G. Van der Horst

Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro

Excised group II introns in yeast mitochondria appear as covalently closed circles under the electron microscope. We show that these circular molecules are branched and resemble the lariats arising through splicing of nuclear pre-mRNAs in yeast and higher eukaryotes. One member of this intron class (aI5c in the gene for cytochrome c oxidase subunit I) is capable of self-splicing in vitro, giving correct exon-exon ligation and resulting in the appearance of both linear and lariat forms of the excised intron. Nuclease digestion of the latter molecules reveals the presence of a complex oligonucleotide with the probable structure AGU, which thus resembles the branch point formed in the spliceosome-dependent reactions undergone by nuclear pre-mRNAs. Unlike group I introns, this group II intron is not demonstrably dependent on GTP for self-splicing and circularization of the isolated, linear intron is not observed. A model accounting for these observations is presented.

Characterization of the promoter of the large ribosomal RNA gene in yeast mitochondria and separation of mitochondrial RNA polymerase into two different functional components.

We have characterized a DNA sequence that functions in recognition of the promoter of the mitochondrial large rRNA gene by the yeast mtRNA polymerase. Promoter‐containing DNA fragments were mutagenized and used as templates to study initiation of transcription in vitro with a partially purified mtRNA polymerase preparation. Deletion mutants, in which increasing stretches of DNA were removed from regions flanking the promoter, define a short area essential for correct initiation of transcription. It virtually coincides with a highly conserved stretch of nine nucleotides that is found immediately upstream of all transcriptional start sites described thus far. Two different point mutations within this nonanucleotide sequence drastically reduce promoter function. Conversely a single point mutation that results in the formation of a nonanucleotide sequence 99 nucleotides upstream of the large rRNA gene leads to a new, efficient transcription initiation site. MtRNA polymerase can be resolved into two different components by chromatography on Blue Sepharose: one retaining the capacity to synthesize RNA, the other conferring the correct specificity of initiation to the catalytic component.

Interlocked RNA circle formation by a self-splicing yeast mitochondrial group I intron

RNA containing the aI3 group I intron of the yeast mitochondrial gene encoding cytochrome oxidase subunit I shows self-splicing in vitro. The excised intron, comprising 1514 nucleotides, is partially split into an upstream portion, containing the intronic reading frame, and a downstream portion, containing the typical group I conserved sequence elements. Full-length intron RNA and intron parts occur in linear and circular form. In the transesterification reactions leading to circle formation, only the guanosine nucleotide added during splicing is removed. Reincubation of isolated, complete circular intron RNA under self-splicing conditions leads to formation of free subintronic RNA circles. Under similar conditions, purified linear intron RNA gives rise to a number of circular and linear products, one of which consists of interlocked subintronic RNA circles. These observations suggest that the intron RNA possesses a dynamic structure in which subtle alterations in folding result in the formation of RNA products with different topology.

New RNA-mediated reactions by yeast mitochondrial group I introns.

The group I self‐splicing reaction is initiated by attack of a guanosine nucleotide at the 5′ splice site of intron‐containing precursor RNA. When precursor RNA containing a yeast mitochondrial group I intron is incubated in vitro under conditions of self‐splicing, guanosine nucleotide attack can also occur at other positions: (i) the 3′ splice site, resulting in formation of a 3′ exon carrying an extra added guanosine nucleotide at its 5′ end; (ii) the first phosphodiester bond in precursor RNA synthesized from the SP6 bacteriophage promoter, leading to substitution of the first 5′‐guanosine by a guanosine nucleotide from the reaction mixture; (iii) the first phosphodiester bond in already excised intron RNA, resulting in exchange of the 5′ terminal guanosine nucleotide for a guanosine nucleotide from the reaction mixture. An identical sequence motif (5′‐GAA‐3′) occurs at the 3′ splice site, the 5′ end of SP6 precursor RNA and at the 5′ end of excised intron RNA. We propose that the aberrant reactions can be explained by base‐pairing of the GAA sequence to the Internal Guide Sequence. We suggest that these reactions are mediated by the same catalytic centre of the intron RNA that governs the normal splicing reactions.

RNA processing in yeast mitochondria: characterization of mit- mutants disturbed in the synthesis of subunit I of cytochrome c oxidase

SummaryMit− mutants disturbed in the synthesis of cytochrome c oxidase subunit I lack the mRNA for this protein and accumulate longer RNAs still containing intron sequences. We have analyzed the patterns of transcripts occurring in several such mutants in an attempt to define a pathway of processing events and to demarcate intron-sequences involved in RNA splicing. We find that processing does not follow a strictly ordered pathway and, in contrast to the situation for the cytochrome b gene, that a block in the processing of an intron does not necessarily lead to a block in the processing of introns downstream. Although in some cases, this may result from overlapping specificities of intronic-URF encoded RNA maturases, an internal start of translation on precursor RNAs seems more likely.M5-16, a mutant deleted for a large part of the central portion of the subunit I gene exhibits delayed processing and a highly simplified pattern of intermediates. The lengths of these indicate that maturation of the mRNA for subunit I involves processing, as well as splicing.