ABSTRACT

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.Trypanosomatids are ancient eukaryotes that cycle between invertebrate vectors and mammalian hosts, causing a wide range of diseases. Leishmania parasites exist as extracellular flagellated promastigotes in the alimentary canal of female flies, and upon transfer to the mammalian host, they enter macrophages and cells of the immune system, transforming into amastigotes. Leishmania parasites are exposed to a broad range of environmental conditions, and stage differentiation is triggered by changes in temperature and pH (22,57).Trypanosomatids are characterized by a variety of unique molecular features, including polycistronic transcription of protein-coding genes (48, 60) and trans splicing (37), whereby a small leader RNA of 39 nucleotides, denoted spliced leader RNA (SL RNA), is spliced onto the 5Ј ends of all mRNAs, providing the cap structure. The trypanosomatid cap is a highly modified structure that, in addition to m 7

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 a, b, or g position of the 59,59-triphosphate chain. Three of them were also modified with methyl groups at the 29-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 b-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 g 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 a position (i.e., m 7 Gppp S G and m 2 7,29-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 59-O-(1-thiodiphosphate) (GDPaS). The analogs resistant to DcpS act as potent inhibitors of in vitro protein synthesis in rabbit reticulocyte lysates.

All eukaryotic mRNAs possess a 5-cap (m 7GpppN) 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.

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