Identification of a Chloroplast Ribonucleoprotein Complex Containing Trans-splicing Factors, Intron RNA, and Novel Components
2013; Elsevier BV; Volume: 12; Issue: 7 Linguagem: Inglês
10.1074/mcp.m112.026583
ISSN1535-9484
AutoresJessica Jacobs, Christina Marx, Vera Kock, Olga Reifschneider, Benjamin Fränzel, Christoph Krisp, Dirk Wolters, Ulrich Kück,
Tópico(s)ATP Synthase and ATPases Research
ResumoMaturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. A yeast-two hybrid screen and co-immunoprecipitation identified chloroplast-localized Raa4-binding protein 1 (Rab1), which specifically binds tscA RNA from the tripartite psaA group II intron. The yeast-two hybrid system provides evidence in support of direct interactions between Rab1 and four trans-splicing factors. Our findings contribute to our knowledge of chloroplast multisubunit ribonucleoprotein complexes and are discussed in support of the generally accepted view that group II introns are the ancestors of the eukaryotic spliceosomal introns. Maturation of chloroplast psaA pre-mRNA from the green alga Chlamydomonas reinhardtii requires the trans-splicing of two split group II introns. Several nuclear-encoded trans-splicing factors are required for the correct processing of psaA mRNA. Among these is the recently identified Raa4 protein, which is involved in splicing of the tripartite intron 1 of the psaA precursor mRNA. Part of this tripartite group II intron is the chloroplast encoded tscA RNA, which is specifically bound by Raa4. Using Raa4 as bait in a combined tandem affinity purification and mass spectrometry approach, we identified core components of a multisubunit ribonucleoprotein complex, including three previously identified trans-splicing factors (Raa1, Raa3, and Rat2). We further detected tscA RNA in the purified protein complex, which seems to be specific for splicing of the tripartite group II intron. A yeast-two hybrid screen and co-immunoprecipitation identified chloroplast-localized Raa4-binding protein 1 (Rab1), which specifically binds tscA RNA from the tripartite psaA group II intron. The yeast-two hybrid system provides evidence in support of direct interactions between Rab1 and four trans-splicing factors. Our findings contribute to our knowledge of chloroplast multisubunit ribonucleoprotein complexes and are discussed in support of the generally accepted view that group II introns are the ancestors of the eukaryotic spliceosomal introns. Intron-containing genes from prokaryotic or organellar genomes carry either group I or group II introns, each of which has distinct features. The splicing mechanism of group II introns and the secondary structures of their presumed active sites were used as early arguments for the hypothesis that this class of introns represents the ancestors of eukaryotic spliceosomal introns (1Sharp P.A. "Five easy pieces".Science. 1991; 254: 663Crossref PubMed Scopus (170) Google Scholar, 2Lambowitz A.M. Zimmerly S. Group II introns: mobile ribozymes that invade DNA.Cold Spring Harb. Perspect. Biol. 2011; 3: a003616Crossref PubMed Scopus (309) Google Scholar). It was further assumed that group II introns invaded the eukaryotic nucleus and subsequently proliferated at various genomic sites, leading to the degeneration of the catalytic intron structure into small nuclear RNAs (snRNAs). 1The abbreviations used are:CRMchloroplast RNA-splicing and ribosome maturationEMSAelectrophoretic mobility shift assayMudPITmultidimensional protein identification technologyNi-NTAnickel-nitrilotriacetic acidqRT-PCRquantitative real-time PCRSDsynthetic dropoutsnRNAsmall nuclear RNATAPtandem affinity purification. 1The abbreviations used are:CRMchloroplast RNA-splicing and ribosome maturationEMSAelectrophoretic mobility shift assayMudPITmultidimensional protein identification technologyNi-NTAnickel-nitrilotriacetic acidqRT-PCRquantitative real-time PCRSDsynthetic dropoutsnRNAsmall nuclear RNATAPtandem affinity purification. This assumption was supported by the observation of naturally occurring variants of group II introns that are split into two or more pieces (3Glanz S. Kück U. Trans-splicing of organelle introns—a detour to continuous RNAs.BioEssays. 2009; 31: 921-934Crossref PubMed Scopus (57) Google Scholar), reminiscent of eukaryotic spliceosomal RNA (1Sharp P.A. "Five easy pieces".Science. 1991; 254: 663Crossref PubMed Scopus (170) Google Scholar). Group II intron RNAs are characterized by six conserved domains, and tertiary interactions among these domains generate the compact native and catalytic complex. Some of these group II intron domains have been shown to act in trans on the splicing of other introns that lack the corresponding domain (4Jarrell K.A. Peebles C.L. Dietrich R.C. Romiti S.L. Perlman P.S. Group II intron self-splicing. Alternative reaction conditions yield novel products.J. Biol. Chem. 1988; 263: 3432-3439Abstract Full Text PDF PubMed Google Scholar). In vivo, various RNA-binding proteins promote the formation of catalytically active intron RNA. In contrast to the nuclear spliceosome, which acts generally on a broad range of nuclear-encoded pre-mRNAs, proteins involved in organellar intron splicing seem to more efficiently stabilize the active three-dimensional RNA structure in vivo. chloroplast RNA-splicing and ribosome maturation electrophoretic mobility shift assay multidimensional protein identification technology nickel-nitrilotriacetic acid quantitative real-time PCR synthetic dropout small nuclear RNA tandem affinity purification. chloroplast RNA-splicing and ribosome maturation electrophoretic mobility shift assay multidimensional protein identification technology nickel-nitrilotriacetic acid quantitative real-time PCR synthetic dropout small nuclear RNA tandem affinity purification. Several splicing factors in higher plants, such as the chloroplast RNA-splicing and ribosome maturation (CRM) domain protein CRS1, as well as the pentatricopeptide repeat proteins OTP51 and PPR4, have been reported to be involved in the splicing of single transcripts (5Stern D.B. Goldschmidt-Clermont M. Hanson M.R. Chloroplast RNA metabolism.Annu. Rev. Plant Biol. 2010; 61: 125-155Crossref PubMed Scopus (338) Google Scholar, 6Barkan A. Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold.Plant Physiol. 2011; 155: 1520-1532Crossref PubMed Scopus (223) Google Scholar). Nonetheless, there are splicing factors that carry out functions on a broad range of transcripts, including CRS2 and its associated proteins CAF1 and CAF2, and WTF1, a splicing factor containing a plant organelle RNA-recognition domain (5Stern D.B. Goldschmidt-Clermont M. Hanson M.R. Chloroplast RNA metabolism.Annu. Rev. Plant Biol. 2010; 61: 125-155Crossref PubMed Scopus (338) Google Scholar, 6Barkan A. Expression of plastid genes: organelle-specific elaborations on a prokaryotic scaffold.Plant Physiol. 2011; 155: 1520-1532Crossref PubMed Scopus (223) Google Scholar). Sedimentation and co-fractionation experiments in, for example, maize have demonstrated that these proteins are part of large multiprotein and ribonucleoprotein complexes with their cognate RNAs (5Stern D.B. Goldschmidt-Clermont M. Hanson M.R. Chloroplast RNA metabolism.Annu. Rev. Plant Biol. 2010; 61: 125-155Crossref PubMed Scopus (338) Google Scholar, 7Kroeger T.S. Watkins K.P. Friso G. van Wijk K.J. Barkan A. A plant-specific RNA-binding domain revealed through analysis of chloroplast group II intron splicing.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 4537-4542Crossref PubMed Scopus (98) Google Scholar). In addition, these complexes resemble the nuclear spliceosome in which the snRNAs associate with more than 200 proteins (8Sperling J. Azubel M. Sperling R. Structure and function of the pre-mRNA splicing machine.Structure. 2008; 16: 1605-1615Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). The unicellular green alga Chlamydomonas reinhardtii is also known to contain high molecular weight complexes containing splicing factors (9Rivier C. Goldschmidt-Clermont M. Rochaix J.D. Identification of an RNA-protein complex involved in chloroplast group II intron trans-splicing in.Chlamydomonas reinhardtii. EMBO J. 2001; 20: 1765-1773Crossref Scopus (72) Google Scholar, 10Perron K. Goldschmidt-Clermont M. Rochaix J.D. A multiprotein complex involved in chloroplast group II intron splicing.RNA. 2004; 10: 704-711Crossref PubMed Scopus (36) Google Scholar). In this alga, the chloroplast-encoded psaA gene, which encodes a major subunit of photosystem I, is split into three independently transcribed exons. Splicing of the psaA pre-mRNAs requires the assembly of two group IIB introns (11Kück U. Choquet Y. Schneider M. Dron M. Bennoun P. Structural and transcriptional analysis of two homologous genes for the P700 chlorophyll α-apoproteins in Chlamydomonas reinhardtii: evidence for in vivo trans-splicing.EMBO J. 1987; 6: 2185-2195Crossref PubMed Google Scholar). For psaA intron 1, the catalytically active intron structure is fragmented into three chloroplast-encoded intron sequences, including the core tscA RNA (12Goldschmidt-Clermont M. Choquet Y. Girard-Bascou J. Michel F. Schirmer-Rahire M. Rochaix J.D. A small chloroplast RNA may be required for trans-splicing in.Chlamydomonas reinhardtii. Cell. 1991; 65: 135-143Scopus (136) Google Scholar). The tscA RNA is required in order to form the active intron structure, because it complements the tripartite intron by contributing domains D2 and D3, as well as parts of D1 and D4 (Fig. 1A). Several photosynthetic mutants have been identified that are deficient in the splicing of either intron 1 or intron 2, or both, or in the processing of tscA RNA. At least 14 nuclear loci are involved in trans-splicing, with six splicing factors identified to date (13Goldschmidt-Clermont M. Girard-Bascou J. Choquet Y. Rochaix J.D. Trans-splicing mutants of.Chlamydomonas reinhardtii. Mol. Gen. Genet. 1990; 223: 417-425Crossref PubMed Scopus (111) Google Scholar). Two of them, Raa3 and Raa4, are directly involved in the correct splicing of intron 1, and Raa1, Rat1, and Rat2 are essential for the processing of tscA RNA from a polycistronic precursor, a prerequisite for intron 1 splicing. Besides its function in processing tscA RNA, Raa1 plays a role in splicing the second psaA intron. A further protein involved in splicing the second psaA intron is Raa2. Except for Rat1, which is significantly homologous to the NAD+-binding domain of poly (ADP-ribose) polymerases, and Raa2, which shows similarities to pseudouridine synthases, all other psaA-splicing factors display only slight sequence homologies to other known proteins (14Glanz S. Jacobs J. Kock V. Mishra A. Kück U. Raa4 is a trans-splicing factor that specifically binds chloroplast tscA intron RNA.Plant J. 2012; 69: 421-431Crossref PubMed Scopus (14) Google Scholar, 15Jacobs J. Glanz S. Bunse-Grassmann A. Kruse O. Kück U. RNA trans-splicing: identification of components of a putative chloroplast spliceosome.Eur. J. Cell. Biol. 2010; 89: 932-939Crossref PubMed Scopus (16) Google Scholar). So far little is known about the protein–protein interactions and the overall composition of the organellar ribonucleoprotein complexes involved in psaA trans-splicing. In this study, we identified basic subunits of a multipartite complex that contains four functional trans-splicing factors. To identify the components of this complex, we used a multifaceted approach combining tandem affinity purification (TAP), mass spectrometry, and yeast two-hybrid screening. We applied different environmental conditions (light, dark, anaerobiosis) to define true and essential subunits of the basic splicing complex that are present under various conditions. Further, we detected a novel intron RNA binding protein that interacts with at least four splicing factors. The protein–RNA complex described here points toward a chloroplast multisubunit splicing complex specific for a tripartite group II intron that is reminiscent of the nuclear spliceosome. C. reinhardtii strains and growth conditions are listed in supplemental Table S1. For TAP, C. reinhardtii cultures were grown in tris-acetate phosphate medium in the light. For the induction of anaerobic conditions, a concentrated and shaded C. reinhardtii culture was flushed with argon as described elsewhere (16Hemschemeier A. Melis A. Happe T. Analytical approaches to photobiological hydrogen production in unicellular green algae.Photosynth. Res. 2009; 102: 523-540Crossref PubMed Scopus (153) Google Scholar). Hydrogenase activity was measured as described elsewhere (16Hemschemeier A. Melis A. Happe T. Analytical approaches to photobiological hydrogen production in unicellular green algae.Photosynth. Res. 2009; 102: 523-540Crossref PubMed Scopus (153) Google Scholar). For dark adaptation, cells were dark incubated for 2 h. The nuclear transformation of algal cells was carried out according to the glass-bead method (17Kindle K.L. High-frequency nuclear transformation of.Chlamydomonas reinhardtii. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 1228-1232Crossref PubMed Scopus (808) Google Scholar) with 5 μg circular or hydrolyzed DNA. Procedures for standard molecular techniques were performed as reported elsewhere (14Glanz S. Jacobs J. Kock V. Mishra A. Kück U. Raa4 is a trans-splicing factor that specifically binds chloroplast tscA intron RNA.Plant J. 2012; 69: 421-431Crossref PubMed Scopus (14) Google Scholar, 18Glanz S. Bunse A. Wimbert A. Balczun C. Kück U. A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in.Chlamydomonas reinhardtii. Nucleic Acids Res. 2006; 34: 5337-5351Crossref PubMed Scopus (16) Google Scholar). Escherichia coli strain XL1-blue MRF′ served as the host for general plasmid construction and maintenance (19Jerpseth B. Greener A. Short J.M. Viola J. Kretz P.L. XL1-Blue MRF′ E. coli cells: McrA-, McrCB-, McrF-, Mrr-, HsdR- derivative of XL1-Blue cells.Strateg. Mol. Biol. 1992; 5: 81-83Google Scholar). S. cerevisiae strain PJ69–4A (20James P. Halladay J. Craig E.A. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar) was used for homologous recombination as described by Colot et al. (21Colot H.V. Park G. Turner G.E. Ringelberg C. Crew C.M. Litvinkova L. Weiss R.L. Borkovich K.A. Dunlap J.C. A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 10352-10357Crossref PubMed Scopus (860) Google Scholar). The transformation of yeast cells was done by means of electroporation according to the method of Becker and Lundblad (22Becker D.M. Lundblad V. Introduction of DNA into Yeast Cells.Current Protocols in Molecular Biology. 2001; 27: 13.7.1-13.7.10Google Scholar) in a Multiporator (Eppendorf, Hamburg, Germany) at 1.5 kV. Transformants were selected for tryptophan or leucine prototrophy. DNA extraction was performed using the E.N.Z.A. Plasmid Miniprep Kit I (Peqlab Biotechnologie, Erlangen, Germany) after treatment with glass beads. C. reinhardtii total RNA was prepared as described elsewhere (11Kück U. Choquet Y. Schneider M. Dron M. Bennoun P. Structural and transcriptional analysis of two homologous genes for the P700 chlorophyll α-apoproteins in Chlamydomonas reinhardtii: evidence for in vivo trans-splicing.EMBO J. 1987; 6: 2185-2195Crossref PubMed Google Scholar). PCR and RT-PCR experiments were performed as described elsewhere (18Glanz S. Bunse A. Wimbert A. Balczun C. Kück U. A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in.Chlamydomonas reinhardtii. Nucleic Acids Res. 2006; 34: 5337-5351Crossref PubMed Scopus (16) Google Scholar). One-step RT-PCR was performed with the OneStep RT-PCR Kit from Qiagen (Hilden, Germany) according to the manufacturer's instructions. Recombinant plasmids and oligonucleotides used for PCR experiments, protein synthesis, or generation of transgenic algal strains are listed in supplemental Table S2 and supplemental Table S3, respectively. If necessary, suitable restriction sites for cloning were added to oligonucleotides. TAP eluates containing nucleic acids were purified via phenol/chloroform/isoamyl alcohol (25:24:1) extraction and precipitation at −20 °C. Genomic DNA was removed by means of DNase I treatment for 25 min at 25 °C. 1 μl of each 44-μl sample was subjected to One-Step qRT-PCR (KAPA Sybr Fast ABI Prism, Peqlab, Erlangen, Germany) using gene-specific oligonucleotides (supplemental Table S3). As a control for successful DNaseI treatment, each reverse transcription was carried out twice, once with and once without reverse transcriptase. qRT-PCR was performed in an ABI 5700 (Applied Biosystems, Foster City, CA) with a One-Step qRT-PCR Kit containing SybrGreen and ROX (KAPA Sybr Fast ABI Prism, Peqlab) in a volume of 20 μl. Each reaction was carried out in triplicate with an oligonucleotide primer at a concentration of 10 μm. Primers were selected to have melting temperatures of 56 °C to 61 °C and to yield amplicons of 147 to 185 bp. PCR conditions were as follows: 42 °C for 5 min, 95 °C for 1 min, and 40 cycles of 95 °C for 5 s and 60 °C for 20 s, followed by a melting curve analysis. Amplicon size was verified using gel electrophoresis. Primer pair efficiencies and expression ratios were calculated as described elsewhere (23Nowrousian M. Ringelberg C. Dunlap J.C. Loros J.J. Kúck U. Cross-species microarray hybridization to identify developmentally regulated genes in the filamentous fungus.Sordaria macrospora. Mol. Genet. Genomics. 2005; 273: 137-149Crossref PubMed Scopus (90) Google Scholar). Each qRT-PCR experiment was done with two biologically independent samples. To construct the Raa4 two-hybrid plasmids, cDNA fragments coding for amino acids 48–610 and 609–1143 were amplified (primers: for_Y2H1, rev_Y2H2; for_Y2H3, rev_Y2H4) and ligated in pDrive or pBIIKS+. After restriction with EcoRI and BamHI, the resulting fragment was cloned into EcoRI and BamHI sites of pGADT7 resulting in plasmids pGADT7_Raa4-A and pGADT7_Raa4-B. The full-length version of Raa4 (pGADT7_Raa4-FL) was obtained after digestion of pBIIKS+_ Raa4-B with SrfI and BamHI and ligation of the resulting fragment in SrfI and BamHI restricted pGADT7_Raa4-A. Rab1 yeast-two hybrid vectors were generated as follows: DNA fragments coding for amino acids 51–725 and 668–1216 were amplified from cDNA (primers: OVK48, OVK49; OVK50, OVK51) and cloned into EcoRI and BamHI restriction sites of pGADT7 resulting in pGADT7_Rab1-A and pGADT7_Rab1-B. For the generation of two-hybrid vectors carrying Raa3 subfragments, RAA3 fragments coding for amino acids 674–1298, 70–675, 1296–1783, and 674–1783 were amplified from cDNA (primers: for_Raa3-1, rev_Raa3-2; for_Raa3–3, rev_Raa3–3; for_Raa3-1, rev_Raa3-1; for_Raa3-2, rev_Raa3-2) and inserted into EcoRI and SalI restriction sites of vector pGBKT7 resulting in plasmids pGBKT7_Raa3-A, pGBKT7_Raa3-B, pGBKT7_Raa3-C, and pGBKT7_Raa3-D. For the Raa3 full-length construct (pGBKT7_Raa3-FL), cDNA was amplified (primers: for_Raa3-3, rev_Raa3-2) and cloned in EcoRI and BamHI sites of pGBKT7. In order to construct RAT2 two-hybrid plasmids, cDNA was amplified in three fragments (primers: for_pGADT7_Rat2, rev_Rat2_F1; for_Rat2_F2, rev_Rat2_F2; for_Rat2_F3, rev_pGADT7_Rat2) that overlapped each other and the pGADT7 cloning site. Full-length cDNA of RAT2 (pGADT7_Rat2-FL) was obtained via homologous recombination in S. cerevisiae PJ69–4A. Two fragments, RAT2-A and RAT2-B, coding for amino acids 1–682 and 683–1376 were amplified from pGADT7_Rat2-FL (primers: for_pGADT7_Rat2, rev_Rat2-Y2H-F1; for_Rat2-Y2H-F2, rev_pGADT7_Rat2) and introduced in pGADT7 via homologous recombination. All RAT2 fragments (Rat2-FL, Rat2-A, and Rat2-B) were cloned in pGBKT7 via NdeI and the compatible restriction sites XhoI and SalI resulting in pGBKT7_Rat2-FL, pGBKT7_Rat2-A, and pGBKT7_Rat2-B. For the generation of yeast two-hybrid vectors containing the RAA1-A fragment, cDNA of RAA1 was amplified in two fragments (primers: for_pGADT7_Raa1, rev_pGADT7_Raa1; for_Raa1-F3–3, rev_Raa1-F3–3). The two fragments showed regions overlapping each other and the pGADT7 cloning site and were introduced to pGADT7 by means of homologous recombination. RAA1-A was inserted into the NdeI and BamHI restriction site of pGBKT7. For the construction of His6::Raa4M, an 884-bp fragment of RAA4 cDNA was amplified via PCR (primers: for_Raa4-M2 and rev_Raa4-M). After ligation into pTOPO and hydrolysis of the resulting plasmid with BamHI and HindIII, the 870-bp fragment was ligated into pQE30 cut with BamHI and HindIII, resulting in plasmid pQE30_Raa4-M2. For cloning of the Rab1::One-STrEP-tag fusion construct, RAB1 cDNA was amplified (primers: OVK_29, OVK_30) and cloned in pASG-IBA3 via StarGate® combinatorial cloning according to the manufacturer's instructions (IBA GmbH, Göttingen, Germany). For the generation of Rab1cTP::cGFP, a genomic fragment was amplified using primers Rab1_cTP_for and Rab1_cTPlong_rev and cloned in pDrive. The resulting plasmid was restricted with NheI and cloned in NheI cut pCr1g resulting in pCr1g_Rab1cTP. The cTAP gene was amplified from pUC57 using oligonucleotides Taptag1 and Taptag1 and cloned into plasmid pCrg1 (18Glanz S. Bunse A. Wimbert A. Balczun C. Kück U. A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in.Chlamydomonas reinhardtii. Nucleic Acids Res. 2006; 34: 5337-5351Crossref PubMed Scopus (16) Google Scholar) via BglII restriction sites resulting in plasmid pCM10. For the generation of an Raa4::TAP tag fusion construct, RAA4 was amplified in two fragments (primers Raa4-A1, Raa4-A2 and Raa4-B1, Raa4-B2) from BAC subclone 2539_1A (14Glanz S. Jacobs J. Kock V. Mishra A. Kück U. Raa4 is a trans-splicing factor that specifically binds chloroplast tscA intron RNA.Plant J. 2012; 69: 421-431Crossref PubMed Scopus (14) Google Scholar). Fragment RAA4-B was cloned via XbaI restriction sites in pCM10 resulting in plasmid pCM12. Fragment RAA4-A was cut with PmeI and cloned in PmeII restricted pCM12 resulting in plasmid pCM13. For deletion of the median PmeI restricition site, pCM13 was restricted with MauBI and SrfI. The resulting 1.2-kb fragment was replaced with the corresponding fragment from plasmid 2539_1A resulting in plasmid pCM15, which comprises the genomic sequence of RAA4 fused to the TAP tag gene under control of the artificial RBCS2/HSP70 tandem promoter. For the construction of an RbcS1::TAP tag fusion construct, RBCS1 was amplified from genomic DNA (primers: RbcS1_NheI_1, RbcS1_NheI_2) and cloned in pDrive. RBCS1 was then introduced into plasmid pCM10 using restriction site NheI resulting in plasmid pCM18. The fluorescence emissions of transformed C. reinhardtii cells were analyzed via laser scanning confocal fluorescence microscopy using a Zeiss LSM 510 META microscopy system (Carl Zeiss, Jena, Germany) based on an Axiovert inverted microscope. cGFP and plastids were excited with the 488-nm line of an argon-ion laser. The fluorescence emission was selected by band pass filter BP505–530 and long pass filter LP560, respectively, using beam splitters HFT UV/488/543/633 and NFT545 as described elsewhere (18Glanz S. Bunse A. Wimbert A. Balczun C. Kück U. A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in.Chlamydomonas reinhardtii. Nucleic Acids Res. 2006; 34: 5337-5351Crossref PubMed Scopus (16) Google Scholar). For the heterologous synthesis of RAA4 and RAB1, E. coli BL21(DE3) was transformed with the respective plasmids (pQE30_Raa4-M, pASG-IBA3_Rab1). Protein production was performed in 0.5 l LB medium containing 100 μg ml−1 ampicilline. Fusion proteins were isolated from inclusion bodies according to the procedure described by Steinle et al. (24Steinle A. Li P. Morris D.L. Groh V. Lanier L.L. Strong R.K. Spies T. Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family.Immunogenetics. 2001; 53: 279-287Crossref PubMed Scopus (391) Google Scholar) as described in Ref. 14Glanz S. Jacobs J. Kock V. Mishra A. Kück U. Raa4 is a trans-splicing factor that specifically binds chloroplast tscA intron RNA.Plant J. 2012; 69: 421-431Crossref PubMed Scopus (14) Google Scholar. The purification of refolded recombinant proteins was performed according to the manufacturer's instructions (GE Healthcare, Freiburg, Germany; Qiagen, Hilden, Germany). For RNA mobility shift assays, uniformly 32P-UTP-labeled run-off transcripts served as substrate RNAs and were generated by the in vitro transcription of plasmids as given in supplemental Table S2. In vitro transcription and EMSAs were performed as previously reported (14Glanz S. Jacobs J. Kock V. Mishra A. Kück U. 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Unlabeled competitor RNAs and nonspecific competitor RNA derived from plasmid pBSIIKS+ (Stratagene, La Jolla, CA) were synthesized as described elsewhere (18Glanz S. Bunse A. Wimbert A. Balczun C. Kück U. A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in.Chlamydomonas reinhardtii. Nucleic Acids Res. 2006; 34: 5337-5351Crossref PubMed Scopus (16) Google Scholar, 26Bunse A.A. Nickelsen J. Kück U. Intron-specific RNA binding proteins in the chloroplast of the green alga.Chlamydomonas reinhardtii. Biochim. Biophys. Acta. 2001; 1519: 46-54Crossref PubMed Scopus (15) Google Scholar). Recombinant His-tagged cNAPL protein or GST-tagged Raa4 were used as controls and were purified as described elsewhere (14Glanz S. Jacobs J. Kock V. Mishra A. Kück U. Raa4 is a trans-splicing factor that specifically binds chloroplast tscA intron RNA.Plant J. 2012; 69: 421-431Crossref PubMed Scopus (14) Google Scholar, 18Glanz S. Bunse A. Wimbert A. Balczun C. Kück U. 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Pazour G. Purton S. Ral J.P. Riano-Pachon D.M. Riekhof W. Rymarquis L. Schroda M. Stern D. Umen J. Willows R. Wilson N. Zimmer S.L. Allmer J. Balk J. Bisova K. Chen C.J. Elias M. Gendler K. Hauser C. Lamb M.R. Ledford H. Long J.C. Minagawa J. Page M.D. Pan J. Pootakham W. Roje S. Rose A. Stahlberg E. Terauchi A.M. Yang P. Ball S. Bowler C. Dieckmann C.L. Gladyshev V.N. Green P. Jorgensen R. Mayfield S. Mueller-Roeber B. Rajamani S. Sayre R.T. Brokstein P. Dubchak I. Goodstein D. Hornick L. Huang Y.W. Jhaveri J. Luo Y. Martinez D. Ngau W.C. Otillar B. Poliakov A. Porter A. Szajkowski L. Werner G. Zhou K. Grigoriev I.V. Rokhsar D.S. Grossman A.R. The Chlamydomonas genome reveals the evolution of key animal and plant functions.Science. 2007; 318: 245-250Crossref PubMed Scopus (1943) Google Scholar). Basic Local Alignment Search Tool (BLAST) searches were performed using NCBI's BLAST Server. Isoelectric point and sequence masses were calculated by the program Clone Manager 9 Professional Edition (Scientific & Educational Software, Cary, NC). Secondary structure analysis was performed using version IV of the GOR secondary structure prediction method (28Garnier J. Gibrat J.F. Robson B. GOR method for predicting protein secondary structure from amino acid sequence.Methods Enzymol. 1996; 266: 540-553Crossref PubMed Google Scholar). Protein motifs were predicted with Motif Scan (29Pagni M. Ioannidis V. Cerutti L. Zahn-Zabal M. Jongeneel C.V. Hau J. Martin O. Kuznetsov D. Falquet L. MyHits: improvements to an interactive resource for analyzing protein sequences.Nucleic Acids Res. 2007; 35: 433-437Crossref PubMed Scopus (159) Google Scholar). For the identification of RNA binding
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