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. 2006;34(18):5312-24.
doi: 10.1093/nar/gkl598. Epub 2006 Sep 29.

Post-transcriptional control of nuclear-encoded cytochrome oxidase subunits in Trypanosoma brucei: evidence for genome-wide conservation of life-cycle stage-specific regulatory elements

Matthew Mayho  1 , Katelyn Fenn, Paul Craddy, Susan Crosthwaite, Keith Matthews
Affiliations

Post-transcriptional control of nuclear-encoded cytochrome oxidase subunits in Trypanosoma brucei: evidence for genome-wide conservation of life-cycle stage-specific regulatory elements

Matthew Mayho et al. Nucleic Acids Res. 2006.

Abstract

Trypanosomes represent an excellent model for the post-transcriptional regulation of gene expression because their genome is organized into polycistronic transcription units. However, few signals governing developmental stage-specific expression have been identified, with there being no compelling evidence for widespread conservation of regulatory motifs. As a tool to search for common regulatory sequences we have used the nuclear-encoded components of the cytochrome oxidase (COX) complex of the trypanosome respiratory chain. Components of this complex represent a form of post-transcriptional operon because trypanosome mitochondrial activity is unusual in being developmentally programmed. By genome analysis we identified the genes for seven components of the COX complex. Each mRNA exhibits bloodstream stage-specific instability, which is not mediated by the RNA silencing pathway but which is alleviated by cycloheximide. Reporter assays have identified regulatory regions within the 3'-untranslated regions of three COX mRNAs operating principally at the translational level, but also via mRNA stability. Interrogation of the mapped regions via oligonucleotide frequency scoring provides evidence for genome-wide conservation of regulatory sequences among a large cohort of procyclic-enriched transcripts. Analysis of the co-regulated subunits of a stage-specific enzyme is therefore a novel approach to uncover cryptic regulatory sequences controlling gene expression at the post-transcriptional level.

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Figures

Figure 1
Figure 1
(A) Mitochondrial localization of identified COX subunits. For each subunit, the coding region was inserted into a trypanosome expression vector such that the expressed protein included a C-terminal Ty1 epitope tag. Transient transfection of each construct into procyclic forms resulted in expression of an epitope-tagged protein which, in each case, localized to the mitochondrion of the procyclic form (panels C–H). This localization also matched the staining observed with the mitochondrial vital dye, Mitotracker red (panel B). A phase contrast image of the cells in panel B stained with DAPI is shown in panel A, this visualizing the position of the cell nucleus and the mitochondrial genome (kinetoplast). (B) Northern blots demonstrating the relative expression of the identified COX subunit transcripts in bloodstream (B) or procyclic (P) form trypanosomes. In each case, a section of the ethidium stained gel is shown (containing the rRNAs) to demonstrate relative loading.
Figure 2
Figure 2
Stability of COX subunit mRNAs in bloodstream and procyclic form trypanosomes. The upper panels show representative northern blots of COX V transcripts from cells untreated (−) or treated (+) with 5 µg/ml actinomycin D. RNA was isolated at the times shown. The lower panels show a quantitation of all COX subunit mRNAs in each life-cycle stage in the presence of actinomycin D, with each data point representing the level of mRNA with respect to its abundance before the addition of actinomycin D. Control transcripts of TbZFP3 (constitutively expressed) and aldolase (bloodstream enriched) are also shown.
Figure 3
Figure 3
(A) Mapping of the site of polyadenylation for two COX subunit mRNAs (COX V and IX) in bloodstream (open circles) or procyclic forms (closed circles). The sites of polyadenylation are distributed, but there is no consistent difference between the sites used in each life-cycle stage. (B) mRNA abundance of COX V and COX IX in wild-type bloodstream forms of T.brucei brucei STIB 247 and in two independently derived Ago null mutants of the same strain (35).
Figure 4
Figure 4
mRNA abundance of COX subunit mRNAs after treatment of bloodstream or procyclic form trypanosomes with cycloheximide. The upper panels show representative northern blots of COX V transcripts from bloodstream (BSF) or procyclic forms (PCF) untreated (−) or treated (+) with 50 µg/ml cycloheximide. RNA was isolated at the times shown. The lower panels show a quantification of all COX subunit mRNAs in each life-cycle stage in the presence of cycloheximide, with each data point representing the level of mRNA with respect to its abundance before the addition of cycloheximide. Control transcripts of TbZFP3 (constitutively expressed) and aldolase (bloodstream-enriched) are also shown.
Figure 5
Figure 5
(A) Investigation of regulatory signals within COX V, VI and IX 3′-UTRs. The intergenic region for each COX subunit was positioned downstream of the CAT gene, this being encompassed within a construct comprising a phleomycin resistance gene and targeted to integrate into the tubulin gene array. The site of polyadenylation is indicated within the intergenic region (denoted ‘A’). At least two stable cell lines were generated for each construct and two independent CAT assays performed on cell extracts from each cell line. (B) Resulting CAT expression for each COX intergenic region deletion are shown as a percentage of the CAT expression from an identical construct comprising a deleted aldolase gene 3′-UTR, which provides constitutive expression in bloodstream and procyclic forms. The CAT mRNA abundance for each construct is shown at the extreme right, as a percentage of the CAT mRNA abundance derived from constructs comprising each intact COX subunit intergenic region (IR). These values were derived from northern blots, representatives of which are shown in Figure 6.
Figure 6
Figure 6
(A) Northern blots of CAT mRNA provided with successive deletions of the COX V, COX VI or COX IX intergenic regions. In each case the rRNA region of the ethidium stained gel is provided to demonstrate relative loading. (B) Northern blots of CAT mRNA derived from cell lines in which the CAT gene is provided with the COX IX intergenic region (9IR) or a deletion (COX IX Δ4, ‘9Δ4’) which results in altered mRNA abundance. All cell lines were exposed to actinomycin D at t = 0 and RNA isolated at time points thereafter in order to follow transcript decay. The relative abundance of CAT mRNA is denoted beneath each lane. (C) Northern blot of CAT mRNA derived from cells transfected with constructs comprising either the intact intergenic region for COX V or a deleted derivative (COX VΔ4; ‘5Δ4’). In each case cells were either untreated (−) or treated (+) with cycloheximide, with mRNA being isolated 4 h after the addition of drug. CAT mRNA abundance is denoted beneath each lane.
Figure 7
Figure 7
(A) CAT activity generated by constructs in which either the COX IXΔ4 (‘9Δ4’) or COX VΔ2 (‘5Δ2’) regions are placed in front of the aldolase 3′-UTR, with 5Δ2 being either in forward or reverse orientation, or in multiple copies. Values are expressed as a percentage of the protein derived from a construct with the aldolase 3′-UTR alone. (B) Northern blot of CAT mRNA derived from the cell lines in which either the COX IXΔ4 (‘9Δ4’) or COX VΔ2 (‘5Δ2’) regions are placed in front of the aldolase 3′-UTR, with 5Δ2 being either in forward (F) or reverse (R) orientation, or in multiple copies (x3). mRNA quantification is expressed as a percentage of a cell line with the aldolase 3′-UTR alone, these being normalized to relative loading as determined by rRNA levels.
Figure 8
Figure 8
(A) Alignment of the EP procyclin 26mer element with the corresponding region in the COX V 3′-UTR (the [TATTTTTT] element is underlined). (B) Structure prediction for the 26mer core element in EP procyclin and COX V 3′-UTRs. In the upper structures, an m-fold prediction (47) for the 26mer element in the procyclin 3′-UTR (14) is used to model the related region in the COX V 3′-UTR. The lower structures represent the centroid consensus derived by s-fold structure prediction (43) covering the same region. Asterisks indicate the UAUUUUUU RNA sequence of the core element. (C) Frequency (expressed as a percentage of genes within each group) of the 26mer core element [TATTTTTT] in the 300 nt downstream from all genes in the trypanosome genome, transcripts enriched in bloodstream forms (BSF-enriched), or transcripts enriched in procyclic forms (PCF-enriched). Expression data were derived from microarray data kindly provided by Christine Clayton, University of Heidelberg.
Figure 9
Figure 9
(A) The 3′-UTR of COX transcripts conserve the oligonucleotide consensus UAG (G) UA (G/U). The 3′-UTR of COX subunits identified in T.brucei and by bioinformatic interrogation of the incomplete T.congolense genome were analysed for overrepresented oligonucleotide sequences. The occurrence of variants of the identified consensus is shown for each transcript, some genes containing multiple representatives. (B) The frequency of predicted mitochondrially located proteins containing the conserved oligonucleotide in either bloodstream or procyclic-enriched transcripts. Mitochondrial location was determined by a combination of PSORT II and manual analysis.
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References

    1. Moore M.J. From birth to death: the complex lives of eukaryotic mRNAs. Science. 2005;309:1514–1518. - PubMed
    1. Gerber A.P., Herschlag D., Brown P.O. Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS Biol. 2004;2:E79. - PMC - PubMed
    1. van Helden J., Andre B., Collado-Vides J. Extracting regulatory sites from the upstream region of yeast genes by computational analysis of oligonucleotide frequencies. J. Mol. Biol. 1998;281:827–842. - PubMed
    1. Jacobs Anderson J.S., Parker R. Computational identification of cis-acting elements affecting post-transcriptional control of gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 2000;28:1604–1617. - PMC - PubMed
    1. Vickerman K. Developmental cycles and biology of pathogenic trypanosomes. Br. Med. Bull. 1985;41:105–114. - PubMed

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