Leishmania Ribosomal Protein (RP) paralogous genes compensate each other's expression maintaining protein native levels.

In the protozoan parasite Leishmania, most genes encoding for ribosomal proteins (RPs) are present as two or more copies in the genome. However, their untranslated regions (UTRs) are predominantly divergent and might be associated with a distinct regulation of the expression of paralogous genes. Herein, we investigated the expression profiles of two RPs (S16 and L13a) encoded by duplicated genes in Leishmania major. The genes encoding for the S16 protein possess identical coding sequences (CDSs) and divergent UTRs, whereas the CDSs of L13a diverge by two amino acids and by their UTRs. Using CRISPR/Cas9 genome editing, we generated knockout (Δ) and endogenously tagged transfectants for each paralog of L13a and S16 genes. Combining tagged and Δ cell lines we found evidence of differential expression of both RPS16 and RPL13a isoforms throughout parasite development, with one isoform consistently more abundant than its respective copy. In addition, compensatory expression was observed for each paralog upon deletion of the corresponding isoform, suggesting functional conservation between these proteins. This differential expression pattern relates to post-translational processes, given compensation occurs at the level of the protein, with no alterations detected at transcript level. Ribosomal profiles for RPL13a indicate a standard behavior for these paralogues suggestive of interaction with heavy RNA-protein complexes, as already reported for other RPs in trypanosomatids. We identified paralog-specific bound to their 3'UTRs which may be influential in regulating paralog expression. In support, we identified conserved cis-elements within the 3'UTRs of RPS16 and RPL13a; cis-elements exclusive to the UTR of the more abundant paralog or to the less abundant ones were identified.

Impact of inherent biases built into proteomic techniques: Proximity labeling and affinity capture compared.

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

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.

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.

RNA Binding Proteins and Gene Expression Regulation in Trypanosoma cruzi.

The regulation of gene expression in trypanosomatids occurs mainly at the post-transcriptional level. In the case of Trypanosoma cruzi, the characterization of messenger ribonucleoprotein (mRNP) particles has allowed the identification of several classes of RNA binding proteins (RBPs), as well as non-canonical RBPs, associated with mRNA molecules. The protein composition of the mRNPs as well as the localization and functionality of the mRNAs depend on their associated proteins. mRNPs can also be organized into larger complexes forming RNA granules, which function as stress granules or P-bodies depending on the associated proteins. The fate of mRNAs in the cell, and consequently the genes expressed, depends on the set of proteins associated with the messenger molecule. These proteins allow the coordinated expression of mRNAs encoding proteins that are related in function, resulting in the formation of post-transcriptional operons. However, the puzzle posed by the combinatorial association of sets of RBPs with mRNAs and how this relates to the expressed genes remain to be elucidated. One important tool in this endeavor is the use of the CRISPR/CAS system to delete genes encoding RBPs, allowing the evaluation of their effect on the formation of mRNP complexes and associated mRNAs in the different compartments of the translation machinery. Accordingly, we recently established this methodology for T. cruzi and deleted the genes encoding RBPs containing zinc finger domains. In this manuscript, we will discuss the data obtained and the potential of the CRISPR/CAS methodology to unveil the role of RBPs in T. cruzi gene expression regulation.

Downregulation of the protein synthesis machinery is a major regulatory event during early adipogenic differentiation of human adipose-derived stromal cells.

Commitment of adult stem cells involves the activation of specific gene networks regulated from transcription to protein synthesis. Here, we used ribosome profiling to identify mRNAs regulated at the translational level, through both differential association to polysomes and modulation of their translational rates. We observed that translational regulation during the differentiation of human adipose-derived stromal cells (hASCs, also known as adipose-derived mesenchymal stem cells), a subset of which are stem cells, to adipocytes was a major regulatory event. hASCs showed a significant reduction of whole protein synthesis after adipogenic induction and a downregulation of the expression and translational efficiency of ribosomal proteins. Additionally, focal adhesion and cytoskeletal proteins were downregulated at the translational level. This negative regulation of the essential biological functions of hASCs resulted in a reduction in cell size and the potential of hASCs to migrate. We analyzed whether the inactivation of key translation initiation factors was involved in this observed major repression of translation. We showed that there was an increase in the hypo phosphorylated forms of 4E-BP1, a negative regulator of translation, during early adipogenesis. Our results showed that extensive translational regulation occurred during the early stage of the adipogenic differentiation of hASCs.

Protein and mRNA content of TcDHH1-containing mRNPs in Trypanosoma cruzi.

In trypanosomatids, the regulation of gene expression occurs mainly at the post-transcriptional level. Previous studies have revealed nontranslated mRNA in the Trypanosoma cruzi cytoplasm. Previously, we have identified and cloned the TcDHH1 protein, a DEAD box RNA helicase. It has been reported that Dhh1 is involved in multiple RNA-related processes in various eukaryotes. It has also been reported to accumulate in stress granules and processing bodies of yeast, animal cells, Trypanosoma brucei and T. cruzi. TcDHH1 is localized to discrete cytoplasmic foci that vary depending on the life cycle status and nutritional conditions. To study the composition of mRNPs containing TcDHH1, we carried out immunoprecipitation assays with anti-TcDHH1 using epimastigote lysates. The protein content of mRNPs was determined by MS and pre-immune serum was used as control. We also carried out a ribonomic approach to identify the mRNAs present within the TcDHH1 immunoprecipitated complexes. For this purpose, competitive microarray hybridizations were performed against negative controls, the nonprecipitated fraction. Our results showed that mRNAs associated with TcDHH1 in the epimastigote stage are those mainly expressed in the other forms of the T. cruzi life cycle. These data suggest that mRNPs containing TcDHH1 are involved in mRNA metabolism, regulating the expression of at least epimastigote-specific genes.

Proteomic analysis reveals the dynamic association of proteins with translated mRNAs in Trypanosoma cruzi.

Gene regulation is mainly post-transcriptional in trypanosomatids. The stability of mRNA and access to polysomes are thought to be tightly regulated, allowing Trypanosoma cruzi to adapt to the different environmental conditions during its life cycle. Post-transcriptional regulation requires the association between mRNAs and certain proteins to form mRNP complexes. We investigated the dynamic association between proteins and mRNAs, using poly(T) beads to isolate and characterize proteins and protein complexes bound to poly-A+ mRNAs. The protein content of these fractions was analyzed by mass spectrometry (LC-MS/MS). We identified 542 protein component of the mRNP complexes associated with mRNAs. Twenty-four of the proteins obtained were present in all fractions, whereas some other proteins were exclusive to a particular fraction: epimastigote polysomal (0.37%) and post-polysomal (2.95%) fractions; stress polysomal (13.8%) and post-polysomal (40.78%) fractions. Several proteins known to be involved in mRNA metabolism were identified, and this was considered important as it made it possible to confirm the reliability of our mRNP isolation approach. This procedure allowed us to have a first insight into the composition and dynamics of mRNPs in T. cruzi.

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi.

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185bp long and encodes a 44.3kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.