Characterization of RBP9 and RBP10, two developmentally regulated RNA-binding proteins in Trypanosoma brucei.
ABSTRACT: The fate of an mRNA is determined by its interaction with proteins and small RNAs within dynamic complexes called ribonucleoprotein complexes (mRNPs). In Trypanosoma brucei and related kinetoplastids, responses to internal and external signals are mainly mediated by post-transcriptional processes. Here, we used proximity-dependent biotin identification (BioID) combined with RNA-seq to investigate the changes resulting from ectopic expression of RBP10 and RBP9, two developmentally regulated RNA-binding proteins (RBPs). Both RBPs have reduced expression in insect procyclic forms (PCFs) compared with bloodstream forms (BSFs). Upon overexpression in PCFs, both proteins were recruited to cytoplasmic foci, co-localizing with the processing body marker SCD6. Further, both RBPs altered the transcriptome from a PCF- to a BSF-like pattern. Notably, upon expression of BirA*-RBP9 and BirA*-RBP10, BioID yielded more than 200 high confidence protein interactors (more than 10-fold enriched); 45 (RBP9) and 31 (RBP10) were directly related to mRNA metabolism. This study validates the use of BioID for investigating mRNP components but also illustrates the complexity of mRNP function.
Project description:Gene expression regulation in trypanosomes differs from other eukaryotes due to absence of transcriptional regulation for most of their genes. RNA-binding proteins (RBPs) associate with mRNAs and other regulatory proteins to form ribonucleoprotein complexes (mRNPs), which play a major role in post-transcriptional regulation. Here, we show that RBP9 is a cytoplasmic RBP in Trypanosoma cruzi with one RNA-recognition motif (RRM). The RBP9 sedimentation profile in a sucrose gradient indicated its presence in cytoplasmic translational complexes, suggesting its involvement in translation regulation. Taking this result as a motivation, we used shotgun proteomics and RNA-seq approaches to assess the core of the RBP9-mRNP complex. In epimastigotes in exponential growth, the complex was composed mostly by RBPs involved in RNA metabolism, such as ZC3H39, UBP1/2, NRBD1, and ALBA3/4. When parasites were subjected to nutritional stress, our analysis identified regulatory RBPs and the translation initiation factors eIF4E5, eIF4G5, eIF4G1, and eIF4G4. The RNA-seq results showed that RBP9-mRNP complex regulates transcripts encoding some RBPs - e.g. RBP5, RBP6, and RBP10 -, and proteins involved in metabolic processes. Therefore, we argue that RBP9 is part of cytoplasmic mRNPs complexes associated with mRNA metabolism and translation regulation in T. cruzi.
Project description:High spatial-resolution confocal photoluminescence (PL) measurements have been performed on a series of semi-polar (11-22) InGaN light emitting diodes (LEDs) with emission wavelengths up to yellow. These LED samples have been grown on our high crystal quality semi-polar GaN templates which feature periodically distributed basal stacking faults (BSFs), which facilitates the study of the influence of BSFs on their optical performance. Scanning confocal PL measurements have been performed across BSFs regions and BSF-free regions. For the blue LED, both the emission intensity and the emission wavelength exhibit a periodic behavior, matching the periodic distribution of BSFs. Furthermore, the BSF regions show a longer emission wavelength and a reduced emission intensity compared with the BSF-free regions. However, with increasing indium content, this periodic behavior in both emission intensity and emission wavelength becomes weaker and weaker. When the indium content (and correspondingly, wavelength) increases up to achieve yellow emission, only random fluctuations have been observed. It is worth highlighting that the influence of BSFs on the optical properties of semi-polar InGaN LEDs is different from the role of dislocations which normally act as non-radiative recombination centers.
Project description:The unicellular eukaryote Trypanosoma brucei relies heavily on posttranscriptional regulatory mechanisms, as pol II transcription is polycistronic. The parasite has six isoforms of the cap-binding translation initiation factor eIF4E, and five eiF4Gs (PMID: 29077018), which potentially allow for differential mRNA target selection in order to fine-tune translation. EIF4E3 and EIF4E4 appear to be general initiation factors; we are investigating the EIF4E proteins that are presumed to have more specialized functions, i.e., EIF4E1, EIF4E2, EIF4E5, and EIF4E6. EIF4E1 interacts with 4E-interacting protein (4EIP) and not with any EIF4G; they functionally resemble mammalian 4E-HP and GIGYF2. 4EIP suppresses translation and provokes mRNA degradation. 4EIP and EIF4E1 are dispensable in slender “bloodstream forms” (BSFs), which multiply in mammals, but 4EIP is required for translation suppression in the growth-arrested “stumpy” BSF (PMID: 30124912). Meanwhile EIF4E1, but not 4EIP, is required for survival of “procyclic forms” (PCFs), which grow in Tsetse. Tethered EIF4E1 is suppressive only when 4EIP is present. We are investigating whether it can, without an EIF4G, activate translation in BSFs/PCFs, as well as how target mRNAs are selected in absence and presence of 4EIP, based on the proteins it associates with. Like EIF4E1, EIF4E2 does not interact with any EIF4G protein, but was found in association with a homolog of the histone mRNA stem-loop-binding protein, called SLBP2, in PCFs (PMID: 29288414). The protein binding partners in BSFs have not been addressed this far. EIF4E3, EIF4E4, EIF4E5, and EIF4E6 all stimulate expression when tethered. They interact with different EIF4G homologs, and each is essential in at least one life-cycle stage, indicating that each serves a distinct role. We have now evidence that EIF4E6 interacts specifically, not only with EIF4G5, but also with a previously characterized stimulatory complex containing MKT1, PBP1, and LSM12, which we aimed to confirm by this quantitative mass spectrometry approach. The complex is recruited to mRNAs via sequence-specific RNA-binding proteins (PMID: 24470144), offering a novel mechanism for specific translation activation by the EIF4E6-EIF4G5 complex. EIF4E5 on the other hand associates with either EIF4G1 or EIF4G2 to exert its functions. It is dispensable in the BSF, but essential in PCFs, where knock-down results in a motility-related phenotype. Furthermore, previous studies identified several 14-3-3 homologs to be associated with EIF4E5 in complex with either EIF4G1 or EIF4G2 in PCFs (PMID: 24962368). In the course of this study, PTP-tagged versions of EIF4E1+/- 4EIP (BSF, PCF), EIF4E2, EIF4E5, and EIF4E6 (BSF only) were pulled down and bound proteins were analyzed by quantitative mass spectrometry, with EIF4E3-PTP (BSF) and GFP-PTP (BSF, PCF) serving as controls.
Project description:III-nitride compound semiconductors are breakthrough materials regarding device applications. However, their heterostructures suffer from very high threading dislocation (TD) densities that impair several aspects of their performance. The physical mechanisms leading to TD nucleation in these materials are still not fully elucidated. An overlooked but apparently important mechanism is their heterogeneous nucleation on domains of basal stacking faults (BSFs). Based on experimental observations by transmission electron microscopy, we present a concise model of this phenomenon occurring in III-nitride alloy heterostructures. Such domains comprise overlapping intrinsic I<sub>1</sub> BSFs with parallel translation vectors. Overlapping of two BSFs annihilates most of the local elastic strain of their delimiting partial dislocations. What remains combines to yield partial dislocations that are always of screw character. As a result, TD nucleation becomes geometrically necessary, as well as energetically favorable, due to the coexistence of crystallographically equivalent prismatic facets surrounding the BSF domain. The presented model explains all observed BSF domain morphologies, and constitutes a physical mechanism that provides insight regarding dislocation nucleation in wurtzite-structured alloy epilayers.
Project description:Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly. In particular, we expressed BirA* fused to the well characterized NMD factors UPF1, UPF2 and SMG5 and detected by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) the streptavidin-purified biotinylated proteins. While the identified already known interactors confirmed the usefulness of BioID, we also found new potentially important interactors that have escaped previous detection by co-IP, presumably because they associate only weakly and/or very transiently with the NMD machinery. Our results suggest that SMG5 only transiently contacts the UPF1-UPF2-UPF3 complex and that it provides a physical link to the decapping complex. In addition, BioID revealed among others CRKL and EIF4A2 as putative novel transient interactors with NMD factors, but whether or not they have a function in NMD remains to be elucidated.
Project description:Hydrocarbon bioremediation in anoxic sediment layers is still challenging not only because it involves metabolic pathways with lower energy yields but also because the production of biosurfactants that contribute to the dispersion of the pollutant is limited by oxygen availability. This work aims at screening populations of culturable hydrocarbonoclastic and biosurfactant (BSF) producing bacteria from deep sub-seafloor sediments (mud volcanos from Gulf of Cadiz) and estuarine sub-surface sediments (Ria de Aveiro) for strains with potential to operate in sub-oxic conditions. Isolates were retrieved from anaerobic selective cultures in which crude oil was provided as sole carbon source and different supplements were provided as electron acceptors. Twelve representative isolates were obtained from selective cultures with deep-sea and estuary sediments, six from each. These were identified by sequencing of 16S rRNA gene fragments belonging to Pseudomonas, Bacillus, Ochrobactrum, Brevundimonas, Psychrobacter, Staphylococcus, Marinobacter and Curtobacterium genera. BSF production by the isolates was tested by atomized oil assay, surface tension measurement and determination of the emulsification index. All isolates were able to produce BSFs under aerobic and anaerobic conditions, except for isolate DS27 which only produced BSF under aerobic conditions. These isolates presented potential to be applied in bioremediation or microbial enhanced oil recovery strategies under conditions of oxygen limitation. For the first time, members of Ochrobactrum, Brevundimonas, Psychrobacter, Staphylococcus, Marinobacter and Curtobacterium genera are described as anaerobic producers of BSFs.
Project description:The discovery and validation of protein-protein interactions provides a knowledge base that is critical for defining protein networks and how they underpin the biology of the cell. Identification of protein interactions that are highly transient, or sensitive to biochemical disruption, can be very difficult. This challenge has been met by proximity labeling methods which generate reactive species that chemically modify neighboring proteins. The most widely used proximity labeling method is BioID, which features a mutant biotin ligase BirA(Arg118Gly), termed BirA*, fused to a protein of interest. Here, we explore how amino acid substitutions at Arg118 affect the biochemical properties of BirA. We found that relative to wild-type BirA, the Arg118Lys substitution both slightly reduced biotin affinity and increased the release of reactive biotinyl-5'-AMP. BioID using a BirA(Arg118Lys)-Lamin A fusion enabled identification of PCNA as a lamina-proximal protein in HEK293T cells, a finding that was validated by immunofluorescence microscopy. Our data expand on the concept that proximity labeling by BirA fused to proteins of interest can be modulated by amino acid substitutions that affect biotin affinity and the release of biotinyl-5'-AMP.
Project description:To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.
Project description:Proximity-dependent biotinylation strategies have emerged as powerful tools to characterize the subcellular context of proteins in living cells. The popular BioID approach employs an abortive E. coli biotin ligase mutant (R118G; denoted as BirA*), which when fused to a bait protein enables the covalent biotinylation of endogenous proximal polypeptides. This approach has been mainly applied to the study of protein proximity in immortalized mammalian cell lines. To expand the application space of BioID, here we describe a set of lentiviral vectors that enable the inducible expression of BirA*-tagged bait fusion proteins for performing proximity-dependent biotinylation in diverse experimental systems. We benchmark this highly adaptable toolkit across immortalized and primary cell systems, demonstrating the ease, versatility and robustness of the system. We also provide guidelines to perform BioID using these reagents.
Project description:The formation of mRNPs controls the interaction of the translation and degradation machinery with individual mRNAs. The yeast Scd6 protein and its orthologs regulate translation and mRNA degradation in yeast, C. elegans, D. melanogaster, and humans by an unknown mechanism. We demonstrate that Scd6 represses translation by binding the eIF4G subunit of eIF4F in a manner dependent on its RGG domain, thereby forming an mRNP repressed for translation initiation. Strikingly, several other RGG domain-containing proteins in yeast copurify with eIF4E/G and we demonstrate that two such proteins, Npl3 and Sbp1, also directly bind eIF4G and repress translation in a manner dependent on their RGG motifs. These observations identify the mechanism of Scd6 function through its RGG motif and indicate that eIF4G plays an important role as a scaffolding protein for the recruitment of translation repressors.