Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure

T. brucei survival relies on the expression of mitochondrial genes, most of which require RNA editing to become translatable. In trypanosomes, RNA editing involves the insertion and deletion of uridylates, a developmentally regulated process directed by guide RNAs (gRNAs) and catalyzed by the editosome, a complex of proteins. The pathway for mRNA/gRNA complex formation and assembly with the editosome is still unknown. Work from our laboratory has suggested that distinct mRNA/gRNA complexes anneal to form a conserved core structure that may be important for editosome assembly. The secondary structure for the apocytochrome b (CYb) pair has been previously determined and is consistant with our model of a three-helical structure. Here, we used cross-linking and solution structure probing experiments to determine the structure of the ATPase subunit 6 (A6) mRNA hybridized to its cognate gA6-14 gRNA in different stages of editing. Our results indicate that both unedited and partially edited A6/gA6-14 pairs fold into a three-helical structure similar to the previously characterized CYb/gCYb-558 pair. These results lead us to conclude that at least two mRNA/gRNA pairs with distinct editing sites and distinct primary sequences fold to a three-helical secondary configuration that persists through the first few editing events.     

Non-linear effects of macromolecular crowding on enzymatic activity of multi-copper oxidase

Enzymes catalyze biochemical reactions in highly crowded environments where the amount of macromolecules may occupy up to 40% of the volume. Here we report how cell-like conditions tune catalytic parameters for the monomeric multi-copper oxidase, Saccharomyces cerevisiae Fet3p, in vitro. At low amounts of crowding agent, we detect increases in both of K_M (weaker substrate binding) and k_cat (improved catalytic efficiency), whereas at higher crowding levels, both parameters were reduced. Presence of crowding agents does not affect Fet3p structural content but increases thermal resistance. The observations are compatible with ordering of a non-optimal substrate-binding site and restricted internal dynamics as a result of excluded volume effects making the protein less structurally ‘strained’.     

Combined Effects of Agitation,Macromolecular Crowding,and Interfaces on Amyloidogenesis

Amyloid formation and accumulation is a hallmark of protein misfolding diseases and is associated with diverse pathologies including type II diabetes and Alzheimer''s disease (AD). In vitro, amyloidogenesis is widely studied in conditions that do not simulate the crowded and viscous in vivo environment. A high volume fraction of most biological fluids is occupied by various macromolecules, a phenomenon known as macromolecular crowding. For some amyloid systems (e.g. α-synuclein) and under shaking condition, the excluded volume effect of macromolecular crowding favors aggregation, whereas increased viscosity reduces the kinetics of these reactions. Amyloidogenesis can also be catalyzed by hydrophobic-hydrophilic interfaces, represented by the air-water interface in vitro and diverse heterogeneous interfaces in vivo (e.g. membranes). In this study, we investigated the effects of two different crowding polymers (dextran and Ficoll) and two different experimental conditions (with and without shaking) on the fibrilization of amyloid-β peptide, a major player in AD pathogenesis. Specifically, we demonstrate that, during macromolecular crowding, viscosity dominates over the excluded volume effect only when the system is spatially non homogeneous (i.e. an air-water interface is present). We also show that the surfactant activity of the crowding agents can critically influence the outcome of macromolecular crowding and that the structure of the amyloid species formed may depend on the polymer used. This suggests that, in vivo, the outcome of amyloidogenesis may be affected by both macromolecular crowding and spatial heterogeneity (e.g. membrane turn-over). More generally, our work suggests that any factors causing changes in crowding may be susceptibility factors in AD.     

In vivo testing of a tobacco plastid DNA segment for guide RNA function in psbL editing

A C-to-U RNA editing event creates a functional initiation codon for translation of the psbL mRNA in tobacco plastids. Small trans-acting guide RNAs (gRNAs) have been shown to be involved in editing site selection in kinetoplastid mitochondria. A computer search of the tobacco plastid genome (ptDNA) identified such a putative gRNA, a 14-nucleotide sequence motif that is complementary to the psbL mRNA, including the A nucleotide required to direct the C-to-U change. The critical A nucleotide of the putative gRNA gene was changed to G by plastid transformation. We report here that the introduced mutation did not abolish psbL editing. Since no other region of the plastid genome contains significant complementarity to the psbL editing site we suggest that, if gRNAs serve as trans-acting factors for plastid psbL mRNA editing, they either have only a limited complementarity to the editing site, or are encoded in the nuclear genome.     

Crowding Effects on Association Reactions at Membranes

The effect of macromolecular crowding on the binding of ligands to a receptor near membranes is studied using Brownian dynamics simulations. The receptor is modeled as a reactive patch on a hard surface and the ligands and crowding agents are modeled as spheres that interact via a steep repulsive interaction potential. When a ligand collides with the patch, it reacts with probability p_rxn. The association rate constant (k_∞) can be decomposed into contributions from diffusion-limited (k_D) and reaction-limited (k_R) rates, i.e., 1/k_∞ = 1/k_D + 1/k_R. The simulations show that k_D is a nonmonotonic function of the volume fraction of crowding agents for receptors of small sizes. k_R is always an increasing function of the volume fraction of crowding agents, and the association rate constant k_∞ determined from both contributions has a qualitatively different dependence on the macromolecular crowding for high and low values of the reaction probability p_rxn. The simulation results are used to predict the velocity of the membrane protrusion driven by actin filament elongation. Based on the simple model where the protrusive force on the membrane is generated by the intercalation of actin monomers between the membrane and actin filament ends, we predict that crowding increases the local concentration of actin monomers near the filament ends and hence accelerates the membrane protrusion.     

Macromolecular Crowding Modulates Folding Mechanism of α/β Protein Apoflavodoxin

Protein dynamics in cells may be different from those in dilute solutions in vitro, because the environment in cells is highly concentrated with other macromolecules. This volume exclusion because of macromolecular crowding is predicted to affect both equilibrium and kinetic processes involving protein conformational changes. To quantify macromolecular crowding effects on protein folding mechanisms, we investigated the folding energy landscape of an α/β protein, apoflavodoxin, in the presence of inert macromolecular crowding agents, using in silico and in vitro approaches. By means of coarse-grained molecular simulations and topology-based potential interactions, we probed the effects of increased volume fractions of crowding agents (ϕ_c) as well as of crowding agent geometry (sphere or spherocylinder) at high ϕ_c. Parallel kinetic folding experiments with purified Desulfovibro desulfuricans apoflavodoxin in vitro were performed in the presence of Ficoll (sphere) and Dextran (spherocylinder) synthetic crowding agents. In conclusion, we identified the in silico crowding conditions that best enhance protein stability, and discovered that upon manipulation of the crowding conditions, folding routes experiencing topological frustrations can be either enhanced or relieved. Our test-tube experiments confirmed that apoflavodoxin''s time-resolved folding path is modulated by crowding agent geometry. Macromolecular crowding effects may be a tool for the manipulation of protein-folding and function in living cells.     

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based “mix and measure” hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.     

Size-dependent studies of macromolecular crowding on the thermodynamic stability,structure and functional activity of proteins: in vitro and in silico approaches

BackgroundThe environment inside cells in which proteins fold and function are quite different from that of the dilute buffer solutions often used during in vitro experiments. The presence of large amounts of macromolecules of varying shapes, sizes and compositions makes the intracellular milieu extremely crowded.Scope of reviewThe overall concentration of macromolecules ranges from 50 to 400 g l^− 1, and they occupy 10–40% of the total cellular volume. These differences in solvent conditions and the level of crowdedness resulting in excluded volume effects can have significant consequences on proteins' biophysical properties. A question that arises is: how important is it to examine the roles of shape, size and composition of macromolecular crowders in altering the biological properties of proteins? This review article aims at focusing, gathering and summarizing all of the research investigations done by means of in vitro and in silico approaches taking into account the size-dependent influence of the crowders on proteins' properties.Major conclusionsAltogether, the internal architecture of macromolecular crowding environment including size, shape and concentration of crowders, appears to be playing an extremely important role in causing changes in the biological processes. Most often the small sized crowders have been found more effective crowding agents. However, thermodynamic stability, structure and functional activity of proteins have been governed by volume exclusion as well as soft (chemical) interactions.General significanceThe article provides an understanding of importance of internal architecture of the cellular environment in altering the biophysical properties of proteins.     

Modulation of Calmodulin Plasticity by the Effect of Macromolecular Crowding

In vitro biochemical reactions are most often studied in dilute solution, a poor mimic of the intracellular space of eukaryotic cells, which are crowded with mobile and immobile macromolecules. Such crowded conditions exert volume exclusion and other entropic forces that have the potential to impact chemical equilibria and reaction rates. In this article, we used the well-characterized and ubiquitous molecule calmodulin (CaM) and a combination of theoretical and experimental approaches to address how crowding impacts CaM's conformational plasticity. CaM is a dumbbell-shaped molecule that contains four EF hands (two in the N-lobe and two in the C-lobe) that each could bind Ca²⁺, leading to stabilization of certain substates that favor interactions with other target proteins. Using coarse-grained molecular simulations, we explored the distribution of CaM conformations in the presence of crowding agents. These predictions, in which crowding effects enhance the population of compact structures, were then confirmed in experimental measurements using fluorescence resonance energy transfer techniques of donor- and acceptor-labeled CaM under normal and crowded conditions. Using protein reconstruction methods, we further explored the folding-energy landscape and examined the structural characteristics of CaM at free-energy basins. We discovered that crowding stabilizes several different compact conformations, which reflects the inherent plasticity in CaM's structure. From these results, we suggest that the EF hands in the C-lobe are flexible and can be thought of as a switch, while those in the N-lobe are stiff, analogous to a rheostat. New combinatorial signaling properties may arise from the product of the differential plasticity of the two distinct lobes of CaM in the presence of crowding. We discuss the implications of these results for modulating CaM's ability to bind Ca²⁺ and target proteins.     

Assembly of mitochondrial ribonucleoprotein complexes involves specific guide RNA (gRNA)-binding proteins and gRNA domains but does not require preedited mRNA.

RNA editing in kinetoplastids probably employs a macromolecular complex, the editosome, that is likely to include the guide RNAs (gRNAs) which specify the edited sequence. Specific ribonucleoprotein (RNP) complexes which form in vitro with gRNAs (H. U. Göringer, D. J. Koslowsky, T. H. Morales, and K. D. Stuart, Proc. Natl. Acad. Sci. USA, in press) are potential editosomes or their precursors. We find that several factors are important for in vitro formation of these RNP complexes and identify specific gRNA-binding proteins present in the complexes. Preedited mRNA promotes the in vitro formation of the four major gRNA-containing RNP complexes under some conditions but is required for the formation of only a subcomponent of one complex. The 5' gRNA sequence encompassing the RYAYA and anchor regions and the 3' gRNA oligo(U) tail are both important in complex formation, since their deletion results in a dramatic decrease of some complexes and the absence of others. UV cross-linking experiments identify several proteins which are in contact with gRNA and preedited mRNA in mitochondrial extracts. Proteins of 25 and 90 kDa are highly specific for gRNAs, and the 90-kDa protein binds specifically to gRNA oligo(U) tails. The gRNA-binding proteins exhibit a differential distribution between the four in vitro-formed complexes. These experiments reveal several proteins potentially involved in RNA editing and indicate that multiple recognition elements in gRNAs are used for complex formation.     

REH2C Helicase and GRBC Subcomplexes May Base Pair through mRNA and Small Guide RNA in Kinetoplastid Editosomes

Mitochondrial mRNAs in Trypanosoma brucei undergo extensive insertion and deletion of uridylates that are catalyzed by the RNA editing core complex (RECC) and directed by hundreds of small guide RNAs (gRNAs) that base pair with mRNA. RECC is largely RNA-free, and accessory mitochondrial RNA-binding complex 1 (MRB1) variants serve as scaffolds for the assembly of mRNA-gRNA hybrids and RECC. However, the molecular steps that create higher-order holoenzymes (“editosomes”) are unknown. Previously, we identified an RNA editing helicase 2-associated subcomplex (REH2C) and showed that REH2 binds RNA. Here we showed that REH2C is an mRNA-associated ribonucleoprotein (mRNP) subcomplex with editing substrates, intermediates, and products. We isolated this mRNP from mitochondria lacking gRNA-bound RNP (gRNP) subcomplexes and identified REH2-associated cofactors 1 and 2 (^H2F1 and ^H2F2). ^H2F1 is an octa-zinc finger protein required for mRNP-gRNP docking, pre-mRNA and RECC loading, and RNP formation with a short synthetic RNA duplex. REH2 and other eukaryotic DEAH/RHA-type helicases share a conserved regulatory C-terminal domain cluster that includes an oligonucleotide-binding fold. Recombinant REH2 and ^H2F1 constructs associate in a purified complex in vitro. We propose a model of stepwise editosome assembly that entails controlled docking of mRNP and gRNP modules via specific base pairing between their respective mRNA and gRNA cargo and regulatory REH2 and ^H2F1 subunits of the novel mRNP that may control specificity checkpoints in the editing pathway.     

RNA Binding and Core Complexes Constitute the U-Insertion/Deletion Editosome

Enzymes embedded into the RNA editing core complex (RECC) catalyze the U-insertion/deletion editing cascade to generate open reading frames in trypanosomal mitochondrial mRNAs. The sequential reactions of mRNA cleavage, U-addition or removal, and ligation are directed by guide RNAs (gRNAs). We combined proteomic, genetic, and functional studies with sequencing of total and complex-bound RNAs to define a protein particle responsible for the recognition of gRNAs and pre-mRNA substrates, editing intermediates, and products. This approximately 23-polypeptide tripartite assembly, termed the RNA editing substrate binding complex (RESC), also functions as the interface between mRNA editing, polyadenylation, and translation. Furthermore, we found that gRNAs represent only a subset of small mitochondrial RNAs, and yet an inexplicably high fraction of them possess 3′ U-tails, which correlates with gRNA''s enrichment in the RESC. Although both gRNAs and mRNAs are associated with the RESC, their metabolic fates are distinct: gRNAs are degraded in an editing-dependent process, whereas edited mRNAs undergo 3′ adenylation/uridylation prior to translation. Our results demonstrate that the well-characterized editing core complex (RECC) and the RNA binding particle defined in this study (RESC) typify enzymatic and substrate binding macromolecular constituents, respectively, of the ∼40S RNA editing holoenzyme, the editosome.     

The Oligonucleotide Binding (OB)-Fold Domain of KREPA4 Is Essential for Stable Incorporation into Editosomes

Most mitochondrial mRNAs in trypanosomatid parasites require uridine insertion/deletion RNA editing, a process mediated by guide RNA (gRNA) and catalyzed by multi-protein complexes called editosomes. The six oligonucleotide/oligosaccharide binding (OB)-fold proteins (KREPA1-A6), are a part of the common core of editosomes. They form a network of interactions among themselves as well as with the insertion and deletion sub-complexes and are essential for the stability of the editosomes. KREPA4 and KREPA6 proteins bind gRNA in vitro and are known to interact directly in yeast two-hybrid analysis. In this study, using several approaches we show a minimal interaction surface of the KREPA4 protein that is required for this interaction. By screening a series of N- and C-terminally truncated KREPA4 fragments, we show that a predicted α-helix of KREPA4 OB-fold is required for its interaction with KREPA6. An antibody against the KREPA4 α-helix or mutations of this region can eliminate association with KREPA6; while a peptide fragment corresponding to the α-helix can independently interact with KREPA6, thereby supporting the identification of KREPA4-KREPA6 interface. We also show that the predicted OB-fold of KREPA4; independent of its interaction with gRNA, is responsible for the stable integration of KREPA4 in the editosomes, and editing complexes co-purified with the tagged OB-fold can catalyze RNA editing. Therefore, we conclude that while KREPA4 interacts with KREPA6 through the α-helix region of its OB-fold, the entire OB-fold is required for its integration in the functional editosome, through additional protein-protein interactions.