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.     

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.     

Identification of domains in apobec-1 complementation factor required for RNA binding and apolipoprotein-B mRNA editing

The C-to-U editing of apolipoprotein-B (apo-B) mRNA is catalyzed by an enzyme complex that recognizes an 11-nt mooring sequence downstream of the editing site. A minimal holoenzyme that edits apo-B mRNA in vitro has been defined. This complex contains apobec-1, the catalytic subunit, and apobec-1 complementation factor (ACF), the RNA-binding subunit that binds to the mooring sequence. Here, we show that ACF binds with high affinity to single-stranded but not double-stranded apo-B mRNA. ACF contains three nonidentical RNA recognition motifs (RRM) and a unique C-terminal auxiliary domain. In many multi-RRM proteins, the RRMs mediate RNA binding and an auxiliary domain functions in protein-protein interactions. Here we show that ACF does not fit this simple model. Based on deletion mutagenesis, the RRMs in ACF are necessary but not sufficient for binding to apo-B mRNA. Amino acids in the pre-RRM region are required for complementing activity and RNA binding, but not for interaction with apobec-1. The C-terminal 196 amino acids are not absolutely essential for function. However, further deletion of an RG-rich region from the auxiliary domain abolished complementing activity, RNA binding, and apobec-1 interaction. The auxiliary domain alone did not bind apobec-1. Although all three RRMs are required for complementing activity and apobec-1 interaction, the individual motifs contribute differently to RNA binding. Point mutations in RRM1 or RRM2 decreased the Kd for apo-B mRNA by two orders of magnitude whereas mutations in RRM3 reduced binding affinity 13-fold. The pairwise expression of RRM1 with RRM2 or RRM3 resulted in moderate affinity binding.     

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.     

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.     

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.     

The solution structure of REF2-I reveals interdomain interactions and regions involved in binding mRNA export factors and RNA

The RNA binding and export factor (REF) family of mRNA export adaptors are found in several nuclear protein complexes including the spliceosome, TREX, and exon junction complexes. They bind RNA, interact with the helicase UAP56/DDX39, and are thought to bridge the interaction between the export factor TAP/NXF1 and mRNA. REF2-I consists of three domains, with the RNA recognition motif (RRM) domain positioned in the middle. Here we dissect the interdomain interactions of REF2-I and present the solution structure of a functionally competent double domain (NM; residues 1-155). The N-terminal domain comprises a transient helix (N-helix) linked to the RRM by a flexible arm that includes an Arg-rich region. The N-helix, which is required for REF2-I function in vivo, overlaps the highly conserved REF-N motif and, together with the adjacent Arg-rich region, interacts transiently with the RRM. RNA interacts with REF2-I through arginine-rich regions in its N- and C-terminal domains, but we show that it also interacts weakly with the RRM. The mode of interaction is unusual for an RRM since it involves loops L1 and L5. NMR signal mapping and biochemical analysis with NM indicate that DDX39 and TAP interact with both the N and RRM domains of REF2-I and show that binding of these proteins and RNA will favor an open conformation for the two domains. The proximity of the RNA, TAP, and DDX39 binding sites on REF2-I suggests their binding may be mutually exclusive, which would lead to successive ligand binding events in the course of mRNA export.     

Highly selective actions of HuR in antagonizing AU-rich element-mediated mRNA destabilization

Human RNA-binding protein HuR, a nucleocytoplasmic shuttling protein, is a ubiquitously expressed member of the family of Hu proteins, which consist of two N-terminal RNA recognition motifs (RRM1 and RRM2), a hinge region, and a C-terminal RRM (RRM3). Although in vitro experiments showed indiscriminate binding of Hu proteins synthesized in bacterial systems to many different AU-rich elements (AREs), in vivo studies have pointed to a cytoplasmic role for HuR protein in antagonizing the rapid decay of some specific ARE-containing mRNAs, depending on physiological situations. By ectopically overexpressing HuR and its mutant derivatives in NIH 3T3 cells to mimic HuR upregulation of specific ARE-containing mRNAs in other systems, we have examined the in vivo ARE-binding specificity of HuR and dissected its functionally critical domains. We show that in NIH 3T3 cells, HuR stabilizes reporter messages containing only the c-fos ARE and not other AREs. Two distinct binding sites were identified within the c-fos ARE, the 5' AUUUA-containing domain and the 3' U-stretch-containing domain. These actions of HuR are markedly different from those of another ARE-binding protein, hnRNP D (also termed AUF1), which in vivo recognizes AUUUA repeats found in cytokine AREs and can exert both stabilizing and destabilizing effects. Further experiments showed that any combination of two of the three RRM domains of HuR is sufficient for strong binding to the c-fos ARE in vitro and to exert an RNA stabilization effect in vivo comparable to that of intact HuR and that the hinge region containing nucleocytoplasmic shuttling signals is dispensable for the stabilization effect of HuR. Our data suggest that the ARE-binding specificity of HuR in vivo is modulated to interact only with and thus regulate specific AREs in a cell type- and physiological state-dependent manner.     

The structure of the C-terminal domain of the largest editosome interaction protein and its role in promoting RNA binding by RNA-editing ligase L2

Trypanosomatids, such as the sleeping sickness parasite Trypanosoma brucei, contain a ～ 20S RNA-editing complex, also called the editosome, which is required for U-insertion/deletion editing of mitochondrial mRNAs. The editosome contains a core of 12 proteins including the large interaction protein A1, the small interaction protein A6, and the editing RNA ligase L2. Using biochemical and structural data, we identified distinct domains of T. brucei A1 which specifically recognize A6 and L2. We provide evidence that an N-terminal domain of A1 interacts with the C-terminal domain of L2. The C-terminal domain of A1 appears to be required for the interaction with A6 and also plays a key role in RNA binding by the RNA-editing ligase L2 in trans. Three crystal structures of the C-terminal domain of A1 have been elucidated, each in complex with a nanobody as a crystallization chaperone. These structures permitted the identification of putative dsRNA recognition sites. Mutational analysis of conserved residues of the C-terminal domain identified Arg703, Arg731 and Arg734 as key requirements for RNA binding. The data show that the editing RNA ligase activity is modulated by a novel mechanism, i.e. by the trans-acting RNA binding C-terminal domain of A1.     

A deletion site editing endonuclease in Trypanosoma brucei

RNA editing in Trypanosoma brucei inserts and deletes uridines in mitochondrial mRNAs by a series of enzymatic steps that are catalyzed by a multiprotein complex, the editosome. KREPB1 and two related editosome proteins KREPB2 and KREPB3 contain motifs that suggest endonuclease and RNA/protein interaction functions. Repression of KREPB1 expression in procyclic forms by RNAi inhibited growth, in vivo editing, and in vitro endoribonucleolytic cleavage of deletion substrates. However, cleavage of insertion substrates and the exoUase, TUTase, and ligase catalytic activities of editing were retained by 20S editosomes. Repression of expression of an ectopic KREPB1 allele in bloodstream forms lacking both endogenous alleles or exclusive expression of KREPB1 with point mutations in the putative RNase III catalytic domain also blocked growth, in vivo editing, and abolished cleavage of deletion substrates, without affecting the other editing steps. These data indicate that KREPB1 is an endoribonuclease that is specific for RNA editing deletion sites.     

Apo raver1 structure reveals distinct RRM domain orientations

Raver1 is a multifunctional protein that modulates both alternative splicing and focal adhesion assembly by binding to the nucleoplasmic splicing repressor polypyrimidine tract protein (PTB) or to the cytoskeletal proteins vinculin and α‐actinin. The amino‐terminal region of raver1 has three RNA recognition motif (RRM1, RRM2, and RRM3) domains, and RRM1 interacts with the vinculin tail (Vt) domain and vinculin mRNA. We previously determined the crystal structure of the raver1 RRM1–3 domains in complex with Vt at 2.75 Å resolution. Here, we report crystal structure of the unbound raver1 RRM1–3 domains at 2 Å resolution. The apo structure reveals that a bound sulfate ion disrupts an electrostatic interaction between the RRM1 and RRM2 domains, triggering a large relative domain movement of over 30°. Superposition with other RNA‐bound RRM structures places the sulfate ion near the superposed RNA phosphate group suggesting that this is the raver1 RNA binding site. While several single and some tandem RRM domain structures have been described, to the best of our knowledge, this is the second report of a three‐tandem RRM domain structure.     

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.     

Two-hybrid cloning identifies an RNA-binding protein, GRY-RBP, as a component of apobec-1 editosome

ApoB mRNA editing is mediated by an editosome complex with apobec-1 as its catalytic component. By yeast two-hybrid cloning using apobec-1 as bait we identified a 69.6-kDa RNA binding protein, GRY-RBP, that contains 3 RNA-recognition motifs (RRMs) as a novel apobec-1 associating protein. GRY-RBP may be an alternatively spliced species of NASP1, a protein of known function. GRY-RBP was shown to bind to apobec-1, the catalytic component of apoB mRNA editosome, in vivo and in vitro. Immunodepletion using a monospecific rabbit antibody abolished editing in apobec-1 expressing HepG2 S-100 extracts. GRY-RBD interacted with apobec-1 through its C-terminus. It contains three RRM (RNA recognition motifs) domains that are homologous to those found in human ACF (apobec-1 complementation factor). Phylogeny analysis of the RRM domain-containing proteins indicates that GRY-RBP clusters with hnRNP-R, ACF, and ABBP-1 (another apobec-1 binding protein). In addition to its involvement with apobec-1 editosome, the suggested cellular functions of GRY-RBD and its structural homologues include RNA transport and RNA secondary structure stabilization.     

The La motif and the RNA recognition motifs of human La autoantigen contribute individually to RNA recognition and subcellular localization

The human La autoantigen (hLa) protein is a predominantly nuclear phosphoprotein that contains three potential RNA binding domains referred to as the La motif and the RNA recognition motifs RRMs 1 and 2. With this report, we differentiated the contribution of its three RNA binding domains to RNA binding by combining in vitro and in vivo assays. Also, surface plasmon resonance technology was used to generate a model for the sequential contribution of the RNA binding domains to RNA binding. The results indicated that the La motif may contribute to specificity rather than affinity, whereas RRM1 is indispensable for association with pre-tRNA and hY1 RNA. Furthermore, RRM2 was not crucial for the interaction with various RNAs in vivo, although needed for full-affinity binding in vitro. Moreover, earlier studies suggest that RNA binding by hLa may direct its subcellular localization. As shown previously for RRM1, deletion of RNP2 sequence in RRM1 alters nucleolar distribution of hLa, not observed after deletion of the La motif. Here we discuss a model for precursor RNA binding based on a sequential association process mediated by RRM1 and the La motif.