The Role of Cytoplasmic mRNA Cap-Binding Protein Complexes in Trypanosoma brucei and Other Trypanosomatids.

Trypanosomatid protozoa are unusual eukaryotes that are well known for having unusual ways of controlling their gene expression. The lack of a refined mode of transcriptional control in these organisms is compensated by several post-transcriptional control mechanisms, such as control of mRNA turnover and selection of mRNA for translation, that may modulate protein synthesis in response to several environmental conditions found in different hosts. In other eukaryotes, selection of mRNA for translation is mediated by the complex eIF4F, a heterotrimeric protein complex composed by the subunits eIF4E, eIF4G, and eIF4A, where the eIF4E binds to the 5'-cap structure of mature mRNAs. In this review, we present and discuss the characteristics of six trypanosomatid eIF4E homologs and their associated proteins that form multiple eIF4F complexes. The existence of multiple eIF4F complexes in trypanosomatids evokes exquisite mechanisms for differential mRNA recognition for translation

A new reporter cell line for studies with proteasome inhibitors in Trypanosoma brucei.

A Trypanosoma brucei cell line is described that produces a visual readout of proteasome activity. The cell line contains an integrated transgene encoding an ubiquitin-green fluorescent protein (GFP) fusion polypeptide responsive to the addition of proteasome inhibitors. A modified version of T. brucei ubiquitin unable to be recognized by deubiquitinases (UbG76V) was fused to eGFP and constitutively expressed. The fusion protein is unstable but addition of the proteasome inhibitor lactacystin stabilizes it and leads to visually detectable GFP. This cell line can be widely used to monitor the efficiency of inhibitor treatment through detection of GFP accumulation in studies involving proteasome-mediated proteolysis, screening of proteasome inhibitors or other events related to the ubiquitin-proteasome pathway

Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei

Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G subunits, is responsible for the canonical selection of mRNAs required for the initiation of mRNA translation. The best-known complexes implicated in translation in trypanosomatids are based on two related pairs of eIF4E and eIF4G subunits (EIF4E3/EIF4G4 and EIF4E4/EIF4G3), whose functional distinctions remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, specifically co-precipitate with each complex. Highlights with the EIF4E4/EIF4G3 pair include RBP23, PABP1, EIF4AI and the CRK1 kinase. Co-precipitated partners with the EIF4E3/EIF4G4 pair are more diverse and include DRBD2, PABP2 and different zinc-finger proteins and RNA helicases. EIF4E3/EIF4G4 are essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes

In silico characterization of multiple genes encoding the GP63 virulence protein from Leishmania braziliensis: identification of sources of variation and putative roles in immune evasion

Abstract Background The leishmaniasis are parasitic diseases caused by protozoans of the genus Leishmania, highly divergent eukaryotes, characterized by unique biological features. To survive in both the mammalian hosts and insect vectors, these pathogens make use of a number of mechanisms, many of which are associated with parasite specific proteases. The metalloprotease GP63, the major Leishmania surface antigen, has been found to have multiple functions required for the parasite’s survival. GP63 is encoded by multiple genes and their copy numbers vary considerably between different species and are increased in those from the subgenus Viannia, including L. braziliensis. Results By comparing multiple sequences from Leishmania and related organisms this study sought to characterize paralogs in silico, evaluating their differences and similarities and the implications for the GP63 function. The Leishmania GP63 genes are encoded on chromosomes 10, 28 and 31, with the genes from the latter two chromosomes more related to genes found in insect or plant parasites. Those from chromosome 10 have experienced independent expansions in numbers in Leishmania, especially in L. braziliensis. These could be clustered in three groups associated with different mRNA 3′ untranslated regions as well as distinct C-terminal ends for the encoded proteins, with presumably distinct expression patterns and subcellular localizations. Sequence variations between the chromosome 10 genes were linked to intragenic recombination events, mapped to the external surface of the proteins and predicted to be immunogenic, implying a role against the host immune response. Conclusions Our results suggest a greater role for the sequence variation found among the chromosome 10 GP63 genes, possibly related to the pathogenesis of L. braziliensis and closely related species within the mammalian host. They also indicate different functions associated to genes mapped to different chromosomes. For the chromosome 10 genes, variable subcellular localizations were found to be most likely associated with multiple functions and target substrates for this versatile protease

Identification of divergent Leishmania (Viannia) braziliensis ecotypes derived from a geographically restricted area through whole genome analysis.

Leishmania braziliensis, the main etiological agent of cutaneous leishmaniasis (CL) in Latin America, is characterized by major differences in basic biology in comparison with better-known Leishmania species. It is also associated with a high phenotypic and possibly genetic diversity that need to be more adequately defined. Here we used whole genome sequences to evaluate the genetic diversity of ten L. braziliensis isolates from a CL endemic area from Northeastern Brazil, previously classified by Multi Locus Enzyme Electrophoresis (MLEE) into ten distinct zymodemes. These sequences were first mapped using the L. braziliensis M2904 reference genome followed by identification of Single Nucleotide Polymorphisms (SNPs). A substantial level of diversity was observed when compared with the reference genome, with SNP counts ranging from ~95,000 to ~131,000 for the different isolates. When the genome data was used to infer relationship between isolates, those belonging to zymodemes Z72/Z75, recovered from forested environments, were found to cluster separately from the others, generally associated with more urban environments. Among the remaining isolates, those from zymodemes Z74/Z106 were also found to form a separate group. Phylogenetic analyses were also performed using Multi-Locus Sequence Analysis from genes coding for four metabolic enzymes used for MLEE as well as the gene sequence coding for the Hsp70 heat shock protein. All 10 isolates were firmly identified as L. braziliensis, including the zymodeme Z26 isolate previously classified as Leishmania shawi, with the clustering into three groups confirmed. Aneuploidy was also investigated but found in general restricted to chromosome 31, with a single isolate, from zymodeme Z27, characterized by extra copies for other chromosomes. Noteworthy, both Z72 and Z75 isolates are characterized by a much reduced heterozygosity. Our data is consistent with the existence of distinct evolutionary groups in the restricted area sampled and a substantial genetic diversity within L. braziliensis

RNA secondary structure and nucleotide composition of the conserved hallmark sequence of Leishmania SIDER2 retroposons are essential for endonucleolytic cleavage and mRNA degradation

We have reported previously that Short Interspersed Degenerate Retroposons of the SIDER2 subfamily, largely located within 3'UTRs of Leishmania transcripts, promote rapid turnover of mRNAs through endonucleolytic cleavage within the highly conserved second tandem 79-nt hallmark sequence (79-nt SII). Here, we used site-directed mutagenesis and in silico RNA structural studies to delineate the cis-acting requirements within 79-nt SII for cleavage and mRNA degradation. The putative cleavage site(s) and other nucleotides predicted to alter the RNA secondary structure of 79-nt SII were either deleted or mutated and their effect on mRNA turnover was monitored using a gene reporter system. We found that short deletions of 8-nt spanning the two predicted cleavage sites block degradation of SIDER2-containing transcripts, leading to mRNA accumulation. Furthermore, single or double substitutions of the dinucleotides targeted for cleavage as well as mutations altering the predicted RNA secondary structure encompassing both cleavage sites also prevent mRNA degradation, confirming that these dinucleotides are the bona fide cleavage sites. In line with these results, we show that stage-regulated SIDER2 inactivation correlates with the absence of endonucleolytic cleavage. Overall, these data demonstrate that both cleavage sites within the conserved 79-nt SII as well as RNA folding in this region are essential for SIDER2-mediated mRNA decay, and further support that SIDER2-harboring transcripts are targeted for degradation by endonucleolytic cleavage

Mutating the cleavage sites and or altering the predicted RNA secondary structure of the second 79-nt SIDER2 signature encompassing the cleavage sites block mRNA decay.

<p>Northern blot hybridization of total RNA extracted from recombinant <i>L</i>. <i>major</i> strains expressing LUC-3810 3'UTR (WT), LUC-3810 3'UTRΔSI+II (ΔSI,II), LUC-3810 3'UTRM1 (M1), LUC-3810 3'UTRM2 (M2), LUC-3810 3'UTRM3 (M3), LUC-3810 3'UTRM4 (M4), LUC-3810 3'UTRM5 (M5), LUC-3810 3'UTRM6 (M6), LUC-3810 3'UTRM6-1 (M6-1) and LUC-3810 3'UTRM6-2 (M6-2). A radiolabeled probe corresponding to the <i>LUC</i> gene was used for hybridization to assess changes in <i>LUC</i> mRNA expression levels. The alpha-tubulin (α-tub) probe was used as an RNA loading control. An ethidium bromide staining visualizing rRNA is also shown as an additional loading control. The graph displayed in the lower panel represents the normalized fold differences in <i>LUC</i> mRNA accumulation in M1 to M6-2 mutants relative to the WT strain. Values were normalized based on RNA loading (<i>LUC</i> and alpha-tubulin hybridization signal intensities were quantified using Phosphorimager) and plasmid copy number, varying among the different LUC-transfectants (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180678#pone.0180678.s002" target="_blank">S1 Fig</a>). The mean with standard deviations of three independent experiments is shown here. The asterisk (*) indicates significant changes in <i>LUC</i> mRNA accumulation upon normalization.</p

SIDER2 can be targeted for endonucleolytic cleavage when outside its 3'UTR context.

<p>(A) Schematic representation of the <i>in vivo</i> cleaved <i>L</i>. <i>major</i> LUC-3810 3′UTR SIDER2-containing mRNA. I and II represent the two conserved 79-nt signature sequences at the 5′end of all SIDER2 retroposons. Depending on the cleavage position within 79-nt SII (2 cleavage sites, Cl1 and Cl2, have been mapped previously in the 3810 SIDER2 element [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180678#pone.0180678.ref014" target="_blank">14</a>]), two fragments of 151-nt (1A) and 103-nt (1B) can be detected by primer extension analysis using a 5′-end labeled primer (P1) complementary to an RNA sequence at ~160-nt downstream. IR, intergenic region downstream of the LmjF.36.3810 3′UTR. (B) Primer extension assay with total RNA extracted from <i>L</i>. <i>major</i> LUC-3810 3′UTR, LUC-3810 SIDER2 (SIDER2 alone) and from <i>L</i>. <i>infantum</i> LUC-4000 3′UTR SIDER2-containing mRNAs using primer 1. Two specific extension fragments of 151-nt and 103-nt were resolved on 8% denaturating acrylamide gel corresponding to both cleavage sites 1 and 2, respectively. Their sizes were estimated with a radiolabeled DNA marker (M) are in agreement with previous results [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180678#pone.0180678.ref014" target="_blank">14</a>]. For the <i>L</i>. <i>infantum</i> LUC-4000 3′UTR SIDER2-containing mRNA, promastigotes (Pro) and axenic amastigotes (Ama) were used for primer extension to determine whether preferential decay of the <i>L</i>. <i>infantum</i> LinJ.36.4000 mRNA in promastigotes was correlated to stage-regulated cleavage. The primer extension data shown here are representative of at least 3 independent experiments yielding comparable results.</p

Increased accumulation of mRNAs harboring mutations in the second 79-nt SIDER2 signature is associated with the absence of cleavage products.

<p>(A) Schematic representation of the uncleaved and cleaved <i>L</i>. <i>major</i> LmjF.36.3810 SIDER2-harboring 3'UTR. I and II correspond to the tandem 79-nt signature sequences I and II of 3810 SIDER2, conserved in all SIDER2 retroposons (upper panel). (B) Northern blot hybridization of total RNA isolated from recombinant <i>L</i>. <i>major</i> LUC-3810 3'UTR, LUC-3810 3'UTRΔSI+II, LUC-3810 3'UTRM3, LUC-3810 3'UTRM4, LUC-3810 3'UTRM5, and LUC-3810 3'UTRM6 strains. The blot was hybridized with a 500 bp radiolabeled probe corresponding to the 3'-end of the LmjF.36.3810 3'UTR, which detects the uncleaved 3.6 kb LUC-3810 3'UTR transcript as well as a cleavage product of ~1.1 kb. An ethidium bromide staining visualizing rRNA is shown here as loading control. Data shown here are representative of two independent experiments yielding comparable results.</p