REH2 RNA Helicase in Kinetoplastid Mitochondria

Abstract

Regulation of gene expression in kinetoplastid mitochondria is largely post-transcriptional and involves the orchestration of polycistronic RNA processing, 3-terminal maturation, RNA editing, turnover, and translation; however, these processes remain poorly studied. Core editing complexes and their U-insertion/deletion activities are relatively well characterized, and a battery of ancillary factors has recently emerged. This study characterized a novel DExH-box RNA helicase, termed here REH2 (RNA editing associated helicase 2), in unique ribonucleoprotein complexes that exhibit unwinding and guide RNA binding activities, both of which required a double-stranded RNA-binding domain (dsRBD) and a functional helicase motif I of REH2. REH2 complexes and recently identified related particles share a multiprotein core but are distinguished by several differential polypeptides. Finally, REH2 associates transiently, via RNA, with editing complexes, mitochondrial ribosomes, and several ancillary factors that control editing and RNA stability. We propose that these putative higher order structures coordinate mitochondrial gene expression.Unique gene expression mechanisms in kinetoplastid flagellates include U-insertion/deletion RNA editing by concerted cycles of cleavage, U-addition/removal, and ligation that can create hundreds of amino acid codons in most mitochondrial mRNAs (1, 2). The RNA editing core complex (RECC) 4 contains 18 -20 subunits (3-6), although a few subunits seem to exchange in substrate-specific variants of this complex (7). The RECC acronym was recently introduced by Simpson et al. (55). Editing complexes recognize partial helices between pre-mRNA and complementary guide RNAs (gRNAs) initially stabilized by a short "anchor" duplex (6,8,9). Substrate determinants for duplex binding and nuclease specificity (6, 10, 11) and substrate structure in solution (12-14) have been characterized.Several accessory factors, mostly in multisubunit arrays, have been proposed to modulate RNA editing during catalysis, substrate production, or RNA turnover. The MRP complex has RNA annealing activity in vitro and may promote mRNA and gRNA pairing (15, 16). Post-transcriptional mRNA terminal 3Ј-poly(A)/(U) and gRNA 3Ј-poly(U) maturation are mediated by KPAP1 and RET1 complexes (17,18). MRB1, TbRGG1, and GRBC complexes proposed to contain between 14 and 24 proteins (termed here MRB-related complexes) share several components, but their functional relationship remains unclear. Repression of a few common subunits inhibited RNA editing and in some cases also decreased the level of total gRNA. GRBC1 and GRBC2 co-purified with RECC subunits (18 -24). MERS1, MRP, and RBP16 proteins were associated with mRNA stability (23, 25). RBP16 also stimulated RNA insertion in vitro (26,27). DEAD-box mHel61 (also termed REH1) is the only predicted helicase known to impact RNA editing (28). Most of these proteins are likely to have additional roles outside editing. RNA helicases are common across species and typically multifunctional; howev...

An Arginine-Glycine-Rich RNA Binding Protein Impacts the Abundance of Specific mRNAs in the Mitochondria of Trypanosoma brucei

“…These findings are consistent with the recent report that GAP1/2 associates with the REH2 helicase apart from the MRB1 core (53), as well as with our previous data suggesting a similar association of GAP1/2 with the mitochondrial zinc finger protein, MRB6070 (20). Given the interaction of REH2 with the RNA editing machinery and its impact on RNA editing, it is easy to envision a role for GAP1/2-bound gRNAs in association with REH2 (31,52,53). In contrast, our results indicate that TbRGG3 is not involved in RNA editing, and thus its function likely does not involve gRNAs.…”

“…We hypothesized that the T. brucei TbRGG3 protein plays a role in mitochondrial RNA biology due to its reported association with the RNA binding proteins, GAP1/2, TbRGG1, and REH2, as well as the large ribosomal subunit (13,25,31). The arginine-glycinerich nature of TbRGG3 further suggested that it possesses intrinsic RNA binding ability (21,32,33).…”

“…The ability of TbRGG3 to bind RNA and modulate RNA structure implicates TbRGG3 in shaping the mitochondrial transcriptome. Additionally, mass spectrometry studies from several laboratories identified TbRGG3 in association with proteins involved in mitochondrial RNA stability, editing, and translation (13,25,31), pointing to a function for TbRGG3 in mitochondrial RNA metabolism. To examine the role of TbRGG3 in mitochondrial gene expression, we generated PF T. brucei cells in which RNAi silenced TbRGG3 expression in a Tet-regulated manner.…”

“…In addition to harboring a predicted mitochondrial import sequence, TbRGG3 was designated "mitochondria likely" by the mitochondrial proteome study of Panigrahi et al (49) (www .trypsproteome.org). Moreover, TbRGG3 was isolated by tandem affinity purification in association with known mitochondrial proteins GAP1, GAP2, and TbRGG1 (13), RNA editing helicase 2 (REH2) (31), and mitochondrial RPL3 (25). To directly determine the subcellular localization of TbRGG3, we generated PF T. brucei cells expressing TbRGG3 with a 2ϫMyc tag at its C terminus in a Tet-regulatable manner ( Fig.…”

“…Due to these findings, and in keeping with nomenclature of mitochondrial proteins with the sequence character of arginine-glycine-rich stretches, we renamed this protein TbRGG3. TbRGG3 is implicated in mitochondrial RNA biology not only by association with GAP1/2 and TbRGG1 but also through its identification in pulldown of the large ribosomal subunit (25) and the REH2 (RNA editing helicase 2) complex (31). Moreover, the arginine-glycine-rich character of TbRGG3 suggests a function in RNA binding (32,33).…”