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Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons.

Zhou F, Karcher D, Bock R - Plant J. (2007)

Bottom Line: At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur.As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs.We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie (MPI-MP), Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

ABSTRACT
Most plastid genes are part of operons and expressed as polycistronic mRNAs. Many primary polycistronic transcripts undergo post-transcriptional processing in monocistronic or oligocistronic units. At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur. As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs. We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts. Separation of foreign genes by this element facilitates transgene stacking in operons, and thus will help to expand the range of applications of transplastomic technology.

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Related in: MedlinePlus

RNA accumulation in transplastomic lines harboring various candidate processing elements between the nptII and yfp cistrons(a) Accumulation of nptII mRNA. All transplastomic lines accumulate predominantly monocistronic nptII mRNA, in addition to small amounts of di-cistronic nptII–yfp transcripts and other minor RNA species that were not further characterized. Accumulation of monocistronic nptII message in the Nt-pZF73 control lines demonstrates that 3′ processing of nptII mRNA is independent of the presence of the putative processing sequences.(b) Accumulation of yfp mRNA. Significant amounts of (monocistronic) yfp mRNA accumulated only in the Nt-pZF75 lines that harbor the ±25 IEE from the psbT–psbH intergenic spacer. Note that, in addition to the monocistronic yfp and di-cistronic nptII–yfp transcripts, two minor RNA species also accumulate. One of them is approximately 200 bp larger than the monocistronic yfp message, and the other is approximately 200 bp larger than the di-cistronic nptII–yfp transcript. These minor RNA species were not further characterized, but the most probable explanation is that they originate from read-through transcription through trnfM (see Figure 1b), whose antisense transcript can also fold into a stable cloverleaf-like secondary structure and thus act as an RNA processing signal.(c) Mapping of the 5′ and 3′ ends of the monocistronic yfp mRNA in Nt-pZF75 plants. The sequence of a cDNA clone derived from head-to-tail ligated yfp mRNA is shown. The ligation site (i.e. the border between the 3′ end and the 5′ end of the circularized mRNA) is indicated by the dotted vertical line. Note that the 5′ end generated by processing within the psbT–psbH spacer element is identical in all ten cDNA clones and corresponds precisely to the 5′ end of the psbH mRNA (Figure 1b,d). Alternative 3′ ends are indicated by arrowheads, and the number of clones in which the respective termini were found is indicated. A putative transcript-stabilizing stem–loop-type RNA secondary structure within the rps16 3′ UTR is marked by horizontal arrows (interruptions indicate unpaired nucleotides). The stop codon, Shine–Dalgarno sequence and start codon are boxed.
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fig04: RNA accumulation in transplastomic lines harboring various candidate processing elements between the nptII and yfp cistrons(a) Accumulation of nptII mRNA. All transplastomic lines accumulate predominantly monocistronic nptII mRNA, in addition to small amounts of di-cistronic nptII–yfp transcripts and other minor RNA species that were not further characterized. Accumulation of monocistronic nptII message in the Nt-pZF73 control lines demonstrates that 3′ processing of nptII mRNA is independent of the presence of the putative processing sequences.(b) Accumulation of yfp mRNA. Significant amounts of (monocistronic) yfp mRNA accumulated only in the Nt-pZF75 lines that harbor the ±25 IEE from the psbT–psbH intergenic spacer. Note that, in addition to the monocistronic yfp and di-cistronic nptII–yfp transcripts, two minor RNA species also accumulate. One of them is approximately 200 bp larger than the monocistronic yfp message, and the other is approximately 200 bp larger than the di-cistronic nptII–yfp transcript. These minor RNA species were not further characterized, but the most probable explanation is that they originate from read-through transcription through trnfM (see Figure 1b), whose antisense transcript can also fold into a stable cloverleaf-like secondary structure and thus act as an RNA processing signal.(c) Mapping of the 5′ and 3′ ends of the monocistronic yfp mRNA in Nt-pZF75 plants. The sequence of a cDNA clone derived from head-to-tail ligated yfp mRNA is shown. The ligation site (i.e. the border between the 3′ end and the 5′ end of the circularized mRNA) is indicated by the dotted vertical line. Note that the 5′ end generated by processing within the psbT–psbH spacer element is identical in all ten cDNA clones and corresponds precisely to the 5′ end of the psbH mRNA (Figure 1b,d). Alternative 3′ ends are indicated by arrowheads, and the number of clones in which the respective termini were found is indicated. A putative transcript-stabilizing stem–loop-type RNA secondary structure within the rps16 3′ UTR is marked by horizontal arrows (interruptions indicate unpaired nucleotides). The stop codon, Shine–Dalgarno sequence and start codon are boxed.

Mentions: Having successfully generated homoplasmic transplastomic plants with all vectors, we next wished to compare the five constructs with respect to RNA processing and transcript accumulation for the two genes of the operon. We first analyzed transcript pattern and RNA accumulation for nptII, the first cistron of the operon. Surprisingly, when RNA gel blots were hybridized to an nptII-specific probe, identical transcript patterns were detected in all transplastomic lines (Figure 4a): a strongly hybridizing band corresponding in size to the monocistronic nptII message was seen in all lines. In addition, weakly hybridizing larger RNA species were detected, including a transcript of the size of the di-cistronic nptII–yfp RNA (Figure 4a). The same transcript pattern was also present in the Nt-pZF73 control lines that do not harbor a putative processing element, indicating that processing downstream of nptII does not require a specific sequence element. This may suggest that the transcript-stabilizing stem–loop structure downstream of the nptII coding region (taken from the rbcL 3′ UTR) (Figure 2b) is sufficient to mediate faithful 3′ end formation of the nptII mRNA.


Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons.

Zhou F, Karcher D, Bock R - Plant J. (2007)

RNA accumulation in transplastomic lines harboring various candidate processing elements between the nptII and yfp cistrons(a) Accumulation of nptII mRNA. All transplastomic lines accumulate predominantly monocistronic nptII mRNA, in addition to small amounts of di-cistronic nptII–yfp transcripts and other minor RNA species that were not further characterized. Accumulation of monocistronic nptII message in the Nt-pZF73 control lines demonstrates that 3′ processing of nptII mRNA is independent of the presence of the putative processing sequences.(b) Accumulation of yfp mRNA. Significant amounts of (monocistronic) yfp mRNA accumulated only in the Nt-pZF75 lines that harbor the ±25 IEE from the psbT–psbH intergenic spacer. Note that, in addition to the monocistronic yfp and di-cistronic nptII–yfp transcripts, two minor RNA species also accumulate. One of them is approximately 200 bp larger than the monocistronic yfp message, and the other is approximately 200 bp larger than the di-cistronic nptII–yfp transcript. These minor RNA species were not further characterized, but the most probable explanation is that they originate from read-through transcription through trnfM (see Figure 1b), whose antisense transcript can also fold into a stable cloverleaf-like secondary structure and thus act as an RNA processing signal.(c) Mapping of the 5′ and 3′ ends of the monocistronic yfp mRNA in Nt-pZF75 plants. The sequence of a cDNA clone derived from head-to-tail ligated yfp mRNA is shown. The ligation site (i.e. the border between the 3′ end and the 5′ end of the circularized mRNA) is indicated by the dotted vertical line. Note that the 5′ end generated by processing within the psbT–psbH spacer element is identical in all ten cDNA clones and corresponds precisely to the 5′ end of the psbH mRNA (Figure 1b,d). Alternative 3′ ends are indicated by arrowheads, and the number of clones in which the respective termini were found is indicated. A putative transcript-stabilizing stem–loop-type RNA secondary structure within the rps16 3′ UTR is marked by horizontal arrows (interruptions indicate unpaired nucleotides). The stop codon, Shine–Dalgarno sequence and start codon are boxed.
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Related In: Results  -  Collection

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fig04: RNA accumulation in transplastomic lines harboring various candidate processing elements between the nptII and yfp cistrons(a) Accumulation of nptII mRNA. All transplastomic lines accumulate predominantly monocistronic nptII mRNA, in addition to small amounts of di-cistronic nptII–yfp transcripts and other minor RNA species that were not further characterized. Accumulation of monocistronic nptII message in the Nt-pZF73 control lines demonstrates that 3′ processing of nptII mRNA is independent of the presence of the putative processing sequences.(b) Accumulation of yfp mRNA. Significant amounts of (monocistronic) yfp mRNA accumulated only in the Nt-pZF75 lines that harbor the ±25 IEE from the psbT–psbH intergenic spacer. Note that, in addition to the monocistronic yfp and di-cistronic nptII–yfp transcripts, two minor RNA species also accumulate. One of them is approximately 200 bp larger than the monocistronic yfp message, and the other is approximately 200 bp larger than the di-cistronic nptII–yfp transcript. These minor RNA species were not further characterized, but the most probable explanation is that they originate from read-through transcription through trnfM (see Figure 1b), whose antisense transcript can also fold into a stable cloverleaf-like secondary structure and thus act as an RNA processing signal.(c) Mapping of the 5′ and 3′ ends of the monocistronic yfp mRNA in Nt-pZF75 plants. The sequence of a cDNA clone derived from head-to-tail ligated yfp mRNA is shown. The ligation site (i.e. the border between the 3′ end and the 5′ end of the circularized mRNA) is indicated by the dotted vertical line. Note that the 5′ end generated by processing within the psbT–psbH spacer element is identical in all ten cDNA clones and corresponds precisely to the 5′ end of the psbH mRNA (Figure 1b,d). Alternative 3′ ends are indicated by arrowheads, and the number of clones in which the respective termini were found is indicated. A putative transcript-stabilizing stem–loop-type RNA secondary structure within the rps16 3′ UTR is marked by horizontal arrows (interruptions indicate unpaired nucleotides). The stop codon, Shine–Dalgarno sequence and start codon are boxed.
Mentions: Having successfully generated homoplasmic transplastomic plants with all vectors, we next wished to compare the five constructs with respect to RNA processing and transcript accumulation for the two genes of the operon. We first analyzed transcript pattern and RNA accumulation for nptII, the first cistron of the operon. Surprisingly, when RNA gel blots were hybridized to an nptII-specific probe, identical transcript patterns were detected in all transplastomic lines (Figure 4a): a strongly hybridizing band corresponding in size to the monocistronic nptII message was seen in all lines. In addition, weakly hybridizing larger RNA species were detected, including a transcript of the size of the di-cistronic nptII–yfp RNA (Figure 4a). The same transcript pattern was also present in the Nt-pZF73 control lines that do not harbor a putative processing element, indicating that processing downstream of nptII does not require a specific sequence element. This may suggest that the transcript-stabilizing stem–loop structure downstream of the nptII coding region (taken from the rbcL 3′ UTR) (Figure 2b) is sufficient to mediate faithful 3′ end formation of the nptII mRNA.

Bottom Line: At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur.As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs.We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie (MPI-MP), Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

ABSTRACT
Most plastid genes are part of operons and expressed as polycistronic mRNAs. Many primary polycistronic transcripts undergo post-transcriptional processing in monocistronic or oligocistronic units. At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur. As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs. We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts. Separation of foreign genes by this element facilitates transgene stacking in operons, and thus will help to expand the range of applications of transplastomic technology.

Show MeSH
Related in: MedlinePlus