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A conserved U-rich RNA region implicated in regulation of translation in Plasmodium female gametocytes.

Braks JA, Mair GR, Franke-Fayard B, Janse CJ, Waters AP - Nucleic Acids Res. (2007)

Bottom Line: A U-rich, TR-associated element, identified previously in the 3'-UTR of TR-associated transcripts, played an essential role in mediating TR and a similar region could be found in the 5'-UTR shown in this study to be active in TR.The silencing effect of this 5'-UTR was shown to be independent of its position relative to its ORF, as transposition to a location 3' of the ORF did not affect TR.These results demonstrate for the first time in a unicellular organism that the 5' or the 3'-UTR of TR-associated transcripts play an important and conserved role in mediating TR in female gametocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Centre of Infectious Diseases, Leiden University Medical Centre (LUMC), Leiden, The Netherlands.

ABSTRACT
Translational repression (TR) plays an important role in post-transcriptional regulation of gene expression and embryonic development in metazoans. TR also regulates the expression of a subset of the cytoplasmic mRNA population during development of fertilized female gametes of the unicellular malaria parasite, Plasmodium spp. which results in the formation of a polar and motile form, the ookinete. We report the conserved and sex-specific regulatory role of either the 3'- or 5'-UTR of a subset of translationally repressed mRNA species as shown by almost complete inhibition of expression of a GFP reporter protein in the female gametocyte. A U-rich, TR-associated element, identified previously in the 3'-UTR of TR-associated transcripts, played an essential role in mediating TR and a similar region could be found in the 5'-UTR shown in this study to be active in TR. The silencing effect of this 5'-UTR was shown to be independent of its position relative to its ORF, as transposition to a location 3' of the ORF did not affect TR. These results demonstrate for the first time in a unicellular organism that the 5' or the 3'-UTR of TR-associated transcripts play an important and conserved role in mediating TR in female gametocytes.

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TR regulatory sequences exist in the 5′-UTR of pb25. (A) Cartoons of the GFP expression cassettes in reporter constructs for the expression of GFP under control of the promoter/5′-UTRs of ccp2, pb28 and pb25 and the 3′-UTR region of dhfr/ts. (B) GFP expression in purified gametocytes transfected with the expression cassettes as shown in (A). Upper panel: northern analysis using a gfp probe to detect the reporter transcripts and the female gametocyte specific ccp2 probe as an internal control. Bottom panel: western analysis using GFP antibodies. Antiserum against the gametocyte-specific protein Pb47 was used as a control. (C) Relative GFP mRNA and protein expression levels in gametocytes transfected with the expression cassettes as shown in (A). GFP mRNA and protein levels were determined as described for Figure 2E. The GFP protein, mRNA and protein/mRNA level in each parasite line is presented relative to the level obtained with the control reporter strain expressing GFP under control of the ccp2 5′ region and the dhfr/ts 3′-UTR. (D) The two panels each show: Top, cartoons of the transgene introduced into P. berghei and bottom, FACS analysis of purified gametocytes transfected with reporter constructs expressing GFP under control of a 3′-UTR comprising 350 bp of the 5′-UTR of pb25 in the right (5′pb25) and reversed (5′pb25rev) orientation. GFP fluorescence intensity of gametocytes was measured as described in Figure 2C. (E) Northern analysis of steady state levels of mRNA from purified gametocytes transfected with reporter constructs expressing gfp under control of the ccp2 5′ region and the 3′-UTRs of pb28, ccp2 and ccp2 with insertions of the 5′-UTR of pb25. The ccp2 5′ region was used as a probe as described for Figure 2D. (F) Conceptually transcribed sequence of the pb25 upstream region that was inserted into the 3′-UTR of the reporter constructs as described in Figure 5D. The 40nt U-rich TR-associated elements identified by MEME are underlined.
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Figure 5: TR regulatory sequences exist in the 5′-UTR of pb25. (A) Cartoons of the GFP expression cassettes in reporter constructs for the expression of GFP under control of the promoter/5′-UTRs of ccp2, pb28 and pb25 and the 3′-UTR region of dhfr/ts. (B) GFP expression in purified gametocytes transfected with the expression cassettes as shown in (A). Upper panel: northern analysis using a gfp probe to detect the reporter transcripts and the female gametocyte specific ccp2 probe as an internal control. Bottom panel: western analysis using GFP antibodies. Antiserum against the gametocyte-specific protein Pb47 was used as a control. (C) Relative GFP mRNA and protein expression levels in gametocytes transfected with the expression cassettes as shown in (A). GFP mRNA and protein levels were determined as described for Figure 2E. The GFP protein, mRNA and protein/mRNA level in each parasite line is presented relative to the level obtained with the control reporter strain expressing GFP under control of the ccp2 5′ region and the dhfr/ts 3′-UTR. (D) The two panels each show: Top, cartoons of the transgene introduced into P. berghei and bottom, FACS analysis of purified gametocytes transfected with reporter constructs expressing GFP under control of a 3′-UTR comprising 350 bp of the 5′-UTR of pb25 in the right (5′pb25) and reversed (5′pb25rev) orientation. GFP fluorescence intensity of gametocytes was measured as described in Figure 2C. (E) Northern analysis of steady state levels of mRNA from purified gametocytes transfected with reporter constructs expressing gfp under control of the ccp2 5′ region and the 3′-UTRs of pb28, ccp2 and ccp2 with insertions of the 5′-UTR of pb25. The ccp2 5′ region was used as a probe as described for Figure 2D. (F) Conceptually transcribed sequence of the pb25 upstream region that was inserted into the 3′-UTR of the reporter constructs as described in Figure 5D. The 40nt U-rich TR-associated elements identified by MEME are underlined.

Mentions: The pb25 3′-UTR region did not exert TR, thus the possible involvement of 5′-UTRs in TR was assessed. Reporter plasmids were constructed in which gfp was flanked with the 3′ region of dhfr/ts and the 5′ region (this includes the promoter and 5′-UTR regions) of ccp2, pb28 and pb25 (Figure 5A). The 5′ regions of pb28 and ccp2 produced similar levels of steady state gfp mRNA in gametocytes of transgenic parasites but translation was much more efficient with the transcripts containing the pb28 5′-UTR (Figure 5B and C). The GFP protein/mRNA ratio was 14 times higher with the pb28 5′-UTR than with the ccp2 5′-UTR (Figure 5C and supplementary Table S4), indicating the pb28 5′-UTR transcripts were translated more efficiently. Interestingly, parasites expressing gfp with the 5′ region of pb25 produced considerably higher levels of steady state gfp mRNA than those with the ccp2 or pb28 5′ regions (20–25 times higher, Figure 5B and C). However, despite the abundance of gfp transcripts produced by the pb25 promoter, only low levels of GFP were produced (Figure 5B and C) indicating that the pb25 5′-UTR prevents efficient translation and thus mediates TR in gametocytes.Figure 5.


A conserved U-rich RNA region implicated in regulation of translation in Plasmodium female gametocytes.

Braks JA, Mair GR, Franke-Fayard B, Janse CJ, Waters AP - Nucleic Acids Res. (2007)

TR regulatory sequences exist in the 5′-UTR of pb25. (A) Cartoons of the GFP expression cassettes in reporter constructs for the expression of GFP under control of the promoter/5′-UTRs of ccp2, pb28 and pb25 and the 3′-UTR region of dhfr/ts. (B) GFP expression in purified gametocytes transfected with the expression cassettes as shown in (A). Upper panel: northern analysis using a gfp probe to detect the reporter transcripts and the female gametocyte specific ccp2 probe as an internal control. Bottom panel: western analysis using GFP antibodies. Antiserum against the gametocyte-specific protein Pb47 was used as a control. (C) Relative GFP mRNA and protein expression levels in gametocytes transfected with the expression cassettes as shown in (A). GFP mRNA and protein levels were determined as described for Figure 2E. The GFP protein, mRNA and protein/mRNA level in each parasite line is presented relative to the level obtained with the control reporter strain expressing GFP under control of the ccp2 5′ region and the dhfr/ts 3′-UTR. (D) The two panels each show: Top, cartoons of the transgene introduced into P. berghei and bottom, FACS analysis of purified gametocytes transfected with reporter constructs expressing GFP under control of a 3′-UTR comprising 350 bp of the 5′-UTR of pb25 in the right (5′pb25) and reversed (5′pb25rev) orientation. GFP fluorescence intensity of gametocytes was measured as described in Figure 2C. (E) Northern analysis of steady state levels of mRNA from purified gametocytes transfected with reporter constructs expressing gfp under control of the ccp2 5′ region and the 3′-UTRs of pb28, ccp2 and ccp2 with insertions of the 5′-UTR of pb25. The ccp2 5′ region was used as a probe as described for Figure 2D. (F) Conceptually transcribed sequence of the pb25 upstream region that was inserted into the 3′-UTR of the reporter constructs as described in Figure 5D. The 40nt U-rich TR-associated elements identified by MEME are underlined.
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Figure 5: TR regulatory sequences exist in the 5′-UTR of pb25. (A) Cartoons of the GFP expression cassettes in reporter constructs for the expression of GFP under control of the promoter/5′-UTRs of ccp2, pb28 and pb25 and the 3′-UTR region of dhfr/ts. (B) GFP expression in purified gametocytes transfected with the expression cassettes as shown in (A). Upper panel: northern analysis using a gfp probe to detect the reporter transcripts and the female gametocyte specific ccp2 probe as an internal control. Bottom panel: western analysis using GFP antibodies. Antiserum against the gametocyte-specific protein Pb47 was used as a control. (C) Relative GFP mRNA and protein expression levels in gametocytes transfected with the expression cassettes as shown in (A). GFP mRNA and protein levels were determined as described for Figure 2E. The GFP protein, mRNA and protein/mRNA level in each parasite line is presented relative to the level obtained with the control reporter strain expressing GFP under control of the ccp2 5′ region and the dhfr/ts 3′-UTR. (D) The two panels each show: Top, cartoons of the transgene introduced into P. berghei and bottom, FACS analysis of purified gametocytes transfected with reporter constructs expressing GFP under control of a 3′-UTR comprising 350 bp of the 5′-UTR of pb25 in the right (5′pb25) and reversed (5′pb25rev) orientation. GFP fluorescence intensity of gametocytes was measured as described in Figure 2C. (E) Northern analysis of steady state levels of mRNA from purified gametocytes transfected with reporter constructs expressing gfp under control of the ccp2 5′ region and the 3′-UTRs of pb28, ccp2 and ccp2 with insertions of the 5′-UTR of pb25. The ccp2 5′ region was used as a probe as described for Figure 2D. (F) Conceptually transcribed sequence of the pb25 upstream region that was inserted into the 3′-UTR of the reporter constructs as described in Figure 5D. The 40nt U-rich TR-associated elements identified by MEME are underlined.
Mentions: The pb25 3′-UTR region did not exert TR, thus the possible involvement of 5′-UTRs in TR was assessed. Reporter plasmids were constructed in which gfp was flanked with the 3′ region of dhfr/ts and the 5′ region (this includes the promoter and 5′-UTR regions) of ccp2, pb28 and pb25 (Figure 5A). The 5′ regions of pb28 and ccp2 produced similar levels of steady state gfp mRNA in gametocytes of transgenic parasites but translation was much more efficient with the transcripts containing the pb28 5′-UTR (Figure 5B and C). The GFP protein/mRNA ratio was 14 times higher with the pb28 5′-UTR than with the ccp2 5′-UTR (Figure 5C and supplementary Table S4), indicating the pb28 5′-UTR transcripts were translated more efficiently. Interestingly, parasites expressing gfp with the 5′ region of pb25 produced considerably higher levels of steady state gfp mRNA than those with the ccp2 or pb28 5′ regions (20–25 times higher, Figure 5B and C). However, despite the abundance of gfp transcripts produced by the pb25 promoter, only low levels of GFP were produced (Figure 5B and C) indicating that the pb25 5′-UTR prevents efficient translation and thus mediates TR in gametocytes.Figure 5.

Bottom Line: A U-rich, TR-associated element, identified previously in the 3'-UTR of TR-associated transcripts, played an essential role in mediating TR and a similar region could be found in the 5'-UTR shown in this study to be active in TR.The silencing effect of this 5'-UTR was shown to be independent of its position relative to its ORF, as transposition to a location 3' of the ORF did not affect TR.These results demonstrate for the first time in a unicellular organism that the 5' or the 3'-UTR of TR-associated transcripts play an important and conserved role in mediating TR in female gametocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Centre of Infectious Diseases, Leiden University Medical Centre (LUMC), Leiden, The Netherlands.

ABSTRACT
Translational repression (TR) plays an important role in post-transcriptional regulation of gene expression and embryonic development in metazoans. TR also regulates the expression of a subset of the cytoplasmic mRNA population during development of fertilized female gametes of the unicellular malaria parasite, Plasmodium spp. which results in the formation of a polar and motile form, the ookinete. We report the conserved and sex-specific regulatory role of either the 3'- or 5'-UTR of a subset of translationally repressed mRNA species as shown by almost complete inhibition of expression of a GFP reporter protein in the female gametocyte. A U-rich, TR-associated element, identified previously in the 3'-UTR of TR-associated transcripts, played an essential role in mediating TR and a similar region could be found in the 5'-UTR shown in this study to be active in TR. The silencing effect of this 5'-UTR was shown to be independent of its position relative to its ORF, as transposition to a location 3' of the ORF did not affect TR. These results demonstrate for the first time in a unicellular organism that the 5' or the 3'-UTR of TR-associated transcripts play an important and conserved role in mediating TR in female gametocytes.

Show MeSH
Related in: MedlinePlus