RNA processing and expression of an intron-encoded protein in yeast mitochondria: role of a conserved dodecamer sequence.

Zhu, Huanhu; Macreadie, I G; Butow, Ronald A.

doi:10.1128/mcb.7.7.2530

Cited by 24 publications

(29 citation statements)

References 25 publications

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“…The expression level of I-PpoI in Physarum microplasmodium is much lower than that in yeast but higher than that of other group I intron-encoded endonucleases in their natural environments since endonuclease activity cannot be detected in the wild-type situation for some group I introns (16,50,54). Lower expression can be attributed at least in part to more rapid processing of PpLSU3 RNA to remove the full-length excised intron species.…”

Section: Discussionmentioning

confidence: 92%

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I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

Lin

Vogt

1998

Molecular and Cellular Biology

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PpLSU3, a mobile group I intron in the rRNA genes of Physarum polycephalum, also can home into yeast chromosomal ribosomal DNA (rDNA) (D. E. Muscarella and V. M. Vogt, Mol. Cell. Biol. 13:1023-1033, 1993). By integrating PpLSU3 into the rDNA copies of a yeast strain temperature sensitive for RNA polymerase I, we have shown that the I-PpoI homing endonuclease encoded by PpLSU3 is expressed from an RNA polymerase I transcript. We have also developed a method to integrate mutant forms of PpLSU3 as well as the Tetrahymena intron TtLSU1 into rDNA, by expressing I-PpoI in trans. Analysis of I-PpoI expression levels in these mutants, along with subcellular fractionation of intron RNA, strongly suggests that the full-length excised intron RNA, but not RNAs that are further cleaved, serves as or gives rise to the mRNA.Group I introns are a class of RNA elements that share a secondary structure which allows the intron to undergo selfsplicing from the primary transcript (5). While most group I introns are located in the genes of mitochondria and chloroplasts of lower eucaryotes, some are found in nuclear genes. Interestingly, nuclear group I introns reside only in rDNA, the gene encoding rRNA, and when present they occupy all of the ca. 200 rDNA copies typical of eucaryotic organisms. Some group I introns are mobile genetic elements. They encode a site-specific endonuclease that recognizes and cleaves a DNA sequence at or near the intron insertion site of the intronlacking allele. The double-strand break is then repaired by replication of the intron into the intron-lacking allele, thus converting all intron-lacking alleles into intron-containing alleles. This process is termed intron homing due to its high specificity (3).Among the ca. 150 nuclear group I introns reported so far, only three have been shown or have been inferred to be mobile: DiSSU1 from the slime mold Didymium iridis (6, 21, 22), NaSSU1 from the protist Naegleria andersoni and other Naegleria species (8), and PpLSU3 from the slime mold Physarum polycephalum. Originally found in the large-subunit rDNA gene of the Carolina strain (29), PpLSU3 contains the open reading frame (ORF) for the homing endonuclease I-PpoI (for nomenclature of intron-encoded endonucleases, see reference 7) in its 5Ј half and the ribozyme part in its 3Ј half. The sequence of the ribozyme part of PpLSU3 is 70% identical to the Tetrahymena thermophila intron TtLSU1, which is inserted at the same location as PpLSU3, suggesting a common evolutionary origin. Previous work has shown that PpLSU3 RNA not only undergoes self-splicing but also cleaves itself at an internal processing site (IPS), thus separating the I-PpoI ORF and the ribozyme (38). I-PpoI recognizes a 13-to 15-bp DNA sequence in a portion of the large-subunit rRNA gene that is 100% identical in all eucaryotes (9, 52). When a plasmid-borne PpLSU3 is transformed into Saccharomyces cerevisiae, most cells die upon the induction of I-PpoI expression, because I-PpoI makes double-strand breaks in the ca. 120 rDNA repeats on chromosome...

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Section: Discussionmentioning

confidence: 92%

“…Expression is therefore downregulated by splicing of the intron from premRNA. I-SceI, the endonuclease encoded by the intron, is expressed from a minor species derived from cleavage at a certain site (54). Endonucleases of T-even phage group I introns are expressed only at later stages of phage infection (14).…”

Section: Discussionmentioning

confidence: 99%

I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

Lin

Vogt

1998

Molecular and Cellular Biology

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“…The COX2 gene cloned in plasmid pJM2 (41) was modified by oligonucleotide-directed mutagenesis (33) using the Muta-Gene kit (Bio-Rad, Richmond, Calif.) and the helper phage, R408 (Stratagene, La Jolla, Calif.). The sequences of the oligonucleotides used to replace the wild-type COX2 promoter with a BamHI site and to introduce a BglII site 12 bp downstream from the dodecamer processing site (45,68), respectively, are shown with the introduced nucleotides in lowercase letters and the restriction sites underlined: 5 The DNA sequence of the PCR-amplified COX2 gene in pADCOX2-int ( Fig. 1) was determined, and a single G-to-T transversion resulting in a substitution of Val for Gly-138 was discovered.…”

Section: Methodsmentioning

confidence: 99%

T7 RNA polymerase-dependent expression of COXII in yeast mitochondria.

Pinkham¹,

Dudley²,

Mason³

1994

Mol. Cell. Biol.

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An in vivo expression system has been developed for controlling the transcription of individual genes in the mitochondrial genome of Saccharomyces cerevisiae. The bacteriophage T7 RNA polymerase (T7Pol), fused to the COXIV mitchondrial import peptide and expressed under the control of either the GAL1 or the ADH1 promoter, efficiently transcribes a target gene, T7-COX2, in the mitochondrial genome. Cells bearing the T7-COX2 gene, but lacking wild-type COX2, require T7Pol for respiration. Functional expression of T7-COX2 is completely dependent on the COX2-specific translational activator Pet111p, despite additional nucleotides at the 5' end of the T7-COX2 transcript. Expression of mitochondrion-targeted T7Pol at high levels from the GAL1 promoter has no detectable effect on mitochondrial function in rho+ cells lacking the T7-COX2 target gene, but in cells with T7-COX2 integrated into the mitochondrial genome, an equivalent level of T7Pol expression causes severe respiratory deficiency. In comparison with wild-type COX2 expression, steady-state levels of T7-COX2 mRNA increase fivefold when transcription is driven by T7Pol expressed from the ADH1 promoter, yet COXII protein levels and cellular respiration rates decrease by about 50%. This discoordinate expression of mRNA and protein provides additional evidence for posttranscriptional control of COX2 expression.

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“…One, located 3' to the olil gene, is a naturally occurring sequence polymorphism containing a U-C substitution at position 6 (15). The other was identified as a mutant in the w intron which had a UA-*AU mutation at positions 3 o-dodecamer sequence (21). RNA cleavage at the olil dodecamer occurs efficiently in vivo, while the mutation in the X dodecamer apparently prevents cleavage (15,21).…”

Section: Resultsmentioning

confidence: 99%

“…The other was identified as a mutant in the w intron which had a UA-*AU mutation at positions 3 o-dodecamer sequence (21). RNA cleavage at the olil dodecamer occurs efficiently in vivo, while the mutation in the X dodecamer apparently prevents cleavage (15,21). When RNA oligonucleotides with these sequence changes were tested for binding, it was found that the olil variant (Var6 U-*C) bound the activity in the extract with somewhat higher efficiency than the Var oligonucleotide did (Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification of a protein complex that binds to a dodecamer sequence found at the 3' ends of yeast mitochondrial mRNAs.

Min¹,

Zassenhaus²

1993

Mol. Cell. Biol.

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An activity from Saccharomyces cerevisiae mitochondria was identified that specifically bound to a 12-nucleotide sequence, AAUAA(U/C)AUUCUU, that is a site for processing of pre-mRNAs so as to generate the mature 3' ends of mRNAs. Because processing occurs 3' to the end of the dodecamer site, all mRNAs in yeast mitochondria terminate with that sequence. RNase T1 digestion fragments which terminated precisely at their 3' ends with the dodecamer sequence bound the activity, indicating that mRNAs in vivo would be capable of binding. Gel mobility shift analyses using RNA oligonucleotides showed that binding was reduced by a U-to-A substitution at position 3 of the dodecamer sequence; a C-to-A substitution at position 10 eliminated binding. UV cross-linking identified three polypeptides with approximate molecular masses of 19, 60, and 70 kDa as constituents of the binding activity. These estimates included the contribution of the 32P-labeled RNA oligonucleotide used to tag these polypeptides. An oligonucleotide with a UA-->AU substitution at positions 3 and 4 of the dodecamer site formed complexes deficient in the 19-kDa species, suggesting that binding specificity was inherent to the higher-molecular-weight polypeptides. Assembly of the complex at a dodecamer site on an RNA protected sequences located 5' to the dodecamer site from digestion by a nucleoside triphosphate-dependent 3' exoribonuclease found in yeast mitochondria. Since mitochondrial mRNAs terminate with an intact dodecamer sequence, the binding activity may function in the stabilization of mRNAs in addition to 3'-end formation of mRNAs.

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RNA processing and expression of an intron-encoded protein in yeast mitochondria: role of a conserved dodecamer sequence.

Cited by 24 publications

References 25 publications

I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

T7 RNA polymerase-dependent expression of COXII in yeast mitochondria.

Identification of a protein complex that binds to a dodecamer sequence found at the 3' ends of yeast mitochondrial mRNAs.

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