Therapeutic efficacy of the humanized JAA-F11 anti-Thomsen-Friedenreich antibody constructs H2aL2a and H3L3 in human breast and lung cancer xenograft models.

The Thomsen-Friedenreich antigen (TF-Ag-α) is found on ~85% of human carcinomas but is cryptic on normal tissue. The humanized highly specific hJAA-F11-H2aL2a and -H3L3 antibodies target TF-Ag-α without binding to TF-Ag-beta (found on surface glycolipids of some normal cells). The relative affinity of H3L3 is 17 times that of H2aL2a, which would seem to favor superior efficacy, however, increased affinity can result in less tumor penetration. To assess the potential therapeutic efficacy of these antibodies, four human cancer- mouse xenograft models were treated with H2aL2a and H3L3. The tumor xenograft models used were human non-small cell lung cancer, H520, and small cell lung cancer, HTB171 in nude mice and human triple negative breast cancer, MDA-MB-231 and HCC1806 in SCID mice. H2aL2a significantly decreased tumor growth in both breast and both lung cancer models. H2aL2a showed statistically equal or better efficacy than H3L3 and has superior production capabilities. These results suggest that H2aL2a may be superior as a naked antibody, as an antibody drug conjugate or as a radiolabeled antibody, however the higher affinity of H3L3 may lead to better efficacy in bi-specific therapies in which the binding is decreased due to the presence of only one TF-Ag-α binding site.

Preclinical Analysis of JAA-F11, a Specific Anti-Thomsen-Friedenreich Antibody via Immunohistochemistry and In Vivo Imaging.

The tumor specificity of JAA-F11, a novel monoclonal antibody specific for the Thomsen-Friedenreich cancer antigen (TF-Ag-alpha linked), has been comprehensively studied by in vitro immunohistochemical (IHC) staining of human tumor and normal tissue microarrays and in vivo biodistribution and imaging by micro-positron emission tomography imaging in breast and lung tumor models in mice. The IHC analysis detailed herein is the comprehensive biological analysis of the tumor specificity of JAA-F11 antibody performed as JAA-F11 is progressing towards preclinical safety testing and clinical trials. Wide tumor reactivity of JAA-F11, relative to the matched mouse IgG3 (control), was observed in 85% of 1269 cases of breast, lung, prostate, colon, bladder, and ovarian cancer. Staining on tissues from breast cancer cases was similar regardless of hormonal or Her2 status, and this is particularly important in finding a target on the currently untargetable triple-negative breast cancer subtype. Humanization of JAA-F11 was recently carried out as explained in a companion paper "Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and In Vitro Efficacy Analysis" (Neoplasia 19: 716-733, 2017), and it was confirmed that humanization did not affect chemical specificity. IHC studies with humanized JAA-F11 showed similar binding to human breast tumor tissues. In vivo imaging and biodistribution studies in a mouse syngeneic breast cancer model and in a mouse-human xenograft lung cancer model with humanized 124I- JAA-F11 construct confirmed in vitro tumor reactivity and specificity. In conclusion, the tumor reactivity of JAA-F11 supports the continued development of JAA-F11 as a targeted cancer therapeutic for multiple cancers, including those with unmet need.

Humanization of JAA-F11, a Highly Specific Anti-Thomsen-Friedenreich Pancarcinoma Antibody and InVitro Efficacy Analysis.

JAA-F11 is a highly specific mouse monoclonal to the Thomsen-Friedenreich Antigen (TF-Ag) which is an alpha-O-linked disaccharide antigen on the surface of ~80% of human carcinomas, including breast, lung, colon, bladder, ovarian, and prostate cancers, and is cryptic on normal cells. JAA-F11 has potential, when humanized, for cancer immunotherapy for multiple cancer types. Humanization of JAA-F11, was performed utilizing complementarity determining regions grafting on a homology framework. The objective herein is to test the specificity, affinity and biology efficacy of the humanized JAA-F11 (hJAA-F11). Using a 609 target glycan array, 2 hJAA-F11 constructs were shown to have excellent chemical specificity, binding only to TF-Ag alpha-linked structures and not to TF-Ag beta-linked structures. The relative affinity of these hJAA-F11 constructs for TF-Ag was improved over the mouse antibody, while T20 scoring predicted low clinical immunogenicity. The hJAA-F11 constructs produced antibody-dependent cellular cytotoxicity in breast and lung tumor lines shown to express TF-Ag by flow cytometry. Internalization of hJAA-F11 into cancer cells was also shown using a surface binding ELISA and confirmed by immunofluorescence microscopy. Both the naked hJAA-F11 and a maytansine-conjugated antibody (hJAA-F11-DM1) suppressed in vivo tumor progression in a human breast cancer xenograft model in SCID mice. Together, our results support the conclusion that the humanized antibody to the TF-Ag has potential as an adjunct therapy, either directly or as part of an antibody drug conjugate, to treat breast cancer, including triple negative breast cancer which currently has no targeted therapy, as well as lung cancer.

The Major Protein Arginine Methyltransferase in Trypanosoma brucei Functions as an Enzyme-Prozyme Complex.

Prozymes are catalytically inactive enzyme paralogs that dramatically stimulate the function of weakly active enzymes through complex formation. The two prozymes described to date reside in the polyamine biosynthesis pathway of the human parasite Trypanosoma brucei, an early branching eukaryote that lacks transcriptional regulation and regulates its proteome through posttranscriptional and posttranslational means. Arginine methylation is a common posttranslational modification in eukaryotes catalyzed by protein arginine methyltransferases (PRMTs) that are typically thought to function as homodimers. We demonstrate that a major T. brucei PRMT, TbPRMT1, functions as a heterotetrameric enzyme-prozyme pair. The inactive PRMT paralog, TbPRMT1PRO, is essential for catalytic activity of the TbPRMT1ENZ subunit. Mutational analysis definitively demonstrates that TbPRMT1ENZ is the cofactor-binding subunit and carries all catalytic activity of the complex. Our results are the first demonstration of an obligate heteromeric PRMT, and they suggest that enzyme-prozyme organization is expanded in trypanosomes as a posttranslational means of enzyme regulation.

An arginine-glycine-rich RNA binding protein impacts the abundance of specific mRNAs in the mitochondria of Trypanosoma brucei.

In kinetoplastid parasites, regulation of mitochondrial gene expression occurs posttranscriptionally via RNA stability and RNA editing. In addition to the 20S editosome that contains the enzymes required for RNA editing, a dynamic complex called the mitochondrial RNA binding 1 (MRB1) complex is also essential for editing. Trypanosoma brucei RGG3 (TbRGG3) was originally identified through its interaction with the guide RNA-associated proteins 1 and 2 (GAP1/2), components of the MRB1 complex. Both the arginine-glycine-rich character of TbRGG3, which suggests a function in RNA binding, and its interaction with MRB1 implicate TbRGG3 in mitochondrial gene regulation. Here, we report an in vitro and in vivo characterization of TbRGG3 function in T. brucei mitochondria. We show that in vitro TbRGG3 binds RNA with broad sequence specificity and has the capacity to modulate RNA-RNA interactions. In vivo, inducible RNA interference (RNAi) studies demonstrate that TbRGG3 is essential for proliferation of insect vector stage T. brucei. TbRGG3 ablation does not cause a defect in RNA editing but, rather, specifically affects the abundance of two preedited transcripts as well as their edited counterparts. Protein-protein interaction studies show that TbRGG3 associates with GAP1/2 apart from the remainder of the MRB1 complex, as well as with several non-MRB1 proteins that are required for mitochondrial RNA editing and/or stability. Together, these studies demonstrate that TbRGG3 is an essential mitochondrial gene regulatory factor that impacts the stabilities of specific RNAs.

Functional interplay between protein arginine methyltransferases in Trypanosoma brucei.

Arginine methylation is a common posttranslational modification that has far-reaching cellular effects. Trypanosoma brucei is an early-branching eukaryote with four characterized protein arginine methyltransferases (PRMTs), one additional putative PRMT, and over 800 arginine methylated proteins, suggesting that arginine methylation has widespread impacts in this organism. While much is known about the activities of individual T. brucei PRMTs (TbPRMTs), little is known regarding how TbPRMTs function together in vivo. In this study, we analyzed single and selected double TbPRMT knockdowns for the impact on expression of TbPRMTs and global methylation status. Repression of TbPRMT1 caused a decrease in asymmetric dimethylarginine and a marked increase in monomethylarginine that was catalyzed by TbPRMT7, suggesting that TbPRMT1 and TbPRMT7 can compete for the same substrate. We also observed an unexpected and strong interdependence between TbPRMT1 and TbPRMT3 protein levels. This finding, together with the observation of similar methyl landscape profiles in TbPRMT1 and TbPRMT3 repressed cells, strongly suggests that these two enzymes form a functional complex. We show that corepression of TbPRMT6/7 synergistically impacts growth of procyclic-form T. brucei. Our findings also implicate the actions of noncanonical, and as yet unidentified, PRMTs in T. brucei. Together, our studies indicate that TbPRMTs display a functional interplay at multiple levels.

Global proteomic analysis in trypanosomes reveals unique proteins and conserved cellular processes impacted by arginine methylation.

Arginine methylation is a common posttranslational modification with reported functions in transcription, RNA processing and translation, and DNA repair. Trypanosomes encode five protein arginine methyltransferases, suggesting that arginine methylation exerts widespread impacts on the biology of these organisms. Here, we performed a global proteomic analysis of Trypanosoma brucei to identify arginine methylated proteins and their sites of modification. Using an approach entailing two-dimensional chromatographic separation and alternating electron transfer dissociation and collision induced dissociation, we identified 1332 methylarginines in 676 proteins. The resulting data set represents the largest compilation of arginine methylated proteins in any organism to date. Functional classification revealed numerous arginine methylated proteins involved in flagellar function, RNA metabolism, DNA replication and repair, and intracellular protein trafficking. Thus, arginine methylation has the potential to impact aspects of T. brucei gene expression, cell biology, and pathogenesis. Interestingly, pathways with known methylated proteins in higher eukaryotes were identified in this study, but often different components of the pathway were methylated in trypanosomes. Methylarginines were often identified in glycine rich contexts, although exceptions to this rule were detected. Collectively, these data inform on a multitude of aspects of trypanosome biology and serve as a guide for the identification of homologous arginine methylated proteins in higher eukaryotes. BIOLOGICAL SIGNIFICANCE: T. brucei is a protozoan parasite that causes lethal African sleeping sickness in humans and nagana in livestock, thereby imposing a significant medical and economic burden on sub-Saharan Africa. The parasite encounters very different environments as it cycles between mammalian and insect hosts, and must exert cellular responses to these varying milieus. One mechanism by which all cells respond to changing environments is through posttranslational modification of proteins. Arginine methylation is one such modification that can dramatically impact protein-protein and protein-nucleic acid interactions and subcellular localization of proteins. To define the breadth of arginine methylation in trypanosomes and identify target proteins, we performed a global proteomic analysis of arginine methylated proteins in insect stage T. brucei. We identified 1332 methylarginines in 676 proteins, generating the largest compilation of methylarginine containing proteins in any organism to date. Numerous arginine methylated proteins function in RNA and DNA related processes, suggesting this modification can impact T. brucei genome integrity and gene regulation at numerous points. Other processes that appear to be strongly influenced by arginine methylation are intracellular protein trafficking, signaling, protein folding and degradation, and flagellar function. The widespread nature of arginine methylation in trypanosomes highlights its potential to greatly affect parasite biology and pathogenesis.

Proteomic analysis reveals diverse classes of arginine methylproteins in mitochondria of trypanosomes.

Arginine (arg) methylation is a widespread posttranslational modification of proteins that impacts numerous cellular processes such as chromatin remodeling, RNA processing, DNA repair, and cell signaling. Known arg methylproteins arise mostly from yeast and mammals, and are almost exclusively nuclear and cytoplasmic. Trypanosoma brucei is an early branching eukaryote whose genome encodes five putative protein arg methyltransferases, and thus likely contains a plethora of arg methylproteins. Additionally, trypanosomes and related organisms possess a unique mitochondrion that undergoes dramatic developmental regulation and uses novel RNA editing and mitochondrial DNA replication mechanisms. Here, we performed a global mass spectrometric analysis of the T. brucei mitochondrion to identify new arg methylproteins in this medically relevant parasite. Enabling factors of this work are use of a combination digestion with two orthogonal enzymes, an efficient offline two dimensional chromatography separation, and high-resolution mass spectrometry analysis with two complementary activations. This approach led to the comprehensive, sensitive and confident identification and localization of methylarg at a proteome level. We identified 167 arg methylproteins with wide-ranging functions including metabolism, transport, chaperoning, RNA processing, translation, and DNA replication. Our data suggest that arg methylproteins in trypanosome mitochondria possess both trypanosome-specific and evolutionarily conserved modifications, depending on the protein targeted. This study is the first comprehensive analysis of mitochondrial arg methylation in any organism, and represents a significant advance in our knowledge of the range of arg methylproteins and their sites of modification. Moreover, these studies establish T. brucei as a model organism for the study of posttranslational modifications.

Functional characterization of two paralogs that are novel RNA binding proteins influencing mitochondrial transcripts of Trypanosoma brucei.

A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.

Multifunctional G-rich and RRM-containing domains of TbRGG2 perform separate yet essential functions in trypanosome RNA editing.

Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3'-to-5' progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. Interactions between TbRGG2 and MRB1 complex proteins are mediated by both G-rich and RRM-containing domains, depending on the binding partner. Overall, our results are consistent with a model in which the high-affinity RNA binding and RNA-annealing activities of the G-rich domain are essential for RNA editing in vivo. The RRM domain may have key functions involving interactions with the MRB1 complex and/or regulation of the activities of the G-rich domain.

Architecture of the trypanosome RNA editing accessory complex, MRB1.

Trypanosoma brucei undergoes an essential process of mitochondrial uridine insertion and deletion RNA editing catalyzed by a 20S editosome. The multiprotein mitochondrial RNA-binding complex 1 (MRB1) is emerging as an equally essential component of the trypanosome RNA editing machinery, with additional functions in gRNA and mRNA stabilization. The distinct and overlapping protein compositions of reported MRB1 complexes and diverse MRB1 functions suggest that the complex is composed of subcomplexes with RNA-dependent and independent interactions. To determine the architecture of the MRB1 complex, we performed a comprehensive yeast two-hybrid analysis of 31 reported MRB1 proteins. We also used in vivo analyses of tagged MRB1 components to confirm direct and RNA-mediated interactions. Here, we show that MRB1 contains a core complex comprised of six proteins and maintained by numerous direct interactions. The MRB1 core associates with multiple subcomplexes and proteins through RNA-enhanced or RNA-dependent interactions. These findings provide a framework for interpretation of previous functional studies and suggest that MRB1 is a dynamic complex that coordinates various aspects of mitochondrial gene regulation.

Protein arginine methylation in parasitic protozoa.

Protozoa constitute the earliest branch of the eukaryotic lineage, and several groups of protozoans are serious parasites of humans and other animals. Better understanding of biochemical pathways that are either in common with or divergent from those of higher eukaryotes is integral in the defense against these parasites. In yeast and humans, the posttranslational methylation of arginine residues in proteins affects myriad cellular processes, including transcription, RNA processing, DNA replication and repair, and signal transduction. The protein arginine methyltransferases (PRMTs) that catalyze these reactions, which are unique to the eukaryotic kingdom of organisms, first become evident in protozoa. In this review, we focus on the current understanding of arginine methylation in multiple species of parasitic protozoa, including Trichomonas, Entamoeba, Toxoplasma, Plasmodium, and Trypanosoma spp., and discuss how arginine methylation may play important and unique roles in each type of parasite. We mine available genomic and transcriptomic data to inventory the families of PRMTs in different parasites and the changes in their abundance during the life cycle. We further review the limited functional studies on the roles of arginine methylation in parasites, including epigenetic regulation in Apicomplexa and RNA processing in trypanosomes. Interestingly, each of the parasites considered herein has significantly differing sets of PRMTs, and we speculate on the importance of this diversity in aspects of parasite biology, such as differentiation and antigenic variation.

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.

Human papillomavirus E1 and E2 mediated DNA replication is not arrested by DNA damage signalling.

Integration of human papillomaviruses into that of the host promotes genomic instability and progression to cancer; factors that promote integration remain to be fully identified. DNA damage agents can promote double strand breaks during DNA replication providing substrates for integration and we investigated the ability of DNA damage to regulate HPV E1 and E2 mediated DNA replication. Results demonstrate that HPV E1 and E2 replication is not arrested following DNA damage, both in vivo and in vitro, while replication by SV40 Large T antigen is arrested and ATR is the candidate kinase for mediating the arrest. LTAg is a target for PIKK DNA damage signalling kinases, while E1 is not. We propose that the failure of E1 to be targeted by PIKKs allows HPV replication in the presence of DNA damaging agents. Such replication will result in double strand breaks in the viral genome ultimately promoting viral integration and cervical cancer.

TbPRMT6 is a type I protein arginine methyltransferase that contributes to cytokinesis in Trypanosoma brucei.

Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). In Saccharomyces cerevisiae and mammals, this modification affects multiple cellular processes, such as chromatin remodeling leading to transcriptional regulation, RNA processing, DNA repair, and cell signaling. The protozoan parasite Trypanosoma brucei possesses five putative PRMTs in its genome. This is a large number of PRMTs relative to other unicellular eukaryotes, suggesting an important role for arginine methylation in trypanosomes. Here, we present the in vitro and in vivo characterization of a T. brucei enzyme homologous to human PRMT6, which we term TbPRMT6. Like human PRMT6, TbPRMT6 is a type I PRMT, catalyzing the production of monomethylarginine and asymmetric dimethylarginine residues. In in vitro methylation assays, TbPRMT6 utilizes bovine histones as a substrate, but it does not methylate several T. brucei glycine/arginine-rich proteins. As such, it exhibits a relatively narrow substrate specificity compared to other T. brucei PRMTs. Knockdown of TbPRMT6 in both procyclic form and bloodstream form T. brucei leads to a modest but reproducible effect on parasite growth in culture. Moreover, upon TbPRMT6 depletion, both PF and BF exhibit aberrant morphologies indicating defects in cell division, and these defects differ in the two life cycle stages. Mass spectrometry of TbPRMT6-associated proteins reveals histones, components of the nuclear pore complex, and flagellar proteins that may represent TbPRMT6 substrates contributing to the observed growth and morphological defects.

Structure of the Trypanosoma brucei p22 protein, a cytochrome oxidase subunit II-specific RNA-editing accessory factor.

Kinetoplastid RNA (k-RNA) editing is a complex process in the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, that involves the guide RNA-directed insertion and deletion of uridines from precursor-mRNAs to produce mature, translatable mRNAs. k-RNA editing is performed by multiprotein complexes called editosomes. Additional non-editosome components termed k-RNA-editing accessory factors affect the extent of editing of specific RNAs or classes of RNAs. The T. brucei p22 protein was identified as one such accessory factor. Here we show that p22 contributes to cell growth in the procyclic form of T. brucei and functions as a cytochrome oxidase subunit II-specific k-RNA-editing accessory factor. To gain insight into its functions, we solved the crystal structure of the T. brucei p22 protein to 2.0-A resolution. The p22 structure consists of a six-stranded, antiparallel beta-sheet flanked by five alpha-helices. Three p22 subunits combine to form a tight trimer that is primarily stabilized by interactions between helical residues. One side of the trimer is strikingly acidic, while the opposite face is more neutral. Database searches show p22 is structurally similar to human p32, which has a number of functions, including regulation of RNA splicing. p32 interacts with a number of target proteins via its alpha1 N-terminal helix, which is among the most conserved regions between p22 and p32. Co-immunoprecipitation studies showed that p22 interacts with the editosome and the k-RNA accessory protein, TbRGG2, and alpha1 of p22 was shown to be important for the p22-TbRGG2 interaction. Thus, these combined studies suggest that p22 mediates its role in k-RNA editing by acting as an adaptor protein.

Distinct and overlapping functions of MRP1/2 and RBP16 in mitochondrial RNA metabolism.

Mitochondrial RNA metabolism in Trypanosoma brucei is a complex process involving both extensive RNA editing and control of RNA stability. MRP1/2 and RBP16 are two factors that have been implicated in regulating the editing and stability of specific mRNAs. These two factors exhibit similar nonspecific RNA binding and RNA-annealing activities, suggesting that some of their actions may have been previously masked by functional redundancy. Here, we examine the functional interaction of MRP1/2 and RBP16 by separate and simultaneous RNA interference and by overexpressing RBP16 in an MRP1/2-depleted background. Simultaneous depletion of these factors resulted in synthetic lethality in procyclic trypanosomes. Analysis of mitochondrial RNAs in procyclic cells revealed distinct functions for MRP1/2 and RBP16 toward edited apocytochrome b mRNA, redundant functions in stabilization of edited ATPase subunit 6 and cytochrome oxidase subunit 3 mRNAs, and concentration-dependent positive and negative functions for RBP16 toward edited RPS12 mRNAs. While simultaneous MRP1/2-RBP16 depletion had no effect on the growth of bloodstream form cells, massive adverse effects on the levels of almost all mitochondrial RNAs were observed. These studies greatly expand our knowledge regarding the functions of MRP1/2 and RBP16 and suggest that both RNA-specific and life cycle stage-specific factors impact MRP1/2 and RBP16 functions.

A type III protein arginine methyltransferase from the protozoan parasite Trypanosoma brucei.

Arginine methylation is a widespread post-translational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). The ancient protozoan parasite, Trypanosoma brucei, possesses five putative PRMTs, a relatively large number for a single-celled eukaryote. Trypanosomatids lack gene regulation at the level of transcription, instead relying on post-transcriptional control mechanisms that act at the levels of RNA turnover, translation, and editing, all processes that likely involve multiple RNA-binding proteins, which are common targets of arginine methylation. Here, we report the characterization of a trypanosome PRMT, TbPRMT7, which is homologous to human PRMT7. Interestingly, trypanosomatids are the only single-celled eukaryotes known to harbor a PRMT7 homologue. TbPRMT7 differs dramatically from all known metazoan PRMT7 homologues in lacking the second AdoMet binding-like domain that is required for activity of the human enzyme. Nevertheless, bacterially expressed TbPRMT7 exhibits robust methyltransferase activity toward multiple targets in vitro. High resolution ion exchange chromatography analysis of methylated substrates reveals that TbPRMT7 is a type III PRMT, catalyzing the formation of only monomethylarginine, thereby representing the only exclusively type III PRMT identified to date. TbPRMT7 is expressed in both mammalian and insect stage T. brucei and is apparently dispensable for growth in both life cycle stages. The enzyme is cytoplasmically localized and is a component of several higher order complexes in vivo. Together, our studies indicate that TbPRMT7 is a Type III PRMT, and its robust activity and presence in numerous complexes suggest it plays multiple roles during the complex T. brucei life cycle.

TbRGG2, an essential RNA editing accessory factor in two Trypanosoma brucei life cycle stages.

In the mitochondria of kinetoplastid protozoa, including Trypanosoma brucei, RNA editing inserts and/or deletes uridines from pre-mRNAs to produce mature, translatable mRNAs. RNA editing is carried out by several related multiprotein complexes known as editosomes, which contain all of the enzymatic components required for catalysis of editing. In addition, noneditosome accessory factors necessary for editing of specific RNAs have also been described. Here, we report the in vitro and in vivo characterization of the mitochondrial TbRGG2 protein (originally termed TbRGGm) and demonstrate that it acts as an editing accessory factor. TbRGG2 is an RNA-binding protein with a preference for poly(U). TbRGG2 protein levels are up-regulated 10-fold in procyclic form T. brucei compared with bloodstream forms. Nevertheless, the protein is essential for growth in both life cycle stages. TbRGG2 associates with RNase-sensitive and RNase-insensitive mitochondrial complexes, and a small fraction of the protein co-immunoprecipitates with editosomes. RNA interference-mediated depletion of TbRGG2 in both procyclic and bloodstream form T. brucei leads to a dramatic decrease in pan-edited RNAs and in some cases a corresponding increase in the pre-edited RNA. TbRGG2 down-regulation also results in moderate stabilization of never-edited and minimally edited RNAs. Thus, our data are consistent with a model in which TbRGG2 is multifunctional, strongly facilitating the editing of pan-edited RNAs and modestly destabilizing minimally edited and never-edited RNAs. This is the first example of an RNA editing accessory factor that functions in the mammalian infective T. brucei life cycle stage.