs (ARCAs), m(7)3'dGp(3)G and m(2)(7,3'-)(O)Gp(3)G, that cannot be incorporated in the reverse orientation because of modifications at the C3' position of m(7)Guo. In the present study, we have synthesized seven new cap analogs modified in the C2' and C3' positions of m(7)Guo and in the number of phosphate residues, m(2)(7,2'-)(O)Gp(3)G, m(7)2'dGp(3)G, m(7)2'dGp(4)G, m(2)(7,2'-)(O)Gp(4)G, m(2)(7,3'-)(O)Gp(4)G, m(7)Gp(5)G, and m(2)(7,3'-)(O)Gp(5)G. These were analyzed for conformation in solution, binding affinity to eIF4E, inhibition of in vitro translation, degree of reverse capping during in vitro transcription, capping efficiency, and the ability to stimulate cap-dependent translation in vitro when incorporated into mRNA. The results indicate that modifications at C2', like those at C3', prevent reverse incorporation, that tetra- and pentaphosphate cap analogs bind eIF4E and inhibit translation more strongly than their triphosphate counterparts, and that tetraphosphate ARCAs promote cap-dependent translation more effectively than previous cap analogs.