The structure of precursor mRNAs and of excised intron RNAs in chloroplasts of <i>Euglena gracilis</i>

Koller, Barbara; Clarke, Jill; Delius, Hajo

doi:10.1002/j.1460-2075.1985.tb03954.x

Cited by 18 publications

(5 citation statements)

References 35 publications

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“…We interpret these transcripts to be RNA processing intermediates of the 1.0 kb tetracistronic mRNA (band 1) with all four introns (band 3), three introns (band 4), two introns (band 5) and one intron (band 6) present. Since the splicing of Euglena chloroplast transcripts is not an ordered process (Koller et al, 1985), the diffuse bands 4, 5, and 6 most likely represent populations of transcripts with different combinations of introns El, E2, Fl, and F2 present. Unspliced precursors of the hexacistronic psaA-psaB-psbE-psbF-psbL-ORF42…”

Section: Analysis Of Cytochrome B-559 Pre-mrnas and Mrnasmentioning

confidence: 99%

Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene.

Copertino

Hallick

1991

The EMBO Journal

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The psbF gene of chloroplast DNAs encodes the beta‐subunit of cytochrome b‐559 of the photosystem II reaction center. The psbF locus of Euglena gracilis chloroplast DNA has an unusual 1042 nt group II intron that appears to be formed from the insertion of one group II intron into structural domain V of a second group II intron. Using both direct primer extension cDNA sequencing and cDNA cloning and sequencing, we have determined that a 618 nt internal intron is first excised from the 1042 nt intron of psbF pre‐mRNA, resulting in a partially spliced pre‐mRNA containing a 424 nt group II intron with a spliced domain V. The 424 nt intron is then removed to yield the mature psbF mRNA. Therefore, the 1042 nt intron of psbF is a group II intron within another group II intron. We use the term ‘twintron’ to define this new type of genetic element. Intermediates in the splicing pathway were detected by northern hybridization. Splicing of both the internal and external introns occurs via lariat intermediates. Twintron splicing was found to proceed by a sequential pathway, the internal intron being removed prior to the excision of the external intron. A possible mechanism for twintron formation by intron transposition is discussed.

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Section: Analysis Of Cytochrome B-559 Pre-mrnas and Mrnasmentioning

confidence: 99%

Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene.

Copertino

Hallick

1991

The EMBO Journal

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“…Although the splicing rate for most nuclear pre-mRNAs is rapid and is not generally rate limiting for gene expression, unspliced transcripts accumulate to high levels in mitochondria and chloroplasts (Hollingsworth et al, 1984;Collins and Lambowitz, 1985;Koller et al, 1985;Shinozaki et al, 1986;Westhoff and Hermann, 1988;Pel et al, 1992). Therefore, the modulation of organellar splicing rates is likely to impact the abundance of mature transcripts.…”

Section: Introductionmentioning

confidence: 99%

Nuclear mutations that block group II RNA splicing in maize chloroplasts reveal several intron classes with distinct requirements for splicing factors.

1997

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To elucidate mechanisms that regulate chloroplast RNA splicing in multicellular plants, we sought nuclear mutations in maize that result in chloroplast splicing defects. Evidence is presented for two nuclear genes whose function is required for the splicing of group II introns in maize chloroplasts. A mutation in the crsl (for chloroplast RNA gplicing 1) gene blocks the splicing of only the atpF intron, whereas a mutation in the crs2 gene blocks the splicing of many chloroplast introns. In addition, a correlation was observed between the absence of plastid ribosomes and the failure to splice several chloroplast introns. Our results suggest that a chloroplast-encoded factor and a nuclear-encoded factor whose activity requires crs2 function facilitate the splicing of distinct sets of group II introns. These two genetically defined intron sets also differ with regard to intron structure: one set consists of only subgroup IIA introns and the other of only subgroup IIB introns. Therefore, it is likely that distinct splicing factors recognize subgroup-specific features of intron structure or facilitate subgroup-specific aspects of the splicing reaction. Of the 12 pre-mRNA introns in the maize chloroplast genome, only one is normally spliced in both crs2 mutants and in mutants lacking plastid ribosomes, indicating that few, if any, of the group II introns in the chloroplast genome undergo autocatalytic splicing in vivo.

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“…The largest are intron lariat 1 linked to partially spliced forms of exons 2-5. Circular excised intron RNAs for the Euglena psbA gene have been detected previously by electron microscopy (8). We have been unable to obtain any results approaching the resolution or sensitivity of detection with this RNA sample and conventional ID gel methods.…”

mentioning

confidence: 59%

“…The polyacrylamide gradient from left to right across the gel is 8% -3.5%. The interpretations of linear and lariat RNA molecules, indicated at the right, are based on previous analyses of Euglena psbA transcripts (6,7,8). The filled boxes correspond to exons, and the hatched boxes correspond to a mixture of downstream partially spliced and unspliced sequences.…”

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confidence: 99%

Transverse gradient gel electrophoresis: a gel system for resolving complex mixtures of lariat and linear RNA molecules

1990

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In studying the complex RNA processing pathways of pre-mRNAs with multiple introns it is often necessary to resolve RNA splicing intermediates and different topological forms of RNA. Conventional one dimension northern hybridization analysis does not offer enough resolution to discriminate intron lariats from larger unspliced pre-mRNAs. Historically, two gels with different concentrations of acrylamide have been used to distinguish between circular and linear RNA species (1,2). However, these analyses were done on 32P-labelled, in vitro synthesized RNA subjected to in vitro splicing reactions where sensitivity of detection is not a problem and the number of RNA species are limited. Such an analysis on in vivo RNA of genes containing multiple introns is ambiguous due to the many linear and lariatcontaining splicing intermediates and the low concentrations of these intermediates in cellular RNA samples. Detecting lariat RNAs with 2 gels is further complicated with complex RNA mixtures when an RNA band detected on one gel cannot be equated with the comparable band on another gel because the RNAs change migration order. We have therefore developed a new method to discriminate between linear and lariat RNAs in a complex, cellular RNA sample. During transverse linear gradient polyacrylamide gel electrophoresis (TGGE) under denaturing conditions, the electrophoretic mobility of lariatcontaining RNA molecules changes much more dramatically as a function of polyacrylamide concentration than that of linear RNAs. In a 3.5-8% polyacrylamide TGGE, lariat molecules run as downward arcing bands that cross over linear molecules. This feature of TGGE allows one to infer relationships between linear and lariat-containing RNAs based on their band shape across the entire gel. TGGE also offers superior sensitivity of detection due to pattern recognition compared to a ID gel, because very faint signals that might be ignored as background in a ID gel appear as distinct arcs in TGGE.TGGE for RNA electrophoresis is an adaptation of a protein gel procedure (3), suggested by Udo Johanningmeier. The apparatus of Studier (4) was used. Gel plates 14 cm by 16 cm were assembled with 4 mm spacers on the top, bottom, and left side of the plates. With the gel plates held vertically, and the right side on top, the gel was cast with a peristaltic pump (flow rate 4.5 ml/min) from a linear gradient maker with 8% acrylamide/8 M urea in the mixing chamber and 3.5 % acrylamide/8 M urea in the second chamber. The acrylamide:bisacrylamide ratio was 19:1 in both solutions. TEMED (0.02%) and ammonium persulfate (0.04%) were added. The gel was overlaid with butanol and allowed to polymerize for 1 h. The spacer at the top of the gel was then removed, and the right spacer was clamped in place. This resulted in a linear 3.5-8% polyacrylamide gradient gel cast transverse to the direction of electrophoresis. 100 stg of Euglena gracilis chloroplast RNA (5) was resuspended in loading dye (80% formamide, 10 mM EDTA, pH 8.0, 1 mg/ml bromphenol blue, 1 mg/mi xylene cya...

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The structure of precursor mRNAs and of excised intron RNAs in chloroplasts of Euglena gracilis

Cited by 18 publications

References 35 publications

Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene.

Group II twintron: an intron within an intron in a chloroplast cytochrome b-559 gene.

Nuclear mutations that block group II RNA splicing in maize chloroplasts reveal several intron classes with distinct requirements for splicing factors.

Transverse gradient gel electrophoresis: a gel system for resolving complex mixtures of lariat and linear RNA molecules

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