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• W1970146598 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. 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 (1Carnes J. Stuart K.D. Methods Enzymol. 2007; 424: 25-54Crossref PubMed Scopus (15) Google Scholar, 2Cruz-Reyes J. Hernandez A. Smith H.C. Protein-Protein and RNA-Protein Interactions in U-Insertion/Deletion RNA Editing Complexes in RNA and DNA Editing. John Wiley & Sons, Inc., New York2008: 71-98Google Scholar). The RNA editing core complex (RECC) 4The abbreviations used are: RECCRNA editing core complexdsRBDdsRNA-binding domaingRNAguide RNARNPribonucleoproteinRNAiRNA interferenceTAPtandem affinity-purifiedGRBCguide RNA binding complexMRBmitochondrial RNA bindingMNmicrococcal nucleaseGAPguide RNA-associated proteinTEVtobacco etch virus protease. contains 18–20 subunits (3Rusché L.N. Cruz-Reyes J. Piller K.J. Sollner-Webb B. EMBO J. 1997; 16: 4069-4081Crossref PubMed Scopus (142) Google Scholar, 4Panigrahi A.K. Schnaufer A. Stuart K.D. Methods Enzymol. 2007; 424: 3-24Crossref PubMed Scopus (25) Google Scholar, 5Aphasizhev R. Aphasizheva I. Nelson R.E. Gao G. Simpson A.M. Kang X. Falick A.M. Sbicego S. Simpson L. EMBO J. 2003; 22: 913-924Crossref PubMed Scopus (118) Google Scholar, 6Hernandez A. Panigrahi A. Cifuentes-Rojas C. Sacharidou A. Stuart K. Cruz-Reyes J. J. Mol. Biol. 2008; 381: 35-48Crossref PubMed Scopus (14) Google Scholar), although a few subunits seem to exchange in substrate-specific variants of this complex (7Carnes J. Trotter J.R. Peltan A. Fleck M. Stuart K. Mol. Cell. Biol. 2008; 28: 122-130Crossref PubMed Scopus (93) Google Scholar). The RECC acronym was recently introduced by Simpson et al. (55Simpson L. Aphasizhev R. Lukes J. Cruz-Reyes J. Protist. 2009; (in press)Google Scholar). Editing complexes recognize partial helices between pre-mRNA and complementary guide RNAs (gRNAs) initially stabilized by a short “anchor” duplex (6Hernandez A. Panigrahi A. Cifuentes-Rojas C. Sacharidou A. Stuart K. Cruz-Reyes J. J. Mol. Biol. 2008; 381: 35-48Crossref PubMed Scopus (14) Google Scholar, 8Blum B. Bakalara N. Simpson L. Cell. 1990; 60: 189-198Abstract Full Text PDF PubMed Scopus (452) Google Scholar, 9Seiwert S.D. Stuart K. Science. 1994; 266: 114-117Crossref PubMed Scopus (127) Google Scholar). Substrate determinants for duplex binding and nuclease specificity (6Hernandez A. Panigrahi A. Cifuentes-Rojas C. Sacharidou A. Stuart K. Cruz-Reyes J. J. Mol. Biol. 2008; 381: 35-48Crossref PubMed Scopus (14) Google Scholar, 10Cifuentes-Rojas C. Halbig K. Sacharidou A. De Nova-Ocampo M. Cruz-Reyes J. Nucleic Acids Res. 2005; 33: 6610-6620Crossref PubMed Scopus (13) Google Scholar, 11Cifuentes-Rojas C. Pavia P. Hernandez A. Osterwisch D. Puerta C. Cruz-Reyes J. J. Biol. Chem. 2007; 282: 4265-4276Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar) and substrate structure in solution (12Koslowsky D.J. Reifur L. Yu L.E. Chen W. RNA Biol. 2004; 1: 28-34Crossref PubMed Scopus (21) Google Scholar, 13Reifur L. Koslowsky D.J. RNA. 2008; 14: 2195-2211Crossref PubMed Scopus (13) Google Scholar, 14Yu L.E. Koslowsky D.J. RNA. 2006; 12: 1050-1060Crossref PubMed Scopus (13) Google Scholar) have been characterized. RNA editing core complex dsRNA-binding domain guide RNA ribonucleoprotein RNA interference tandem affinity-purified guide RNA binding complex mitochondrial RNA binding micrococcal nuclease guide RNA-associated protein tobacco etch virus protease. 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 (15Müller U.F. Lambert L. Göringer H.U. EMBO J. 2001; 20: 1394-1404Crossref PubMed Scopus (78) Google Scholar, 16Aphasizhev R. Aphasizheva I. Nelson R.E. Simpson L. RNA. 2003; 9: 62-76Crossref PubMed Scopus (93) Google Scholar). Post-transcriptional mRNA terminal 3′-poly(A)/(U) and gRNA 3′-poly(U) maturation are mediated by KPAP1 and RET1 complexes (17Aphasizhev R. Sbicego S. Peris M. Jang S.H. Aphasizheva I. Simpson A.M. Rivlin A. Simpson L. Cell. 2002; 108: 637-648Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 18Etheridge R.D. Aphasizheva I. Gershon P.D. Aphasizhev R. EMBO J. 2008; 27: 1596-1608Crossref PubMed Scopus (84) Google Scholar). 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 (18Etheridge R.D. Aphasizheva I. Gershon P.D. Aphasizhev R. EMBO J. 2008; 27: 1596-1608Crossref PubMed Scopus (84) Google Scholar, 19Fisk J.C. Ammerman M.L. Presnyak V. Read L.K. J. Biol. Chem. 2008; 283: 23016-23025Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 20Acestor N. Panigrahi A.K. Carnes J. Zíková A. Stuart K.D. RNA. 2009; 15: 277-286Crossref PubMed Scopus (48) Google Scholar, 21Hashimi H. Zíková A. Panigrahi A.K. Stuart K.D. Lukes J. RNA. 2008; 14: 970-980Crossref PubMed Scopus (73) Google Scholar, 22Hashimi H. Cicová Z. Novotná L. Wen Y.Z. Lukes J. RNA. 2009; 15: 588-599Crossref PubMed Scopus (70) Google Scholar, 23Weng J. Aphasizheva I. Etheridge R.D. Huang L. Wang X. Falick A.M. Aphasizhev R. Mol. Cell. 2008; 32: 198-209Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 24Panigrahi A.K. Zíková A. Dalley R.A. Acestor N. Ogata Y. Anupama A. Myler P.J. Stuart K.D. Mol. Cell. Proteomics. 2008; 7: 534-545Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). MERS1, MRP, and RBP16 proteins were associated with mRNA stability (23Weng J. Aphasizheva I. Etheridge R.D. Huang L. Wang X. Falick A.M. Aphasizhev R. Mol. Cell. 2008; 32: 198-209Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 25Vondrusková E. van den Burg J. Zíková A. Ernst N.L. Stuart K. Benne R. Lukes J. J. Biol. Chem. 2005; 280: 2429-2438Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). RBP16 also stimulated RNA insertion in vitro (26Pelletier M. Read L.K. RNA. 2003; 9: 457-468Crossref PubMed Scopus (72) Google Scholar, 27Miller M.M. Halbig K. Cruz-Reyes J. Read L.K. RNA. 2006; 12: 1292-1303Crossref PubMed Scopus (30) Google Scholar). DEAD-box mHel61 (also termed REH1) is the only predicted helicase known to impact RNA editing (28Missel A. Souza A.E. Nörskau G. Göringer H.U. Mol. Cell. Biol. 1997; 17: 4895-4903Crossref PubMed Scopus (95) Google Scholar). Most of these proteins are likely to have additional roles outside editing. RNA helicases are common across species and typically multifunctional; however, only a few examples have been studied in mitochondria. This work characterized the protein and RNA interactions of a factor REH2 (Tb927.4.1500) that we initially found in native editing complexes of Trypanosoma brucei. REH2 has a conserved dsRNA-binding (dsRBD) and DExH-helicase domains and forms novel ribonucleoprotein complexes (RNPs) containing helicase activity, gRNA, and a protein array that overlaps with MRB-related complexes. The integrity of REH2 RNPs as well as their helicase and gRNA binding activities require the dsRBD. REH2 associates, via RNA, with RECC, a battery of accessory editing factors, and mitochondrial ribosomes; thereby, we propose that REH2 RNPs are integral components of RNA-linked supramolecular networks that orchestrate the expression of the mitochondrial genome. A TAP-REH2 construct was made by PCR amplification of the entire open reading frame from procyclic genomic DNA using a proofreading thermostable polymerase mix (AccuTaq, Sigma) and cloning into the XhoI and BamHI sites of pLew79-ada-TAP. PCR-based site-directed mutagenesis was performed directly on this plasmid to alter the helicase motif I with oligonucleotides F-REH2-mI and R-REH2-mI and to delete the dsRBD with oligonucleotides F-dsRBD-Δ and R-dsRBD-Δ. An RNAi construct was obtained by cloning an REH2 fragment of 1665 bp into p2T7-177 (29Wickstead B. Ersfeld K. Gull K. Mol. Biochem. Parasitol. 2002; 125: 211-216Crossref PubMed Scopus (204) Google Scholar). All constructs were confirmed by DNA sequencing, linearized with NotI, and transfected in procyclic 29-13 trypanosomes (30Wirtz E. Leal S. Ochatt C. Cross G.A. Mol. Biochem. Parasitol. 1999; 99: 89-101Crossref PubMed Scopus (1098) Google Scholar). REH2-TAP expression and RNAi were induced with tetracycline at 1 μg/ml. Native editing complexes were purified by ion-exchange chromatography from mitochondrial extracts (3Rusché L.N. Cruz-Reyes J. Piller K.J. Sollner-Webb B. EMBO J. 1997; 16: 4069-4081Crossref PubMed Scopus (142) Google Scholar, 6Hernandez A. Panigrahi A. Cifuentes-Rojas C. Sacharidou A. Stuart K. Cruz-Reyes J. J. Mol. Biol. 2008; 381: 35-48Crossref PubMed Scopus (14) Google Scholar), and TAP purifications were performed essentially as reported (31Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Séraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2268) Google Scholar) with some modifications. Sedimentation fractions were obtained from freshly made mitochondria or whole-cell extracts in 10–30% glycerol gradients. Although our protocols to prepare the extracts include DNase I, a sample indicated in the text was subjected to an extra DNase treatment (DNA-free kit, Ambion) prior to sedimentation. Catalase and thyroglobulin were used as ∼10 S and ∼20 S markers, and Western blots of Tbmp45 (formerly termed REAP1) were used to determine the ∼40 S region (32Cruz-Reyes J. Sollner-Webb B. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 8901-8906Crossref PubMed Scopus (95) Google Scholar). Affinity-purified REH2 antibodies were produced against the peptide CSHTPTTSAEAGGDS (Bethyl Laboratories, Inc). Immunoprecipitations of endogenous and ectopic REH2 used antibody-conjugated protein A-Dynabeads (Invitrogen). Ectopic REH2 was specifically immunopurified using anti-rabbit IgG Dynabeads (Invitrogen). All washes were performed at 150 mm KCl. For mass spectrometry analyses, the antibodies were cross-linked to the beads with 25 mm dimethyl pimelimidate in 0.2 m triethanolamine, pH 8.2. The conditions and substrates to assay for full-round (33Cruz-Reyes J. Methods Enzymol. 2007; 424: 107-125Crossref PubMed Scopus (7) Google Scholar) and precleaved (1Carnes J. Stuart K.D. Methods Enzymol. 2007; 424: 25-54Crossref PubMed Scopus (15) Google Scholar) editing were as described. Photoreactive substrates containing a single thio-U and 32P at the editing site were prepared (33Cruz-Reyes J. Methods Enzymol. 2007; 424: 107-125Crossref PubMed Scopus (7) Google Scholar, 34Sacharidou A. Cifuentes-Rojas C. Halbig K. Hernandez A. Dangott L.J. De Nova-Ocampo M. Cruz-Reyes J. RNA. 2006; 12: 1219-1228Crossref PubMed Scopus (12) Google Scholar), and gRNA labeling was performed as reported (35Blum B. Simpson L. Cell. 1990; 62: 391-397Abstract Full Text PDF PubMed Scopus (191) Google Scholar). RNA helicase assays used a dsRNA substrate consisting of the pre-mRNA fragment A6-tag annealed to the cognate gRNA gA6[14] (9Seiwert S.D. Stuart K. Science. 1994; 266: 114-117Crossref PubMed Scopus (127) Google Scholar). The dephosphorylated mRNA was 5′-end-labeled with [γ-32P]ATP and annealed with a 10-fold excess of gRNA in RNA folding buffer (25 mm Tris-HCl, pH 8.0, 250 mm KCl, 10 mm Mg(OAc)2, 0.5 mm EDTA) by incubation at 95 °C for 10 min followed by a gradual return to room temperature over the course of 2 h. The annealed form was purified by native gel electrophoresis in an 8% polyacrylamide gel in 1× TBE supplemented with 10 mm Mg(OAc)2. The standard RNA helicase assay consisted of 50 cps (∼10 fmol) of dsRNA in 25 mm Tris-HCl, 22 mm KCl, 10 mm Mg(OAc)2, 0.5 mm EDTA, 3 mm dithiothreitol, 1 unit/μl RNase inhibitor, 1 mm ATP, 50 ng/μl bovine serum albumin, a 20-fold excess of an unlabeled trap single strand RNA that complements ∼33 bp of gRNA, and 10 μl of beads in a final volume of 20 μl. Reactions were incubated for 30–60 min at 26 °C with constant flicking to mix the beads. This was followed by addition of 4 μl of 6× stop solution as follows: 0.12% xylene cyanol, 0.12% bromphenol blue, 3% SDS, 125 ng/μl proteinase K, 17% glycerol, and incubation was at room temperature for 10 min. Samples were then loaded onto an 8% polyacrylamide gel, 1× TBE supplemented with 10 mm Mg(OAc)2. Protein RNA photocross-linking was performed as described (34Sacharidou A. Cifuentes-Rojas C. Halbig K. Hernandez A. Dangott L.J. De Nova-Ocampo M. Cruz-Reyes J. RNA. 2006; 12: 1219-1228Crossref PubMed Scopus (12) Google Scholar) except that it was scaled up 10-fold. Denaturation of complexes was accomplished by the addition of SDS to 1% final and sequential incubations at 95 °C for 10 min and at 70 °C for 30–60 min. After allowing the sample to reach room temperature, Triton-X-100 was added to 5% and incubated for 10 min at room temperature. Samples were then passed through a gel filtration spin column (Bio-spin 6, Bio-Rad 732 6221) according to the manufacturer's instructions and immunoprecipitated as above. Samples coupled to Dynabeads were treated with the following nucleases as indicated in the text at the given final concentration: RNase A (0.1 unit/μl), T1 (0.125 unit/μl), V1 (0.001/μl), and micrococcal nuclease (0.03 unit/μl) for 60 min in ice. Trichloroacetic acid precipitates were resuspended in digestion buffer (100 mm Tris-HCl, pH 8.5, 8 m urea) and digested by the sequential addition of Lys-C and trypsin proteases as described previously (36Florens L. Carozza M.J. Swanson S.K. Fournier M. Coleman M.K. Workman J.L. Washburn M.P. Methods. 2006; 40: 303-311Crossref PubMed Scopus (235) Google Scholar). Digested samples were fractionated using a five-step on-line separation method during which peptides were eluted directly into an LTQ-Orbitrap mass spectrometer (Thermo Fisher) in which tandem mass spectra were collected (37Washburn M.P. Wolters D. Yates 3rd, J.R. Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4029) Google Scholar, 38Wohlschlegel J.A. Methods Mol. Biol. 2009; 497: 33-49Crossref PubMed Scopus (55) Google Scholar, 39Wolters D.A. Washburn M.P. Yates 3rd, J.R. Anal. Chem. 2001; 73: 5683-5690Crossref PubMed Scopus (1544) Google Scholar). SEQUEST and DTASelect algorithms were used to identify peptide sequences from tandem mass spectra (40Eng J. McCormack A. Yates J. J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5315) Google Scholar, 41Tabb D.L. McDonald W.H. Yates 3rd, J.R. J. Proteome Res. 2002; 1: 21-26Crossref PubMed Scopus (1114) Google Scholar). Proteins were considered present in a sample if at least two peptides were identified per protein using a peptide level false-positive rate of 5% as determined using a decoy data base strategy (42Elias J.E. Gygi S.P. Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2727) Google Scholar). Detailed protocols and oligonucleotide sequences used during this work are available upon request. In a mass spectrometric analysis of native editing complexes purified from T. brucei mitochondria, we detected multiple unique peptides of most RECC subunits and the accessory MRP factors (6Hernandez A. Panigrahi A. Cifuentes-Rojas C. Sacharidou A. Stuart K. Cruz-Reyes J. J. Mol. Biol. 2008; 381: 35-48Crossref PubMed Scopus (14) Google Scholar). However, we also found a single peptide for a 241-kDa protein, termed REH2, with highly conserved DExH-helicase domains, a dsRBD, and an N-terminal mitochondrial import sequence (Fig. 1A). Western blot analyses clearly detected an ∼250-kDa protein in native but not in TAP-isolated editing complexes (Fig. 1B), although a weak signal was apparent in TAP-REL1 complexes (see the middle panel). This suggested a transient interaction of REH2 with RECC that is disrupted during high stringency affinity purifications. Silver staining of native editing complexes did not evidently detect components near 250 kDa suggesting that REH2 may be substoichiometric relative to RECC subunits (Fig. 1C). To confirm the physical interaction of REH2 with editing subunits, we expressed in procyclic trypanosomes the complete 6504-nucleotide REH2 gene with a C-terminal TAP tag (31Rigaut G. Shevchenko A. Rutz B. Wilm M. Mann M. Séraphin B. Nat. Biotechnol. 1999; 17: 1030-1032Crossref PubMed Scopus (2268) Google Scholar) under the control of a tetracycline-inducible promoter (Fig. 2A). REH2 mRNA and protein increased 5- and 2–3-fold, respectively, at day 3 of induction (Fig. 2, B and C), and a slight reduction in cell growth was seen at day 5 (Fig. 2D). Consistent with its predicted mitochondrial localization (supplemental Fig. S1), REH2 was enriched in a mitochondrial lysate (Fig. 2E) (43McManus M.T. Shimamura M. Grams J. Hajduk S.L. RNA. 2001; 7: 167-175Crossref PubMed Scopus (97) Google Scholar). REH2 antibodies reacted well with purified native editing complexes and mitochondrial lysates but weakly with TAP pulldowns due to a partial occlusion of the tag and consequent low purification efficiency from mitochondrial lysates. A small amount of RECC was detected in both TEV (Fig. 3A) and concentrated EGTA eluates (Fig. 3B) but not in mock pulldowns from empty vector control cells (last lane in each panel). Interestingly, this low level of RECC in the eluates was resistant to a pretreatment with RNases A and T1 and micrococcal nuclease (MN). In a converse approach, REH2 was detected in mitochondrial extract pulldowns of several RECC subunits (Fig. 3C). Also, REH2 antibodies immunoprecipitated a small fraction of preisolated native editing complexes after an extensive RNase-MN treatment including the dsRNA-specific RNase V1. However, most RECC remained in the unbound fraction (Fig. 3D), suggesting that relatively few purified complexes were stably bound to REH2.FIGURE 3REH2 co-purification with RECC is largely sensitive to extensive nuclease treatments. REH2 TAP purification and detection of editing subunits before or after (+/−) treatments with RNases A, T1 (and V1 as indicated), and micrococcal nuclease (MN). A, TEV (T) eluates from IgG-Sepharose. B, EGTA (ET) eluates from calmodulin-agarose. Control lanes include native editing complex (N), diluted whole-cell lysate (L) and empty-vector controls (TE or ETE; last lane). EGTA eluates were concentrated by acetone precipitation. Immunoblots of REH2 and editing subunits and auto-adenylylation of REL ligases are shown. ATPases in whole-cell lysates often inhibit the latter activity (e.g. A, lane 2). MP63 may co-migrate with antibody cross-reacting bovine serum albumin added as precipitation carrier (see the ETE control lane). REH2 fragmentation (*) is observed in the eluates. C, mitochondrial lysate immunoprecipitated (IP) pulldowns with antibodies to RECC subunits (MP81, MP63, REL1, and MP42) or a mock reaction without antibodies. D, REH2 pulldowns from purified native editing complexes. E, REH2 pulldowns from mitochondrial (M) lysate (Mito lysate). Mock reactions used pre-immune serum. An unaccounted band near MP52 is visible in the lysate lane.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Besides the above examination of affinity-purified eluates and isolated native editing complexes, we further analyzed the REH2/RECC association in mitochondrial lysates. Importantly, although RECC subunits were present in nuclease-treated REH2 pulldowns, most RECC was released by the treatment (Fig. 3E). This suggests that the transient association observed is largely mediated by RNA. Also, in line with transient contacts, REH2 purifications exhibited some precleaved but not full-round editing activity, which is less sensitive due to a limiting cleavage step (supplemental Fig. S2). Sedimentation analyses of mitochondrial lysates showed significant heterodispersion of REH2 with a broad peak at ∼20–30 S. Western blots of these fractions with REH2 antibodies and the peroxidase anti-peroxidase reagent (to score ectopic REH2 only) showed that the tag does not affect the sedimentation of REH2 (Fig. 4A, top and middle panels). The RECC subunit MP63 was also dispersed, but in contrast to REH2 it was not detected in light fractions (Fig. 4A, bottom panel). Interestingly, REH2 at ∼20 S and >40 S co-immunopurified with RECC subunits (see Fig. 4D). A pretreatment of the mitochondrial lysate with RNases/MN disrupted most high S value REH2 complexes generating a discrete peak at ∼15 S, whereas a significantly sharpened peak of editing complexes remained at ∼20 S (Fig. 4B). As described above, RNase treatment eliminated most REH2 association with RECC (Fig. 3E). To examine the relevance of the conserved dsRBD, we expressed a construct with a deletion of the entire motif (dsRBD-Δ) (see supplemental Fig. S3). Notably, this construct severely compromised cell growth (Fig. 2D) and reduced the sedimentation of both endogenous and ectopic REH2 to an extent comparable with the RNase treatment (Fig. 4C). Finally, we established that DNA does not largely contribute to the observed broad sedimentation of REH2 in a sample treated with DNase (“Experimental Procedures”; data not shown). Thus, REH2 forms heterodisperse particles that include editing complexes and are stabilized by RNA and the dsRBD, as well as relatively low density particles that resist RNase. Weng et al. (23Weng J. Aphasizheva I. Etheridge R.D. Huang L. Wang X. Falick A.M. Aphasizhev R. Mol. Cell. 2008; 32: 198-209Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) recently reported that GRBC complexes, which co-purified with REH2, bind gRNA. We determined whether REH2 immunopurified complexes associate with gRNA, and we further examined the importance of conserved domains of this protein. To this end, we analyzed IgG-Dynabead pulldowns of ectopically expressed REH2 wild type and mutant dsRBD-Δ or motif I (GK-to-AQ) (Fig. 1A). The motif I mutated residues have been associated with ATP binding and hydrolysis in other DExH proteins (44Jankowsky E. Nature. 2007; 449: 999-1000Crossref PubMed Scopus (4) Google Scholar). Although a significant amount of total gRNA co-purified with wild-type REH2, little if any was associated with either mutant (Fig. 5, A and B). It is of interest, however, that relatively large RNA species accumulated in both mutants. Furthermore, as shown by Hashimi et al. (22Hashimi H. Cicová Z. Novotná L. Wen Y.Z. Lukes J. RNA. 2009; 15: 588-599Crossref PubMed Scopus (70) Google Scholar), RNAi down-regulation of REH2 decreased the steady-state levels of gRNA (Fig. 5, C and D). Importantly, REH2 pulldowns from wild-type cells contained gRNA, demonstrating that endogenous REH2 RNPs bind gRNA and that this association is not an artifact of overexpression (Fig. 5, E and F). Thus, gRNA binding by REH2 RNPs requires the dsRBD and wild-type motif I of REH2. We examined the REH2 immunoprecipitated pulldown from mitochondrial extracts for possible RNA helicase activity and RNA photocross-linking. Notably, a model A6 pre-edited mRNA, preannealed with cognate gRNA, was efficiently unwound by the REH2 pulldown in a reaction requiring ATP hydrolysis at its β-γ phosphodiester linkage (Fig. 6A). Although the dsRNA substrate in these assays was gel-isolated, some dissociation is visible in input and mock lanes in the absence of REH2 complexes. A continuous duplex with 3′-overhangs was also unwound (Fig. 6B) but not an identical helix with 5′- or no overhangs (data not shown), consistent with the substrate specificity of the vast majority of SF2 RNA helicases with the exception of DEAD-box proteins (45Bleichert F. Baserga S.J. Mol. Cell. 2007; 27: 339-352Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Importantly, REH2 pulldowns from the dsRBD-Δ and motif I cell lines had no detectable unwinding activity (Fig. 6C). We analyzed the sedimentation distribution of this helicase activity relative to REH2 and the DEAD-box helicase REH1. Interestingly, most helicase activity sedimented in fractions containing REH2 but away from REH1, which localizes at the top of the gradient in fractions 1–3 (Fig. 6, D–F) (28Missel A. Souza A.E. Nörskau G. Göringer H.U. Mol. Cell. Biol. 1997; 17: 4895-4903Crossref PubMed Scopus (95) Google Scholar). An REH2 pulldown of fraction 8 exhibited unwinding activity (data not shown). Together with our above studies of REH2 mutants, these data suggest that REH2 is linked with the observed RNA helicase activity. To examine the possibility that REH2 may directly bind RNA, we cross-linked a protein fraction enriched in RECC (3Rusché L.N. Cruz-Reyes J. Piller K.J. Sollner-Webb B. EMBO J. 1997; 16: 4069-4081Crossref PubMed Scopus (142) Google Scholar) with the pre-mRNA/gRNA substrate described above but substituted with a photoreactive thio-U and 32P at the editing site (34Sacharidou A. Cifuentes-Rojas C. Halbig K. Hernandez A. Dangott L.J. De Nova-Ocampo M. Cruz-Reyes J. RNA. 2006; 12: 1219-1228Crossref PubMed Scopus (12) Google Scholar). This protein fraction produced a cross-link at ∼250 kDa (Fig. 7A, lane 1) that was enriched in a pulldown with REH2 but not REL1 antibodies (lanes 2 and 3). Importantly, REH2 pulldowns of cross-linked reactions that were treated with SDS and increasing temperature to dissociate the RNPs further enriched the ∼250-kDa cross-link (Fig. 7A, lanes 4 and 5), suggesting that the reacting protein is REH2. As a proof of concept for the above denaturation protocol (Fig. 7B), we isolated the RNA photocross-linked RECC subunits REL1 (lane 2) and reported MP63 (lane 3) (6Hernandez A. Panigrahi A. Cifuentes-Rojas C. Sacharidou A. Stuart K. Cruz-Reyes J. J. Mol. Biol. 2008; 381: 35-48Crossref PubMed Scopus (14) Google Scholar, 34Sacharidou A. Cifuentes-Rojas C. Halbig K. Hernandez A. Dangott L.J. De Nova-Ocampo M. Cruz-Reyes J. RNA. 2006; 12: 1219-1228Crossref PubMed Scopus (12) Google Scholar) using specific antibodies against these proteins. Additional studies are needed to confirm that the ∼250-kDa cross-link represents direct binding by REH2, but these data suggest that REH2 contacts the RNA duplex near the photoreactive moiety in the model editing site (≤4 Å) (46Fabre A. Morrison H. Photobiochemistry and Nucleic Acids. John Wiley & Sons, Inc., New York1990: 379-425Google Scholar).FIGURE 7A ∼250-kDa protein in REH2 pulldowns photocross-links with RNA. A, cross-linking of an A6 pre-mRNA/gRNA pair bearing a single photoreactive thio-U at the editing site. Mitochondrial Q-Sepharose fraction enriched with RECC (lane 1) or immunoprecipitation (IP) pulldowns of this material by antibodies to REL1 or REH2 (lanes 2 and 3, respectively). Lanes 4 and 5 are repeats of lane 3 but after a treatment with 0.1% SDS at 70 and 90 °C, respectively, that enriches a cross-link at ∼250 kDa (arrowhead). The REL1 pulldown shows at least four reported cross-linking RECC subunits (6Hernandez A. Panigrahi A. Cifuentes-Rojas C. Sacharidou A. Stuart K. Cruz-Reyes J. J. Mol. Biol. 2008; 381: 35-48Crossref PubMed Scopus (14) Google Scholar, 34Sacharidou A. Cifuentes-Rojas C. Halbig K. Hernandez A. Dangott L.J. De Nova-Ocampo M. Cruz-Reyes J. RNA. 2006; 12: 1219-1228Crossref PubMed Scopus (12) Google Scholar) (see B). B, proof of concept of the denaturation" @default.
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• W1970146598 date "2010-01-01" @default.
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• W1970146598 title "REH2 RNA Helicase in Kinetoplastid Mitochondria" @default.
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