MersV1, Istex, Corpus, bibRecord, 002681

RNA-Mediated RNA Degradation and Chalcone Synthase A Silencing in Petunia

Identifieur interne : 002681 ( Istex/Corpus ); précédent : 002680; suivant : 002682

RNA-Mediated RNA Degradation and Chalcone Synthase A Silencing in Petunia

Auteurs : M. Metzlaff ; M. O'Dell ; P. D Cluster ; R. B Flavell

Source :

Cell [ 0092-8674 ] ; 1997.

RBID : ISTEX:6BA5CD05DB0BDF2B407D6291E69B9E41CB70D256

English descriptors

Teeft :
- Aliquot, Amplification, Annealing, Boehringer mannheim, Cdna, Chalcone, Chalcone synthase, Chsa, Chsa coding region, Chsa endogene, Chsa rnas, Cleavage, Cleavage site, Coding region, Complementarity, Complementary sequences, Cosuppression, Degradation, Endogene, Endogene chsa, Endonucleolytic, Endonucleolytic cleavage, Endonucleolytic cleavages, Floral cosuppression, Flower tissue, Flower tissues, Fragment, High levels, Hybrida, Jorgensen, Leaf tissue, Mrna, Napoli, Nontransgenic, Oligonucleotide, Pairing, Petunia, Petunia hybrida, Plant cell, Posttranscriptional, Posttranscriptional chsa, Primer, Purple flower tissue, Purple flowers, Rna, Rnase, Sequencing, Synthase, Transgene, Transgene chsa, Transgenic, Transgenic lines, Transgenic plants, White flower tissue, White sectors.

Abstract

Abstract: Transgenic Petunia plants with a chsA coding sequence under the control of a 35S promoter sometimes lose endogene and transgene chalcone synthase activity and purple flower pigment through posttranscriptional chsA RNA degradation. In these plants, shorter poly(A)+ and poly(A)− chsA RNAs are found, and a 3′ end–specific RNA fragment from the endogene is more resistant to degradation. The termini of this RNA fragment are located in a region of complementarity between the chsA 3′ coding region and its 3′ untranslated region. Equivalent chsA RNA fragments remain in the white flower tissue of a nontransgenic Petunia variety. We present a model involving cycles of RNA–RNA pairing between complementary sequences followed by endonucleolytic RNA cleavages to describe how RNA degradation is likely to be promoted.

Url:

https://api.istex.fr/ark:/67375/6H6-9K7V8X48-5/fulltext.pdf

DOI: 10.1016/S0092-8674(00)81930-3

Links to Exploration step

ISTEX:6BA5CD05DB0BDF2B407D6291E69B9E41CB70D256

Le document en format XML

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<front><div type="abstract" xml:lang="en">Abstract: Transgenic Petunia plants with a chsA coding sequence under the control of a 35S promoter sometimes lose endogene and transgene chalcone synthase activity and purple flower pigment through posttranscriptional chsA RNA degradation. In these plants, shorter poly(A)+ and poly(A)− chsA RNAs are found, and a 3′ end–specific RNA fragment from the endogene is more resistant to degradation. The termini of this RNA fragment are located in a region of complementarity between the chsA 3′ coding region and its 3′ untranslated region. Equivalent chsA RNA fragments remain in the white flower tissue of a nontransgenic Petunia variety. We present a model involving cycles of RNA–RNA pairing between complementary sequences followed by endonucleolytic RNA cleavages to describe how RNA degradation is likely to be promoted.</div>
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<note type="content">Figure 1: Northern RNA Analysis of Leaf and Flower Tissues of Wild-Type and Selected Transgenic Petunia Plants Ten micrograms of total RNA was separated on a 1% agarose–20% formaldehyde gel, transferred onto a nylon filter, and hybridized with a 32P-chsA-cDNA probe. The size of the chsA transcript and the flower phenotypes are indicated. l, leaves; f, flowers.</note>
<note type="content">Figure 2: RT-PCR Performed on Total RNA Isolated from Leaf and Flower Tissues of Nontransgenic and Selected Transgenic Petunia Plants Endogene and transgene chsA RNA–specific cDNAs were amplified by 18 cycles of PCR. The resulting PCR products were separated on either 1.5% (A) or 2.5% (B and C) agarose gels, transferred onto nylon membrane, and hybridized with a chsA-cDNA probe. The sizes of the RT-PCR products are indicated. V26 is the purple flowering wild-type line, Red Star a nontransgenic line with purple-white patterned flowers, C356 a transgenic line with purple-white patterned flowers, C002 a transgenic line with fully purple flowers, and C001 a transgenic line with all-white flowers. The type of tissue from which the total RNAs were isolated are labeled as follows: pl, leaf tissue of plants with fully purple flowers; pf, purple flower tissue; jl, leaf tissue of plants with purple-white patterned flowers; pjf or wjf, purple or white sectors, respectively, of purple-white patterned flowers; wl, leaf tissue of plants with fully white flowers; wf, white flower tissue; ml, leaf tissue of nontransgenic plants with purple-white patterned flowers; pmf or wmf, purple or white sectors, respectively, of nontransgenic purple-white flowers.</note>
<note type="content">Figure 3: Sequence of Specific RNA Termini Located at the Endogene chsA 3′ End The sequences surrounding identified termini at the endogene chsA 3′ end are shown. The numbers of the first 5′ nucleotide (3490) and the last 3′ nucleotide (3793) of the RNA fragment that are more resistant to degradation in vivo are given according to the numbering in sequence file X14591 of the EMBL/GenBank/DDBJ databases. The stop codon UAA and the secondary poly(A) site are indicated, as are the numbers and location of oligonucleotides used in 3′ end–specific RT-PCR amplifications.</note>
<note type="content">Figure 4: RT-PCR Patterns Obtained for the Endogene chsA 3′ End RT-PCR amplifications were carried out on total RNA isolated from leaf tissue (ml) and purple (pmf) or white (wmf) flower tissue of the nontransgenic Petunia line Red Star. The sizes of the resulting RT-PCR products are indicated. The following oligonucleotide combinations were used for the RT-PCR amplifications: A, G3006/G3398; B, G1607/G3398; C, G3006/G3280; and D, G1607/G3280. For the location of these oligonucleotides see also Figure 3. M, 100 bp marker.</note>
<note type="content">Figure 5: Computer-Based RNA Folding Analysis for the Endogene chsA 3′ End The putative secondary structure of the last 400 nucleotides of the endogene chsA RNA 3′ end was obtained by using the program FOLDRNA (Zuker and Stiegler 1981). The locations of the UAA stop codon and the 5′ and 3′ termini of the 304-base chsA RNA fragment that resists degradation are indicated.</note>
<note type="content">Figure 6: In Vitro Transcription, Annealing, and Endonucleolytic Cleavage of Complementary chsA RNA Regions 32P-labeled in vitro transcripts were either self-annealed or cross-annealed and were separated on a 5% nondenaturing PAA gel. (A) A 32P-labeled sense transcript of the complete chsA 3′ UTR was self-annealed with a 10-fold excess of its unlabeled complementary transcript. (B) An equivalent aliquot of this self-annealed RNA was incubated with dsRNA- specific RNaseIII. (C) A 32P-labeled sense transcript of the complementary region located at the 3′ end of the chsA coding region was annealed with a 10-fold excess of unlabeled sense transcript of the complete chsA 3′ UTR. (D) An equivalent aliquot of the RNA mix used in (C) was treated with RNaseIII.</note>
<note type="content">Figure 7: A Cyclic Model of RNA Degradation Based on Complementary RNA Pairing and Endonucleolytic Cleavage The process is initiated by the local accumulation of aberrant chsA RNAs, which can be any chsA RNA, from endogene or transgene, whose “normal” export from the nucleus and translation are inefficient or prevented because of structural aberrations. The aberrant RNA base pairs with the complementary sequence in the 3′ UTR of an endogene RNA (A). Endonucleolytic cleavages occur at specific sites (short line pairs) to generate two truncated molecules. The truncated endogene RNA can be repolyadenylated and exported from the nucleus to form the observed shorter poly(A)+ endogene RNA. Alternatively, it can pair with the 3′ UTR sequence of another chsA endogene RNA (B). Specific endonucleolytic cleavages of this duplex lead to the observed 304-base RNA and a long chsA RNA lacking its 3′ end. This latter RNA can be repolyadenylated and exported from the nucleus, or it can pair with the 3′ UTR sequence of another chsA endogene RNA (C), as in (B). The 304 bp product from (B) can also pair with another endogene RNA (D) to produce another long RNA lacking its 3′ end. Thus the products of one pairing–cleavage event are potential substrates for two others. The same cycle can also take place between RNAs in the cytoplasm.</note>
<note type="content">Table 1: Truncation Points in the 3′ Region of chsA RNA (A) in Comparison with Two Published Cases of mRNA Truncations (B)</note>
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<abstract xml:lang="en"><p>Abstract: Transgenic Petunia plants with a chsA coding sequence under the control of a 35S promoter sometimes lose endogene and transgene chalcone synthase activity and purple flower pigment through posttranscriptional chsA RNA degradation. In these plants, shorter poly(A)+ and poly(A)− chsA RNAs are found, and a 3′ end–specific RNA fragment from the endogene is more resistant to degradation. The termini of this RNA fragment are located in a region of complementarity between the chsA 3′ coding region and its 3′ untranslated region. Equivalent chsA RNA fragments remain in the white flower tissue of a nontransgenic Petunia variety. We present a model involving cycles of RNA–RNA pairing between complementary sequences followed by endonucleolytic RNA cleavages to describe how RNA degradation is likely to be promoted.</p>
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<ce:abstract-sec><ce:simple-para>Transgenic Petunia plants with a <ce:italic>chsA</ce:italic>
 coding sequence under the control of a 35S promoter sometimes lose endogene and transgene chalcone synthase activity and purple flower pigment through posttranscriptional <ce:italic>chsA</ce:italic>
 RNA degradation. In these plants, shorter poly(A)<ce:sup>+</ce:sup>
 and poly(A)<ce:sup>−</ce:sup>
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 RNAs are found, and a 3′ end–specific RNA fragment from the endogene is more resistant to degradation. The termini of this RNA fragment are located in a region of complementarity between the <ce:italic>chsA</ce:italic>
 3′ coding region and its 3′ untranslated region. Equivalent <ce:italic>chsA</ce:italic>
 RNA fragments remain in the white flower tissue of a nontransgenic Petunia variety. We present a model involving cycles of RNA–RNA pairing between complementary sequences followed by endonucleolytic RNA cleavages to describe how RNA degradation is likely to be promoted.</ce:simple-para>
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<abstract lang="en">Abstract: Transgenic Petunia plants with a chsA coding sequence under the control of a 35S promoter sometimes lose endogene and transgene chalcone synthase activity and purple flower pigment through posttranscriptional chsA RNA degradation. In these plants, shorter poly(A)+ and poly(A)− chsA RNAs are found, and a 3′ end–specific RNA fragment from the endogene is more resistant to degradation. The termini of this RNA fragment are located in a region of complementarity between the chsA 3′ coding region and its 3′ untranslated region. Equivalent chsA RNA fragments remain in the white flower tissue of a nontransgenic Petunia variety. We present a model involving cycles of RNA–RNA pairing between complementary sequences followed by endonucleolytic RNA cleavages to describe how RNA degradation is likely to be promoted.</abstract>
<note type="content">Section title: Article</note>
<note type="content">Figure 1: Northern RNA Analysis of Leaf and Flower Tissues of Wild-Type and Selected Transgenic Petunia Plants Ten micrograms of total RNA was separated on a 1% agarose–20% formaldehyde gel, transferred onto a nylon filter, and hybridized with a 32P-chsA-cDNA probe. The size of the chsA transcript and the flower phenotypes are indicated. l, leaves; f, flowers.</note>
<note type="content">Figure 2: RT-PCR Performed on Total RNA Isolated from Leaf and Flower Tissues of Nontransgenic and Selected Transgenic Petunia Plants Endogene and transgene chsA RNA–specific cDNAs were amplified by 18 cycles of PCR. The resulting PCR products were separated on either 1.5% (A) or 2.5% (B and C) agarose gels, transferred onto nylon membrane, and hybridized with a chsA-cDNA probe. The sizes of the RT-PCR products are indicated. V26 is the purple flowering wild-type line, Red Star a nontransgenic line with purple-white patterned flowers, C356 a transgenic line with purple-white patterned flowers, C002 a transgenic line with fully purple flowers, and C001 a transgenic line with all-white flowers. The type of tissue from which the total RNAs were isolated are labeled as follows: pl, leaf tissue of plants with fully purple flowers; pf, purple flower tissue; jl, leaf tissue of plants with purple-white patterned flowers; pjf or wjf, purple or white sectors, respectively, of purple-white patterned flowers; wl, leaf tissue of plants with fully white flowers; wf, white flower tissue; ml, leaf tissue of nontransgenic plants with purple-white patterned flowers; pmf or wmf, purple or white sectors, respectively, of nontransgenic purple-white flowers.</note>
<note type="content">Figure 3: Sequence of Specific RNA Termini Located at the Endogene chsA 3′ End The sequences surrounding identified termini at the endogene chsA 3′ end are shown. The numbers of the first 5′ nucleotide (3490) and the last 3′ nucleotide (3793) of the RNA fragment that are more resistant to degradation in vivo are given according to the numbering in sequence file X14591 of the EMBL/GenBank/DDBJ databases. The stop codon UAA and the secondary poly(A) site are indicated, as are the numbers and location of oligonucleotides used in 3′ end–specific RT-PCR amplifications.</note>
<note type="content">Figure 4: RT-PCR Patterns Obtained for the Endogene chsA 3′ End RT-PCR amplifications were carried out on total RNA isolated from leaf tissue (ml) and purple (pmf) or white (wmf) flower tissue of the nontransgenic Petunia line Red Star. The sizes of the resulting RT-PCR products are indicated. The following oligonucleotide combinations were used for the RT-PCR amplifications: A, G3006/G3398; B, G1607/G3398; C, G3006/G3280; and D, G1607/G3280. For the location of these oligonucleotides see also Figure 3. M, 100 bp marker.</note>
<note type="content">Figure 5: Computer-Based RNA Folding Analysis for the Endogene chsA 3′ End The putative secondary structure of the last 400 nucleotides of the endogene chsA RNA 3′ end was obtained by using the program FOLDRNA (Zuker and Stiegler 1981). The locations of the UAA stop codon and the 5′ and 3′ termini of the 304-base chsA RNA fragment that resists degradation are indicated.</note>
<note type="content">Figure 6: In Vitro Transcription, Annealing, and Endonucleolytic Cleavage of Complementary chsA RNA Regions 32P-labeled in vitro transcripts were either self-annealed or cross-annealed and were separated on a 5% nondenaturing PAA gel. (A) A 32P-labeled sense transcript of the complete chsA 3′ UTR was self-annealed with a 10-fold excess of its unlabeled complementary transcript. (B) An equivalent aliquot of this self-annealed RNA was incubated with dsRNA- specific RNaseIII. (C) A 32P-labeled sense transcript of the complementary region located at the 3′ end of the chsA coding region was annealed with a 10-fold excess of unlabeled sense transcript of the complete chsA 3′ UTR. (D) An equivalent aliquot of the RNA mix used in (C) was treated with RNaseIII.</note>
<note type="content">Figure 7: A Cyclic Model of RNA Degradation Based on Complementary RNA Pairing and Endonucleolytic Cleavage The process is initiated by the local accumulation of aberrant chsA RNAs, which can be any chsA RNA, from endogene or transgene, whose “normal” export from the nucleus and translation are inefficient or prevented because of structural aberrations. The aberrant RNA base pairs with the complementary sequence in the 3′ UTR of an endogene RNA (A). Endonucleolytic cleavages occur at specific sites (short line pairs) to generate two truncated molecules. The truncated endogene RNA can be repolyadenylated and exported from the nucleus to form the observed shorter poly(A)+ endogene RNA. Alternatively, it can pair with the 3′ UTR sequence of another chsA endogene RNA (B). Specific endonucleolytic cleavages of this duplex lead to the observed 304-base RNA and a long chsA RNA lacking its 3′ end. This latter RNA can be repolyadenylated and exported from the nucleus, or it can pair with the 3′ UTR sequence of another chsA endogene RNA (C), as in (B). The 304 bp product from (B) can also pair with another endogene RNA (D) to produce another long RNA lacking its 3′ end. Thus the products of one pairing–cleavage event are potential substrates for two others. The same cycle can also take place between RNAs in the cytoplasm.</note>
<note type="content">Table 1: Truncation Points in the 3′ Region of chsA RNA (A) in Comparison with Two Published Cases of mRNA Truncations (B)</note>
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RNA-Mediated RNA Degradation and Chalcone Synthase A Silencing in Petunia

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