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CRS1 is a novel group II intron splicing factor that was derived from a domain of ancient origin 总被引:7,自引:0,他引:7
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Protein-dependent group II intron splicing provides a forum for exploring the roles of proteins in facilitating RNA-catalyzed reactions. The maize nuclear gene crs1 is required for the splicing of the group II intron in the chloroplast atpF gene. Here we report the molecular cloning of the crs1 gene and an initial biochemical characterization of its gene product. Several observations support the notion that CRS1 is a bona fide group II intron splicing factor. First, CRS1 is found in a ribonucleoprotein complex in the chloroplast, and cofractionation data provide evidence that this complex includes atpF intron RNA. Second, CRS1 is highly basic and includes a repeated domain with features suggestive of a novel RNA-binding domain. This domain is related to a conserved free-standing open reading frame of unknown function found in both the eubacteria and archaea. crs1 is the founding member of a gene family in plants that was derived by duplication and divergence of this primitive gene. In addition to its previously established role in atpF intron splicing, new genetic data implicate crs1 in chloroplast translation. The chloroplast splicing and translation functions of crs1 may be mediated by the distinct protein products of two crs1 mRNA forms that result from alternative splicing of the crs1 pre-mRNA. 相似文献
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CRS1, a chloroplast group II intron splicing factor, promotes intron folding through specific interactions with two intron domains 总被引:4,自引:0,他引:4
Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1's specificity for the atpF intron in vivo can be explained by CRS1's intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1's intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments. 相似文献
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The chloroplastic DEVH‐box RNA helicase INCREASED SIZE EXCLUSION LIMIT 2 involved in plasmodesmata regulation is required for group II intron splicing
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Nicolas Carlotto Sonia Wirth Nicolas Furman Nazarena Ferreyra Solari Federico Ariel Martin Crespi Ken Kobayashi 《Plant, cell & environment》2016,39(1):165-173
INCREASED SIZE EXCLUSION LIMIT 2 (ISE2) encodes a putative DEVH‐box RNA helicase originally identified through a genetic screening for Arabidopsis mutants altered in plasmodesmata (PD) aperture. Depletion of ISE2 also affects chloroplasts activity, decreases accumulation of photosynthetic pigments and alters expression of photosynthetic genes. In this work, we show the chloroplast localization of ISE2 and decipher its role in plastidic RNA processing and, consequently, PD function. Group II intron‐containing RNAs from chloroplasts exhibit defective splicing in ise2 mutants and ISE2‐silenced plants, compromising plastid viability. Furthermore, RNA immunoprecipitation suggests that ISE2 binds in vivo to several splicing‐regulated RNAs. Finally, we show that the chloroplast clpr2 mutant (defective in a subunit of a plastidic Clp protease) also exhibits abnormal PD function during embryogenesis, supporting the idea that chloroplast RNA processing is required to regulate cell–cell communication in plants. 相似文献
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Keren I Klipcan L Bezawork-Geleta A Kolton M Shaya F Ostersetzer-Biran O 《The Journal of biological chemistry》2008,283(34):23333-23342
CRM (chloroplast RNA splicing and ribosome maturation) is a recently recognized RNA-binding domain of ancient origin that has been retained in eukaryotic genomes only within the plant lineage. Whereas in bacteria CRM domains exist as single domain proteins involved in ribosome maturation, in plants they are found in a family of proteins that contain between one and four repeats. Several members of this family with multiple CRM domains have been shown to be required for the splicing of specific plastidic group II introns. Detailed biochemical analysis of one of these factors in maize, CRS1, demonstrated its high affinity and specific binding to the single group II intron whose splicing it facilitates, the plastid-encoded atpF intron RNA. Through its association with two intronic regions, CRS1 guides the folding of atpF intron RNA into its predicted "catalytically active" form. To understand how multiple CRM domains cooperate to achieve high affinity sequence-specific binding to RNA, we analyzed the RNA binding affinity and specificity associated with each individual CRM domain in CRS1; whereas CRM3 bound tightly to the RNA, CRM1 associated specifically with a unique region found within atpF intron domain I. CRM2, which demonstrated only low binding affinity, also seems to form specific interactions with regions localized to domains I, III, and IV. We further show that CRM domains share structural similarities and RNA binding characteristics with the well known RNA recognition motif domain. 相似文献
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The chloroplast RNA helicase ISE2 is required for multiple chloroplast RNA processing steps in Arabidopsis thaliana 总被引:1,自引:0,他引:1
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Krzysztof Bobik Tyra N. McCray Ben Ernest Jessica C. Fernandez Katharine A. Howell Thomas Lane Margaret Staton Tessa M. Burch‐Smith 《The Plant journal : for cell and molecular biology》2017,91(1):114-131
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An evolutionarily conserved P‐subfamily pentatricopeptide repeat protein is required to splice the plastid ndhA transcript in the moss Physcomitrella patens and Arabidopsis thaliana
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Ayaka Ito Chieko Sugita Mizuho Ichinose Yoshinobu Kato Hiroshi Yamamoto Toshiharu Shikanai Mamoru Sugita 《The Plant journal : for cell and molecular biology》2018,94(4):638-648
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Li Zhang Jingli Chen Liqun Zhang Ying Wei Yajuan Li Xinyun Xu Hui Wu Zhong‐Nan Yang Jirong Huang Fenhong Hu Weihua Huang Yong‐Lan Cui 《植物学报(英文版)》2021,63(11):1952-1966
Chloroplast biogenesis requires the coordinated expression of chloroplast and nuclear genes. Here, we show that EMB1270, a plastid-localized pentatricopeptide repeat (PPR) protein, is required for chloroplast biogenesis in Arabidopsis thaliana. Knockout of EMB1270 led to embryo arrest, whereas a mild knockdown mutant of EMB1270 displayed a virescent phenotype. Almost no photosynthetic proteins accumulated in the albino emb1270 knockout mutant. By contrast, in the emb1270 knockdown mutant, the levels of ClpP1 and photosystem I (PSI) subunits were significantly reduced, whereas the levels of photosystem II (PSII) subunits were normal. Furthermore, the splicing efficiencies of the clpP1.2, ycf3.1, ndhA, and ndhB plastid introns were dramatically reduced in both emb1270 mutants. RNA immunoprecipitation revealed that EMB1270 associated with these introns in vivo. In an RNA electrophoretic mobility shift assay (REMSA), a truncated EMB1270 protein containing the 11 N-terminal PPR motifs bound to the predicted sequences of the clpP1.2, ycf3.1, and ndhA introns. In addition, EMB1270 specifically interacted with CRM Family Member 2 (CFM2). Given that CFM2 is known to be required for splicing the same plastid RNAs, our results suggest that EMB1270 associates with CFM2 to facilitate the splicing of specific group II introns in Arabidopsis. 相似文献
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A ribonuclease III domain protein functions in group II intron splicing in maize chloroplasts 总被引:3,自引:1,他引:2
Watkins KP Kroeger TS Cooke AM Williams-Carrier RE Friso G Belcher SE van Wijk KJ Barkan A 《The Plant cell》2007,19(8):2606-2623
Chloroplast genomes in land plants harbor approximately 20 group II introns. Genetic approaches have identified proteins involved in the splicing of many of these introns, but the proteins identified to date cannot account for the large size of intron ribonucleoprotein complexes and are not sufficient to reconstitute splicing in vitro. Here, we describe an additional protein that promotes chloroplast group II intron splicing in vivo. This protein, RNC1, was identified by mass spectrometry analysis of maize (Zea mays) proteins that coimmunoprecipitate with two previously identified chloroplast splicing factors, CAF1 and CAF2. RNC1 is a plant-specific protein that contains two ribonuclease III (RNase III) domains, the domain that harbors the active site of RNase III and Dicer enzymes. However, several amino acids that are essential for catalysis by RNase III and Dicer are missing from the RNase III domains in RNC1. RNC1 is found in complexes with a subset of chloroplast group II introns that includes but is not limited to CAF1- and CAF2-dependent introns. The splicing of many of the introns with which it associates is disrupted in maize rnc1 insertion mutants, indicating that RNC1 facilitates splicing in vivo. Recombinant RNC1 binds both single-stranded and double-stranded RNA with no discernible sequence specificity and lacks endonuclease activity. These results suggest that RNC1 is recruited to specific introns via protein-protein interactions and that its role in splicing involves RNA binding but not RNA cleavage activity. 相似文献
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Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA 总被引:1,自引:1,他引:0
Grossberger R Mayer O Waldsich C Semrad K Urschitz S Schroeder R 《Nucleic acids research》2005,33(7):2280-2289
Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25°C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding. 相似文献
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We used alpha-32P-GTP labeling of total RNA preparations to identify self-splicing group I introns in Chlamydomonas. Several RNAs become labeled with alpha-32P-GTP, a subset of which is not seen with RNA from a mutant that lacks both copies of the psbA gene. Hybridization of the GTP-labeled RNAs to chloroplast DNA indicates that they originate from the psbA and rrn 23S genes, respectively, the only genes known to contain group I introns in this organism. Introns 1, 2, and 3 of psbA (with flanking exon sequences) were subcloned and transcribed in vitro. The synthetic RNAs were found to self-splice; splicing required Mg2+, GTP, and elevated temperature. In addition, the accuracy of self-splicing was confirmed for introns 1 and 2, and intermediates in the splicing reactions were detected. These results, together with our recent data on the 23S intron, indicate that the ability to self-splice is a general feature of Chlamydomonas group I introns. These findings have significant implications for the mechanism of group I intron splicing and evolution in Chlamydomonas and other chloroplast genomes. 相似文献