Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of translating ribosomes triggering co-translational quality control of newly synthesized polypeptides.

DDX3 is a multifaceted RNA helicase of the DEAD-box family that plays central roles in all aspects of RNA metabolism including translation initiation. Here, we provide evidence that the Leishmania DDX3 ortholog functions in post-initiation steps of translation. We show that genetic depletion of DDX3 slows down ribosome movement resulting in elongation-stalled ribosomes, impaired translation elongation and decreased de novo protein synthesis. We also demonstrate that the essential ribosome recycling factor Rli1/ABCE1 and termination factors eRF3 and GTPBP1 are less recruited to ribosomes upon DDX3 loss, suggesting that arrested ribosomes may be inefficiently dissociated and recycled. Furthermore, we show that prolonged ribosome stalling triggers co-translational ubiquitination of nascent polypeptide chains and a higher recruitment of E3 ubiquitin ligases and proteasome components to ribosomes of DDX3 knockout cells, which further supports that ribosomes are not elongating optimally. Impaired elongation of translating ribosomes also results in the accumulation of cytoplasmic protein aggregates, which implies that defects in translation overwhelm the normal quality controls. The partial recovery of translation by overexpressing Hsp70 supports this possibility. Collectively, these results suggest an important novel contribution of DDX3 to optimal elongation of translating ribosomes by preventing prolonged translation stalls and stimulating recycling of arrested ribosomes.

The Pumilio-domain protein PUF6 contributes to SIDER2 retroposon-mediated mRNA decay in Leishmania.

Leishmania and other trypanosomatid protozoa lack control at the level of transcription initiation and regulate gene expression exclusively post-transcriptionally. We have reported previously that Leishmania harbors a unique class of short interspersed degenerate retroposons (SIDERs) that are predominantly located within 3'UTRs and play a major role in post-transcriptional control. We have shown that members of the SIDER2 subfamily initiate mRNA decay through endonucleolytic cleavage within the second conserved 79-nt signature sequence of SIDER2 retroposons. Here, we have developed an optimized MS2 coat protein tethering system to capture trans-acting factor(s) regulating SIDER2-mediated mRNA decay. Tethering of the MS2 coat protein to a reporter RNA harboring two MS2 stem-loop aptamers and the cognate SIDER2-containing 3'UTR in combination with immunoprecipitation and mass spectrometry analysis led to the identification of RNA-binding proteins with known functions in mRNA decay. Among the candidate SIDER2-interacting proteins that were individually tethered to a SIDER2 reporter RNA, the Pumilio-domain protein PUF6 was shown to enhance degradation and reduce transcript half-life. Furthermore, we showed that PUF6 binds to SIDER2 sequences that include the regulatory 79-nt signature motif, hence contributing to the mRNA decay process. Consistent with a role of PUF6 in SIDER2-mediated decay, genetic inactivation of PUF6 resulted in increased accumulation and higher stability of endogenous SIDER2-bearing transcripts. Overall, these studies provide new insights into regulated mRNA decay pathways in Leishmania controlled by SIDER2 retroposons and propose a broader role for PUF proteins in mRNA decay within the eukaryotic kingdom.

Valosin-containing protein VCP/p97 is essential for the intracellular development of Leishmania and its survival under heat stress.

Valosin-containing protein (VCP)/p97/Cdc48 is one of the best-characterised type II cytosolic AAA+ ATPases most known for their role in ubiquitin-dependent protein quality control. Here, we provide functional insights into the role of the Leishmania VCP/p97 homologue (LiVCP) in the parasite intracellular development. We demonstrate that although LiVCP is an essential gene, Leishmania infantum promastigotes can grow with less VCP. In contrast, growth of axenic and intracellular amastigotes is dramatically affected upon decreased LiVCP levels in heterozygous and temperature sensitive (ts) LiVCP mutants or the expression of dominant negative mutants known to specifically target the second conserved VCP ATPase domain, a major contributor of the VCP overall ATPase activity. Interestingly, these VCP mutants are also unable to survive heat stress, and a ts VCP mutant is defective in amastigote growth. Consistent with LiVCP's essential function in amastigotes, LiVCP messenger ribonucleic acid undergoes 3'Untranslated Region (UTR)-mediated developmental regulation, resulting in higher VCP expression in amastigotes. Furthermore, we show that parasite mutant lines expressing lower VCP levels or dominant negative VCP forms exhibit high accumulation of polyubiquitinated proteins and increased sensitivity to proteotoxic stress, supporting the ubiquitin-selective chaperone function of LiVCP. Together, these results emphasise the crucial role LiVCP plays under heat stress and during the parasite intracellular development.

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania.

Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or "Cos-Seq"), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms.

Phosphorylation and interactions associated with the control of the Leishmania Poly-A Binding Protein 1 (PABP1) function during translation initiation.

The Poly-A Binding Protein (PABP) is a conserved eukaryotic polypeptide involved in many aspects of mRNA metabolism. During translation initiation, PABP interacts with the translation initiation complex eIF4F and enhances the translation of polyadenylated mRNAs. Schematically, most PABPs can be divided into an N-terminal RNA-binding region, a non-conserved linker segment and the C-terminal MLLE domain. In pathogenic Leishmania protozoans, three PABP homologues have been identified, with the first one (PABP1) targeted by phosphorylation and shown to co-immunoprecipitate with an eIF4F-like complex (EIF4E4/EIF4G3) implicated in translation initiation. Here, PABP1 phosphorylation was shown to be linked to logarithmic cell growth, reminiscent of EIF4E4 phosphorylation, and coincides with polysomal association. Phosphorylation targets multiple serine-proline (SP) or threonine-proline (TP) residues within the PABP1 linker region. This is an essential protein, but phosphorylation is not needed for its association with polysomes or cell viability. Mutations which do impair PABP1 polysomal association and are required for viability do not prevent phosphorylation, although further mutations lead to a presumed inactive protein largely lacking phosphorylated isoforms. Co-immunoprecipitation experiments were carried out to investigate PABP1 function further, identifying several novel protein partners and the EIF4E4/EIF4G3 complex, but no other eIF4F-like complex or subunit. A novel, direct interaction between PABP1 and EIF4E4 was also investigated and found to be mediated by the PABP1 MLLE binding to PABP Interacting Motifs (PAM2) within the EIF4E4 N-terminus. The results shown here are consistent with phosphorylation of PABP1 being part of a novel pathway controlling its function and possibly translation in Leishmania.

Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania.

Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment.

An Alba-domain protein contributes to the stage-regulated stability of amastin transcripts in Leishmania.

Leishmania infantum promastigotes differentiate into amastigote forms within the phagolysosome of mammalian macrophages causing visceral leishmaniasis. Delta-amastins belong to a multigenic surface protein family of potential virulence factors that are specifically expressed in the amastigote life cycle stage through distinct regulatory elements in the 3' UTR controlling either mRNA stability or translation. Here, we provide novel insights on trans-acting factors regulating amastin developmental gene expression. Using RNA affinity chromatography with a 300 nt regulatory region within the amastin 3' UTR as bait, we identified an Alba-domain protein of 25 kDa (LiAlba20) as a specific amastin mRNA-binding partner. Genomic depletion of LiAlba20 results in amastin mRNA destabilization specifically in amastigotes, supporting a role of LiAlba20 in amastin gene regulation. As shown by comparative DNA microarray analysis, several delta-amastin transcripts but also other known developmentally regulated transcripts were downregulated in LiAlba20-/- knockout parasites. Inactivation of the second Alba-domain gene, LiAlba13, does not seem to affect amastin mRNA stability in either life stage of the parasite. These data indicate an important role of Alba-domain proteins in the regulation of Leishmania differentially expressed transcripts and open a new field of investigation for better understanding mechanisms contributing to post-transcriptional control in these parasites.

Altered expression of an RBP-associated arginine methyltransferase 7 in Leishmania major affects parasite infection.

Protein arginine methylation is a widely conserved post-translational modification performed by arginine methyltransferases (PRMTs). However, its functional role in parasitic protozoa is still under-explored. The Leishmania major genome encodes five PRMT homologs, including PRMT7. Here we show that LmjPRMT7 expression and arginine monomethylation are tightly regulated in a lifecycle stage-dependent manner. LmjPRMT7 levels are higher during the early promastigote logarithmic phase, negligible at stationary and late-stationary phases and rise once more post-differentiation to intracellular amastigotes. Immunofluorescence and co-immunoprecipitation studies demonstrate that LmjPRMT7 is a cytosolic protein associated with several RNA-binding proteins (RBPs) from which Alba20 is monomethylated only in LmjPRMT7-expressing promastigote stages. In addition, Alba20 protein levels are significantly altered in stationary promastigotes of the LmjPRMT7 knockout mutant. Considering RBPs are well-known mammalian PRMT substrates, our data suggest that arginine methylation via LmjPRMT7 may modulate RBP function during Leishmania spp. lifecycle progression. Importantly, genomic deletion of the LmjPRMT7 gene leads to an increase in parasite infectivity both in vitro and in vivo, while lesion progression is significantly reduced in LmjPRMT7-overexpressing parasites. This study is the first to describe a role of Leishmania protein arginine methylation in host-parasite interactions.

The unique Leishmania EIF4E4 N-terminus is a target for multiple phosphorylation events and participates in critical interactions required for translation initiation.

The eukaryotic initiation factor 4E (eIF4E) recognizes the mRNA cap structure and, together with eIF4G and eIF4A, form the eIF4F complex that regulates translation initiation in eukaryotes. In trypanosomatids, 2 eIF4E homologues (EIF4E3 and EIF4E4) have been shown to be part of eIF4F-like complexes with presumed roles in translation initiation. Both proteins possess unique N-terminal extensions, which can be targeted for phosphorylation. Here, we provide novel insights on the Leishmania infantum EIF4E4 function and regulation. We show that EIF4E4 is constitutively expressed throughout the parasite development but is preferentially phosphorylated in exponentially grown promastigote and amastigote life stages, hence correlating with high levels of translation. Phosphorylation targets multiple serine-proline or threonine-proline residues within the N-terminal extension of EIF4E4 but does not require binding to the EIF4E4's partner, EIF4G3, or to the cap structure. We also report that EIF4E4 interacts with PABP1 through 3 conserved boxes at the EIF4E4 N-terminus and that this interaction is a prerequisite for efficient EIF4E4 phosphorylation. EIF4E4 is essential for Leishmania growth and an EIF4E4 null mutant was only obtained in the presence of an ectopically provided wild type gene. Complementation for the loss of EIF4E4 with several EIF4E4 mutant proteins affecting either phosphorylation or binding to mRNA or to EIF4E4 protein partners revealed that, in contrast to other eukaryotes, only the EIF4E4-PABP1 interaction but neither the binding to EIF4G3 nor phosphorylation is essential for translation. These studies also demonstrated that the lack of both EIF4E4 phosphorylation and EIF4G3 binding leads to a non-functional protein. Altogether, these findings further highlight the unique features of the translation initiation process in trypanosomatid protozoa.

Genome sequencing of the lizard parasite Leishmania tarentolae reveals loss of genes associated to the intracellular stage of human pathogenic species.

The Leishmania tarentolae Parrot-TarII strain genome sequence was resolved to an average 16-fold mean coverage by next-generation DNA sequencing technologies. This is the first non-pathogenic to humans kinetoplastid protozoan genome to be described thus providing an opportunity for comparison with the completed genomes of pathogenic Leishmania species. A high synteny was observed between all sequenced Leishmania species. A limited number of chromosomal regions diverged between L. tarentolae and L. infantum, while remaining syntenic to L. major. Globally, >90% of the L. tarentolae gene content was shared with the other Leishmania species. We identified 95 predicted coding sequences unique to L. tarentolae and 250 genes that were absent from L. tarentolae. Interestingly, many of the latter genes were expressed in the intracellular amastigote stage of pathogenic species. In addition, genes coding for products involved in antioxidant defence or participating in vesicular-mediated protein transport were underrepresented in L. tarentolae. In contrast to other Leishmania genomes, two gene families were expanded in L. tarentolae, namely the zinc metallo-peptidase surface glycoprotein GP63 and the promastigote surface antigen PSA31C. Overall, L. tarentolae's gene content appears better adapted to the promastigote insect stage rather than the amastigote mammalian stage.

Distinctive features of the oropharyngeal microbiome in Inuit of Nunavik and correlations of mild to moderate bronchial obstruction with dysbiosis.

Inuit of Nunavik are coping with living conditions that can influence respiratory health. Our objective was to investigate associations between respiratory health in Inuit communities and their airway microbiome. Oropharyngeal samples were collected during the Qanuilirpitaa? 2017 Inuit Health Survey and subjected to metagenomic analyses. Participants were assigned to a bronchial obstruction group or a control group based on their clinical history and their pulmonary function, as monitored by spirometry. The Inuit microbiota composition was found to be distinct from other studied populations. Within the Inuit microbiota, differences in diversity measures tend to distinguish the two groups. Bacterial taxa found to be more abundant in the control group included candidate probiotic strains, while those enriched in the bronchial obstruction group included opportunistic pathogens. Crossing taxa affiliation method and machine learning consolidated our finding of distinct core microbiomes between the two groups. More microbial metabolic pathways were enriched in the control participants and these were often involved in vitamin and anti-inflammatory metabolism, while a link could be established between the enriched pathways in the disease group and inflammation. Overall, our results suggest a link between microbial abundance, interactions and metabolic activities and respiratory health in the Inuit population.

Leishmania infantum NTPDase1 and NTPDase2 play an important role in infection and nitric oxide production in macrophages.

Leishmania infantum, the causative agent of American Visceral Leishmaniasis (VL), is known for its ability to modulate the host immune response to its own favor. Ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) represents a family of enzymes that hydrolyze nucleotides and are involved in nucleotide-dependent biological processes. L. infantum has two ENTPDases, namely LiNTPDase1 and LiNTPDase2. Here, we used genetic tools to overexpress or abolish the expression of LiNTPDase1 and -2 to assess their role in parasite growth in culture and macrophage infection. While LiNTPDase1 or 2-overexpressing clones showed no morphological or growth changes in promastigotes, LiNTPDase2 overexpression increased macrophage adhesion and infection by 50% and 30%, respectively. The individual LiNTPDase1 and 2 knockout mutants showed lag in growth profile, which was reversed by the addition of adenine and guanine to the culture media. Moreover, the morphology of the knockout mutants even in supplemented media was changed to an amastigote-like form. The double knockout of both genes was lethal and a mechanism of compensation of deletion of one isoform was detected in these mutants. Correspondingly, the absence of LiNTPDase1 or LiNTPDase2 led to a dramatic reduction in in vitro infection (∼90%). Interestingly, nitric oxide production was decreased in both knockout mutants during infection, which suggests that both LiNTPDases can inhibit macrophage responses against the parasite. Overall, our results show important roles of LiNTPDase1 and -2 concerning in vitro macrophage infection and reinforce their use as potential targets to control Leishmania infections.

Analysis of membrane-enriched and high molecular weight proteins in Leishmania infantum promastigotes and axenic amastigotes.

Membrane and high molecular weight (HMW) proteins tend to be underrepresented in proteome analyses. Here, we optimized a protocol designed for the extraction and purification of membranes from the protozoan parasite Leishmania using a combination of serial centrifugation and free-flow zone electrophoresis (ZE-FFE). We also enriched for Leishmania HMW proteins from total extracts using the Gelfree 8100 fractionation system. This allowed the study of expression of both membrane-enriched and HMW proteins in Leishmania infantum promastigotes and amastigotes. We identified 194 proteins with at least one transmembrane domain (TMD) and 171 HMW proteins (≥100 kDa) in the invertebrate promastigote stage and 66 proteins with at least one TMD and 154 HMW proteins in the mammalian amastigote stage. Several of the proteins identified in one of the stages are part of pathways consistent with the known biology of the parasite, with many proteins involved in lipid synthesis, numerous dynein heavy chains, and some surface antigen proteins 2 detected in the promastigote stage. Notably, some proteins involved in transport and proteolysis were detected either in promastigote or amastigote. The present study is using improved proteomic methods for studying membrane-enriched and HMW proteins helping to achieve a better understanding of the parasite life cycle.

Promastigote to amastigote differentiation of Leishmania is markedly delayed in the absence of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation.

The parasitic protozoan Leishmania is the etiological agent of human leishmaniasis worldwide. It undergoes cellular differentiation from the sandfly promastigote form into amastigotes within mammalian macrophages, a process that is essential for its intracellular survival. Here, we characterized the Leishmania infantum PERK eIF2alpha kinase homologue and addressed its role in the parasite's cytodifferentiation. We show that Leishmania PERK is an endoplasmic reticulum (ER) transmembrane protein that largely colocalizes with the ER BiP chaperone. The Leishmania PERK catalytic kinase domain undergoes autohyperphosphorylation and phosphorylates the translation initiation factor 2-alpha subunit (eIF2alpha) in vitro at threonine 166. We also report that PERK is post-translationally regulated specifically in the intracellular stage of the parasite or under ER stress, most likely through extensive autohyperphosphorylation. We have generated a PERK dominant negative mutant overexpressing a truncated PERK protein lacking the N-terminal luminal domain and showed that this mutant is impaired in eIF2alpha phosphorylation in response to ER stress or during amastigote differentiation. Most importantly, we showed that lack of eIF2alpha phosphorylation markedly delays the Leishmania differentiation process towards amastigote forms both in parasites grown axenically or within macrophages. These data highlight the importance of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation in the intracellular development of Leishmania.

Rapid decay of unstable Leishmania mRNAs bearing a conserved retroposon signature 3'-UTR motif is initiated by a site-specific endonucleolytic cleavage without prior deadenylation.

We have previously shown that the Leishmania genome possess two widespread families of extinct retroposons termed Short Interspersed DEgenerated Retroposons (SIDER1/2) that play a role in post-transcriptional regulation. Moreover, we have demonstrated that SIDER2 retroposons promote mRNA degradation. Here we provide new insights into the mechanism by which unstable Leishmania mRNAs harboring a SIDER2 retroposon in their 3'-untranslated region are degraded. We show that, unlike most eukaryotic transcripts, SIDER2-bearing mRNAs do not undergo poly(A) tail shortening prior to rapid turnover, but instead, they are targeted for degradation by a site-specific endonucleolytic cleavage. The main cleavage site was mapped in two randomly selected SIDER2-containing mRNAs in vivo between an AU dinucleotide at the 5'-end of the second 79-nt signature (signature II), which represents the most conserved sequence amongst SIDER2 retroposons. Deletion of signature II abolished endonucleolytic cleavage and deadenylation-independent decay and increased mRNA stability. Interestingly, we show that overexpression of SIDER2 anti-sense RNA can increase sense transcript abundance and stability, and that complementarity to the cleavage region is required for protecting SIDER2-containing transcripts from degradation. These results establish a new paradigm for how unstable mRNAs are degraded in Leishmania and could serve as the basis for a better understanding of mRNA decay pathways in general.

Selective inactivation of SIDER2 retroposon-mediated mRNA decay contributes to stage- and species-specific gene expression in Leishmania.

Despite their high genomic synteny, the Leishmania major and Leishmania infantum species exhibit extensive differences in mRNA expression patterns throughout the parasite's development. Yet, the underlying mechanisms for this species-specific differential gene expression are largely unknown. Here we report that Short Interspersed DEgenerated Retroposons of the SIDER2 subfamily, shown previously to promote rapid mRNA turnover, confer differential regulation of orthologous transcripts resulting in a stage- and species-specific gene expression. We demonstrate that SIDER2-mediated decay of two L. major transcripts encoding a hypothetical protein and an aminomethyltransferase to a similar extent in promastigote and amastigote developmental forms results in a constitutive low expression of the corresponding proteins. In contrast, their L. infantum orthologs are differentially expressed due to the selective inactivation of SIDER2 in intracellular amastigotes. Inactivation of the SIDER2 function blocks the SIDER2-mediated deadenylation-independent decay pathway, and stabilized transcripts are degraded by a slower, deadenylation-dependent mechanism. Sequence variations in SIDER2 retroposons between orthologous transcripts do not contribute to SIDER2 inactivation. Our data suggest that SIDER2 inactivation is 3'-untranslated region context-dependent and that involves possibly species- and stage-specific trans-acting factor(s). These findings further emphasize the important contribution of SIDER retroposons in the control of gene expression across the Leishmania genus.

Leishmania major: Protective capacity of DNA vaccine using amastin fused to HSV-1 VP22 and EGFP in BALB/c mice model.

An intercellular spreading strategy using herpes simplex virus type 1 (HSV-1) VP22 protein is employed to enhance DNA vaccine potency of Leishmania major amastin antigen in BALB/c mice model. We evaluated the immunogenicity and protective efficacy of plasmid DNA vaccines encoding amastin-enhanced green fluorescent protein (EGFP) and VP22-amastin-EGFP. Optimal cell-mediated immune responses were observed in BALB/c mice immunized with VP22-amastin-EGFP as assessed by cytokine gene expression analysis using real time RT-PCR. Vaccination with the VP22-amastin-EGFP fusion construct elicited significantly higher IFN-gamma response upon antigen stimulation of splenocytes from immunized mice compared to amastin as a sole antigen. Mice immunized by VP22-amastin-EGFP showed partial protection following infectious challenge with L. major, as measured by parasite load in spleens. These results suggest that the development of DNA vaccines encoding VP22 fused to a target Leishmania antigen would be a promising strategy to improve immunogenicity and DNA vaccine potency.

Identification of novel proteins and mRNAs differentially bound to the Leishmania Poly(A) Binding Proteins reveals a direct association between PABP1, the RNA-binding protein RBP23 and mRNAs encoding ribosomal proteins.

Poly(A) Binding Proteins (PABPs) are major eukaryotic RNA-binding proteins (RBPs) with multiple roles associated with mRNA stability and translation and characterized mainly from multicellular organisms and yeasts. A variable number of PABP homologues are seen in different organisms however the biological reasons for multiple PABPs are generally not well understood. In the unicellular Leishmania, dependent on post-transcriptional mechanisms for the control of its gene expression, three distinct PABPs are found, with yet undefined functional distinctions. Here, using RNA-immunoprecipitation sequencing analysis we show that the Leishmania PABP1 preferentially associates with mRNAs encoding ribosomal proteins, while PABP2 and PABP3 bind to an overlapping set of mRNAs distinct to those enriched in PABP1. Immunoprecipitation studies combined to mass-spectrometry analysis identified RBPs differentially associated with PABP1 or PABP2, including RBP23 and DRBD2, respectively, that were investigated further. Both RBP23 and DRBD2 bind directly to the three PABPs in vitro, but reciprocal experiments confirmed preferential co-immunoprecipitation of PABP1, as well as the EIF4E4/EIF4G3 based translation initiation complex, with RBP23. Other RBP23 binding partners also imply a direct role in translation. DRBD2, in contrast, co-immunoprecipitated with PABP2, PABP3 and with RBPs unrelated to translation. Over 90% of the RBP23-bound mRNAs code for ribosomal proteins, mainly absent from the transcripts co-precipitated with DRBD2. These experiments suggest a novel and specific route for translation of the ribosomal protein mRNAs, mediated by RBP23, PABP1 and the associated EIF4E4/EIF4G3 complex. They also highlight the unique roles that different PABP homologues may have in eukaryotic cells associated with mRNA translation.

Analysis of stage-specific expression of basic proteins in Leishmania infantum.

Prior analyses of the proteome of the protozoan parasite Leishmania have underrepresented basic proteins. Here, we applied protein fractionation by isoelectric point (pI) using free-flow electrophoresis (FFE) to study stage-specific expression of basic proteins in this pathogen. Overall, we resolved 2469 protein spots in both the flagellated promastigote and the nonmotile amastigote forms in the basic range by two-dimensional gel electrophoresis (2-DE). Highly basic proteins were enriched by FFE fractionation, allowing many to be identified and characterized for the first time by proteomics analysis. Among proteins upregulated in the promastigote stage, we found glycolytic enzymes and flagellar proteins. Proteins upregulated in the amastigote stage included enzymes involved in gluconeogenesis and fatty acid beta-oxidation. In both life stages, many proteins were found in multiple spots or as proteolytic fragments, suggesting that extensive post-translational modification and processing occur. Interestingly, evidence was obtained suggesting that some of these processes may be stage-specific.

Deadenylation-independent stage-specific mRNA degradation in Leishmania.

The life cycle of Leishmania alternates between developmental forms residing within the insect vector (e.g. promastigotes) and the mammalian host (amastigotes). In Leishmania nearly all control of gene expression is post-transcriptional and involves sequences in the 3'-untranslated regions (3'UTRs) of mRNAs. Very little is known as to how these cis-elements regulate RNA turnover and translation rates in trypanosomatids and nothing is known about mRNA degradation mechanisms in Leishmania in particular. Here, we use the amastin mRNA-an amastigote-specific transcript-as a model and show that a approximately 100 nt U-rich element (URE) within its 3'UTR significantly accounts for developmental regulation. RNase-H-RNA blot analysis revealed that a major part of the rapid promastigote-specific degradation of the amastin mRNA is not initiated by deadenylation. This is in contrast to the amastin mRNA in amastigotes and to reporter RNAs lacking the URE, which, in common with most eukaryotic mRNAs studied to-date, are deadenylated before being degraded. Moreover, our analysis did not reveal a role for decapping in the stage-specific degradation of the amastin mRNA. Overall, these results suggest that degradation of the amastin mRNA of Leishmania is likely to be bi-phasic, the first phase being stage-specific and dependent on an unusual URE-mediated pathway of mRNA degradation.