Trypanosoma brucei: functions of RBP16 cold shock and RGG domains in macromolecular interactions

The RNA binding protein RBP16 regulates mitochondrial RNA editing and stability in Trypanosoma brucei. To aid in understanding the biochemical mechanisms of RBP16 function, we analyzed the RNA and protein binding capacity of RBP16 and its individual cold shock (CSD) and RGG domains. Both recombinantly expressed domains possess RNA binding activity. However, the specificity and affinity of RBP16 for gRNA is mediated predominantly through the interaction of the CSD with poly(U). The RGG domain contributes to the association between full length RBP16 and gRNA, as it was required for maximal binding. We further demonstrate that both domains contribute to maximal binding of RBP16 to the mitochondrial p22 protein. However, p22 can interact with the CSD alone and stimulate its gRNA binding activity. Thus, the CSD is primary in RBP16 interactions, while the RGG domain enhances the capacity of the CSD to bind both RNA and protein. These results suggest a model for RBP16 molecular interactions.     

TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites

TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5′ ends of pan-edited RNAs than at their 3′ ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3′ to 5′ progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3′ ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA–RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3′ to 5′ progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs.     

RBP16 is a multifunctional gene regulatory protein involved in editing and stabilization of specific mitochondrial mRNAs in Trypanosoma brucei

RBP16 is a Trypanosoma brucei mitochondrial RNA-binding protein that associates with guide RNAs (gRNAs), mRNAs, and ribosomal RNAs. Based on its inclusion in the multifunctional Y-box protein family and its ability to bind multiple RNA classes, we hypothesized that RBP16 plays a role in diverse aspects of mitochondrial gene regulation. To gain insight into RBP16 function, we generated cells expressing less than 10% of wild-type RBP16 levels by tetracycline-regulated RNA interference (RNAi). Poisoned primer extension analyses revealed that edited, but not unedited, CYb mRNA is reduced by approximately 98% in tetracycline-induced RBP16 RNAi cells, suggesting that RBP16 is critical for CYb RNA editing. The down-regulation of CYb editing in RBP16 RNAi transfectants apparently entails a defect in gRNA utilization, as gCYb[560] abundance is similar in uninduced and induced cells. We observed a surprising degree of specificity regarding the ability of RBP16 to modulate editing, as editing of mRNAs other than CYb is not significantly affected upon RBP16 disruption. However, the abundance of the never edited mitochondrial RNAs COI and ND4 is reduced by 70%-80% in RBP16 RNAi transfectants, indicating an additional role for RBP16 in the stabilization of these mRNAs. Analysis of RNAs bound to RBP16 immunoprecipitated from wild-type cells reveals that RBP16 is associated with multiple gRNA sequence classes in vivo, including those whose abundance and usage appear unaffected by RBP16 disruption. Overall, our results indicate that RBP16 is an accessory factor that regulates the editing and stability of specific populations of mitochondrial mRNAs.     

A novel member of the RNase D exoribonuclease family functions in mitochondrial guide RNA metabolism in Trypanosoma brucei

RNA turnover and RNA editing are essential for regulation of mitochondrial gene expression in Trypanosoma brucei. RNA turnover is controlled in part by RNA 3' adenylation and uridylation status, with trans-acting factors also impacting RNA homeostasis. However, little is known about the mitochondrial degradation machinery or its regulation in T. brucei. We have identified a mitochondrial exoribonuclease, TbRND, whose expression is highly up-regulated in the insect proliferative stage of the parasite. TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain. In vitro, TbRND exhibits 3' to 5' exoribonuclease activity, with specificity toward uridine homopolymers, including the 3' oligo(U) tails of guide RNAs (gRNAs) that provide the sequence information for RNA editing. Several lines of evidence generated from RNAi-mediated knockdown and overexpression cell lines indicate that TbRND functions in gRNA metabolism in vivo. First, TbRND depletion results in gRNA tails extended by 2-3 nucleotides on average. Second, overexpression of wild type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3'-uridylated, are unaffected by TbRND depletion or overexpression. Finally, we show that gRNA binding proteins co-purify with TbRND. In summary, TbRND is a novel 3' to 5' exoribonuclease that appears to have evolved a function highly specific to the mitochondrion of trypanosomes.     

The trypanosome homolog of human p32 interacts with RBP16 and stimulates its gRNA binding activity

RBP16 is a guide RNA (gRNA)-binding protein that was shown through immunoprecipitation experiments to interact with ~30% of total gRNAs in Trypanosoma brucei mitochondria. To gain insight into the biochemical function of RBP16, we used affinity chromatography and immunoprecipitation to identify RBP16 protein binding partners. By these methods, RBP16 does not appear to stably interact with the core editing machinery. However, fractionation of mitochondrial extracts on MBP–RBP16 affinity columns consistently isolated proteins of 12, 16, 18 and 22 kDa that were absent from MBP control columns. We describe here our analysis of one RBP16-associated protein, p22. The predicted p22 protein has significant sequence similarity to a family of multimeric, acidic proteins that includes human p32 and Saccharomyces cerevisiae mam33p. Glutaraldehyde crosslinking of recombinant p22 identified homo-multimeric forms of the protein, further substantiating its homology to p32. We confirmed the p22–RBP16 interaction and demonstrated that the two proteins bind each other directly by ELISA utilizing recombinant p22 and RBP16. p32 family members have been reported to modulate viral and cellular pre-mRNA splicing, in some cases by perturbing interaction of their binding partners with RNA. To determine whether p22 similarly affects the gRNA binding properties of RBP16, we titrated recombinant p22 into UV crosslinking assays. These experiments revealed that p22 significantly stimulates the gRNA binding capacity of RBP16. Thus, p22 has the potential to be a regulatory factor in T.brucei mitochondrial gene expression by modulating the RNA binding properties of RBP16.     

RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction

RBP16 is an abundant RNA binding protein from Trypanosoma brucei mitochondria that affects both RNA editing and stability. We report here experiments aimed at elucidating the mechanism of RBP16 function in RNA editing. In in vitro RNA editing assays, recombinant RBP16 is able to significantly stimulate insertion editing of both CYb and A6 pre-mRNAs. Enhancement of in vitro editing activity occurs at, or prior to, the step of pre-mRNA cleavage, as evidenced by increased accumulation of pre-mRNA 3' cleavage products in the presence of RBP16. Mutated RBP16 that is severely compromised in cold shock domain (CSD)-mediated RNA binding was able to enhance editing to levels comparable to the wild-type protein in some assays at the highest RBP16 levels tested. However, at low RBP16 concentrations or in assays with native, oligo(U)-tail-bearing gRNAs, editing stimulation by mutant RBP16 was somewhat compromised. Together, these results indicate that both the N-terminal CSD and C-terminal RGG RNA binding domains of RBP16 are required for maximal editing stimulation. Finally, the relaxed specificity of RBP16 for stimulation of both CYb and A6 editing in vitro implicates additional specificity factors that account for the strict CYb specificity of RBP16 action in editing in vivo. Our results constitute the first report of any putative RNA editing accessory factor eliciting an effect on editing in vitro. Overall, these results support a novel accessory role for RBP16 in U insertion editing.     

A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing

Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing.     

Structure and function of the native and recombinant mitochondrial MRP1/MRP2 complex from Trypanosoma brucei

The mitochondrial RNA-binding proteins (MRP) 1 and 2 play a regulatory role in RNA editing and putative role(s) in RNA processing in Trypanosoma brucei. Here, we report the purification of a high molecular weight protein complex consisting solely of the MRP1 and MRP2 proteins from the mitochondrion of T. brucei. The MRP1/MRP2 complex natively purified from T. brucei and the one reconstituted in Escherichia coli in vivo bind guide (g) RNAs and pre-mRNAs with dissociation constants in the nanomolar range, and efficiently promote annealing of pre-mRNAs with their cognate gRNAs. In addition, the MRP1/MRP2 complex stimulates annealing between two non-cognate RNA molecules suggesting that along with the cognate duplexes, spuriously mismatched RNA hybrids may be formed at some rate in vivo. A mechanism of catalysed annealing of gRNA/pre-mRNA by the MRP1/MRP2 complex is proposed.     

OB-fold domain of KREPA4 mediates high-affinity interaction with guide RNA and possesses annealing activity

KREPA4, also called MP24, is an essential mitochondrial guide RNA (gRNA)-binding protein with a preference for the 3′ oligo(U) tail in trypanosomes. Structural prediction and compositional analysis of KREPA4 have identified a conserved OB (oligonucleotide/oligosaccharide-binding)-fold at the C-terminal end and two low compositional complexity regions (LCRs) at its N terminus. Concurrent with these predictions, one or both of these regions in KREPA4 protein may be involved in gRNA binding. To test this possibility, deletion mutants of KREPA4 were made and the effects on the gRNA-binding affinities were measured by quantitative electrophoretic mobility shift assays. The gRNA-binding specificities of these mutants were evaluated by competition experiments using gRNAs with U-tail deletions or stem–loop modifications and uridylated nonguide RNAs or heterologous RNA. Our results identified the predicted OB-fold as the functional domain of KREPA4 that mediates a high-affinity interaction with the gRNA oligo(U) tail. An additional contribution toward RNA-binding function was localized to LCRs that further stabilize the binding through sequence-specific interactions with the guide secondary structure. In this study we also found that the predicted OB-fold has an RNA annealing activity, representing the first report of such activity for a core component of the RNA editing complex.     

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.     

Association of Guide RNA Binding Protein gBP21 with Active RNA Editing Complexes in Trypanosoma brucei

RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.     

Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3′-to-5′ in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3′ ends and strain-specific alternative 3′ editing within 3′ UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.     

Arginine methylation regulates mitochondrial gene expression in Trypanosoma brucei through multiple effector proteins

Arginine methylation is a post-translational modification that impacts gene expression in both the cytoplasm and nucleus. Here, we demonstrate that arginine methylation also affects mitochondrial gene expression in the protozoan parasite, Trypanosoma brucei. Down-regulation of the major trypanosome type I protein arginine methyltransferase, TbPRMT1, leads to destabilization of specific mitochondrial mRNAs. We provide evidence that some of these effects are mediated by the mitochondrial RNA-binding protein, RBP16, which we previously demonstrated affects both RNA editing and stability. TbPRMT1 catalyzes methylation of RBP16 in vitro. Further, MALDI-TOF-MS analysis of RBP16 isolated from TbPRMT1-depleted cells indicates that, in vivo, TbPRMT1 modifies two of the three known methylated arginine residues in RBP16. Expression of mutated, nonmethylatable RBP16 in T. brucei has a dominant negative effect, leading to destabilization of a subset of those mRNAs affected by TbPRMT1 depletion. Our results suggest that the specificity and multifunctional nature of RBP16 are due, at least in part, to the presence of differentially methylated forms of the protein. However, some effects of TbPRMT1 depletion on mitochondrial gene expression cannot be accounted for by RBP16 action. Thus, these data implicate additional, unknown methylproteins in mitochondrial gene regulation.