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Operons are a conserved feature of nematode genomes.

Pettitt J, Philippe L, Sarkar D, Johnston C, Gothe HJ, Massie D, Connolly B, Müller B - Genetics (2014)

Bottom Line: The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla.We have nevertheless identified putative operons conserved between Enoplea and Chromadorea.Our data suggest that operons and "spliced leader" (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom j.pettitt@abdn.ac.uk.

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Related in: MedlinePlus

Processing of a synthetic operon containing the T. spiralis cpt-2∼nuaf-3 intercistronic region in C. elegans. (A) Schematic of the structure of sur-5::gfp∼mCherry synthetic operon construct containing the Tsp-cpt-2∼nuaf-3 intercistronic region. The sequence immediately downstream from the Tsp-cpt-2 3′-UTR is shown, illustrating the presence of the Ur motif and the trans-splice acceptor site. Strain PE613 contains an identical construct, but with the ICR replaced with that from between Cel-cpt-2 and prx-14. (B) Detection of operon transcript processing by SL2 trans-splicing. mCherry transcripts trans-spliced to SL2 were detected by reverse transcription of RNA prepared from either PE612 or PE613 animals followed by PCR with an SL2 primer and a primer located in the mCherry coding region (+RT). Primers amplifying gpd-1 were included to control for sample variation. gpd-1 genomic DNA was detected in the −RT control reaction (*) and SL2-ZK1236.7a is a minor product detected in the +RT reactions. Reactions with RNA isolated from N2 wild-type animals were included as control. M is a DNA size standard. (C) Alignment of transgene sequences and SL2-mCherry transcripts confirming correct splice site usage. The beginning of the mCherry open reading frame is countershaded black, the NheI cloning site is countershaded gray, and the SL2 sequences are underlined.
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fig4: Processing of a synthetic operon containing the T. spiralis cpt-2∼nuaf-3 intercistronic region in C. elegans. (A) Schematic of the structure of sur-5::gfp∼mCherry synthetic operon construct containing the Tsp-cpt-2∼nuaf-3 intercistronic region. The sequence immediately downstream from the Tsp-cpt-2 3′-UTR is shown, illustrating the presence of the Ur motif and the trans-splice acceptor site. Strain PE613 contains an identical construct, but with the ICR replaced with that from between Cel-cpt-2 and prx-14. (B) Detection of operon transcript processing by SL2 trans-splicing. mCherry transcripts trans-spliced to SL2 were detected by reverse transcription of RNA prepared from either PE612 or PE613 animals followed by PCR with an SL2 primer and a primer located in the mCherry coding region (+RT). Primers amplifying gpd-1 were included to control for sample variation. gpd-1 genomic DNA was detected in the −RT control reaction (*) and SL2-ZK1236.7a is a minor product detected in the +RT reactions. Reactions with RNA isolated from N2 wild-type animals were included as control. M is a DNA size standard. (C) Alignment of transgene sequences and SL2-mCherry transcripts confirming correct splice site usage. The beginning of the mCherry open reading frame is countershaded black, the NheI cloning site is countershaded gray, and the SL2 sequences are underlined.

Mentions: Analysis of the intercistronic region between Tsp-cpt-2 and nuaf-3 downstream of the polyadenylation signal of Tsp-cpt-2 revealed a clear Ur element and there are several U-rich regions, characteristics of the ICRs in C. elegans operons (Graber et al. 2007). To investigate the possibility that this region is able to function in polycistronic RNA processing, we determined whether the ICR from it could be recognized and processed if heterologously expressed in C. elegans. We generated an artificial operon consisting of sur-5::gfp (Gu et al. 1998) and mCherry genes flanking the ICR from Tsp-cpt-2∼nuaf-3. Transgenic animals carrying this construct expressed nuclear GFP and cytoplasmic mCherry, consistent with the processing of the two coding regions under the direction of the Tsp-cpt-2∼nuaf-3 ICR. We confirmed that this involved trans-splicing to SL2, as expected for polycistronic RNA processing in C. elegans, by showing that we could detect SL2 trans-splicing to the mCherry mRNA in RNA derived from transgenic animals (Figure 4). Thus, the predicted ICR between Tsp-cpt-2 and nuaf-3 is recognized and used as a substrate for polycistronic RNA processing in C. elegans.


Operons are a conserved feature of nematode genomes.

Pettitt J, Philippe L, Sarkar D, Johnston C, Gothe HJ, Massie D, Connolly B, Müller B - Genetics (2014)

Processing of a synthetic operon containing the T. spiralis cpt-2∼nuaf-3 intercistronic region in C. elegans. (A) Schematic of the structure of sur-5::gfp∼mCherry synthetic operon construct containing the Tsp-cpt-2∼nuaf-3 intercistronic region. The sequence immediately downstream from the Tsp-cpt-2 3′-UTR is shown, illustrating the presence of the Ur motif and the trans-splice acceptor site. Strain PE613 contains an identical construct, but with the ICR replaced with that from between Cel-cpt-2 and prx-14. (B) Detection of operon transcript processing by SL2 trans-splicing. mCherry transcripts trans-spliced to SL2 were detected by reverse transcription of RNA prepared from either PE612 or PE613 animals followed by PCR with an SL2 primer and a primer located in the mCherry coding region (+RT). Primers amplifying gpd-1 were included to control for sample variation. gpd-1 genomic DNA was detected in the −RT control reaction (*) and SL2-ZK1236.7a is a minor product detected in the +RT reactions. Reactions with RNA isolated from N2 wild-type animals were included as control. M is a DNA size standard. (C) Alignment of transgene sequences and SL2-mCherry transcripts confirming correct splice site usage. The beginning of the mCherry open reading frame is countershaded black, the NheI cloning site is countershaded gray, and the SL2 sequences are underlined.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4125394&req=5

fig4: Processing of a synthetic operon containing the T. spiralis cpt-2∼nuaf-3 intercistronic region in C. elegans. (A) Schematic of the structure of sur-5::gfp∼mCherry synthetic operon construct containing the Tsp-cpt-2∼nuaf-3 intercistronic region. The sequence immediately downstream from the Tsp-cpt-2 3′-UTR is shown, illustrating the presence of the Ur motif and the trans-splice acceptor site. Strain PE613 contains an identical construct, but with the ICR replaced with that from between Cel-cpt-2 and prx-14. (B) Detection of operon transcript processing by SL2 trans-splicing. mCherry transcripts trans-spliced to SL2 were detected by reverse transcription of RNA prepared from either PE612 or PE613 animals followed by PCR with an SL2 primer and a primer located in the mCherry coding region (+RT). Primers amplifying gpd-1 were included to control for sample variation. gpd-1 genomic DNA was detected in the −RT control reaction (*) and SL2-ZK1236.7a is a minor product detected in the +RT reactions. Reactions with RNA isolated from N2 wild-type animals were included as control. M is a DNA size standard. (C) Alignment of transgene sequences and SL2-mCherry transcripts confirming correct splice site usage. The beginning of the mCherry open reading frame is countershaded black, the NheI cloning site is countershaded gray, and the SL2 sequences are underlined.
Mentions: Analysis of the intercistronic region between Tsp-cpt-2 and nuaf-3 downstream of the polyadenylation signal of Tsp-cpt-2 revealed a clear Ur element and there are several U-rich regions, characteristics of the ICRs in C. elegans operons (Graber et al. 2007). To investigate the possibility that this region is able to function in polycistronic RNA processing, we determined whether the ICR from it could be recognized and processed if heterologously expressed in C. elegans. We generated an artificial operon consisting of sur-5::gfp (Gu et al. 1998) and mCherry genes flanking the ICR from Tsp-cpt-2∼nuaf-3. Transgenic animals carrying this construct expressed nuclear GFP and cytoplasmic mCherry, consistent with the processing of the two coding regions under the direction of the Tsp-cpt-2∼nuaf-3 ICR. We confirmed that this involved trans-splicing to SL2, as expected for polycistronic RNA processing in C. elegans, by showing that we could detect SL2 trans-splicing to the mCherry mRNA in RNA derived from transgenic animals (Figure 4). Thus, the predicted ICR between Tsp-cpt-2 and nuaf-3 is recognized and used as a substrate for polycistronic RNA processing in C. elegans.

Bottom Line: The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla.We have nevertheless identified putative operons conserved between Enoplea and Chromadorea.Our data suggest that operons and "spliced leader" (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.

View Article: PubMed Central - PubMed

Affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom j.pettitt@abdn.ac.uk.

Show MeSH
Related in: MedlinePlus