Self-splicing of a mitochondrial group I intron from the cytochromeb gene of the ascomycetePodospora anserina

Abstract

We have shown that the second intron of the Podospora mitochondrial gene coding for cytochrome b (Cytb 12) splices autocatalytically, using in vitro transcripts generated from the T7 promoter. The reaction takes place at 37 degrees C in the presence of 50 mM TRIS-HCl pH 7.5, 60 mM MgCl2 and 1 mM GTP but shows a low efficiency even at high KCl concentrations of up to 1.2 M. Under these conditions, intron bI2 follows the conventional pathway of group I splicing, and all characteristic products, with regard to both transesterification and hydrolysis, could be identified. Moreover, the intron is capable of undergoing cyclization, thereby releasing the noncoded G and one additional nucleotide (U) from the 5' end. The 5' cleavage site is preceded by the same two nucleotides, indicating a base-pairing at the same site of the internal guide sequence (IGS) for both splicing and cyclization ("one-binding-site model"). In addition, products resulting from site-specific hydrolysis 138 nucleotides downstream of the 5' splice site were detected. Unusually, the shortened intron is also able to form a circular RNA and an alternative sequence that aligns the cyclization site to the catalytic core of the intron must be assumed.

tRNA-dependent cleavage of the ColE1 plasmid-encoded RNA I

“…hammerhead motif ribozyme (Birikh et al, 1997); hairpin motif ribozyme (Fedor, 2000); hepatitis delta virus ribozyme (Been & Wickham, 1997); varkud satellite (VS) ribozyme (Collins, 2002)], and self-splicing RNAs [for example: group I introns (Schmidt et al, 1992) and group II introns (Jacquier, 1996)]. (c) Fig.…”

Transcript mapping and processing of mitochondrial RNA in the chlorophyte alga Prototheca wickerhamii

“…Antisense RNA probes were generated by in vitro transcription of appropriate recombinant plasmids in the presence of a32p-UTP as described by Winkler and Ktick [79]. In vitro splicing experiments followed a protocol based on Schmidt et al [68]. Recombinant plasmids containing the three different cox1 introns and appropriate parts of the flanking exon sequences were used for in vitro transcription [79].…”

“…To determine whether the three cox1 introns from P. wickerhamii display in vitro self-splicing activity, transcripts of introns plus flanking exonic regions were spliced under 'standard conditions' (see Materials and methods). The reaction conditions employed here have already been used successfully for the grouplD intron 2 in the cob gene from the filamentous fungus Podospora anserina, which is structurally closely related to cox1 intron 8 from P. anserina and intron 2 from P. wickerhamii [68,84]. Under these conditions, intron 2 and intron 3 of the P. wickerhamii cox1 gene show in vitro autocatalytic splicing activity, while intron 1 does not (data not shown).…”

“…It has been demonstrated that the spatial arrangement of the intron core, which is defined by conserved helices and loops interacting with certain exon sequences, is sufficient for the splicing process in vitro (reviewed in [ 18,17,50]). Employing in vitro self-splicing reaction conditions established for a structurally related grouplD intron from P. anserina [68], coxl.i2 from Protoheca undergoes in vitro autocatalytic splicing displaying all expected splicing products and intermediates. The data indicate a correlation between the structural and functional properties of grouplD introns among different species, this implying a common ancestor of structurally similar introns.…”

“…Group I introns of the cox 1 gene from P. wickerhamii show structural similarities to corresponding fungal introns [84], and have therefore been chosen for comparative in vitro self-splicing experiments. Two of the three group I introns residing in the coxl gene displayed in vitro selfsplicing activity under conditions defined for splicing of structurally related fungal introns [ 68].…”