Limits...
HuB (elavl2) mRNA is restricted to the germ cells by post-transcriptional mechanisms including stabilisation of the message by DAZL.

Wiszniak SE, Dredge BK, Jensen KB - PLoS ONE (2011)

Bottom Line: Restriction of HuB mRNA to the germ cells is dependent on a number of sequence elements in its 3'UTR, which act to degrade the mRNA in the soma and stabilise it in the germ cells.In addition, we show that the germ cell specific RNA-binding protein DAZL is able to promote HuB mRNA stability and translation in germ cells, and further demonstrate that these activities require a 30 nucleotide element in the 3'UTR.Our study suggests that DAZL specifically binds the HuB 3'UTR and protects the message from degradation and/or enhances HuB translation, leading to the germ cell specific expression of HuB protein.

View Article: PubMed Central - PubMed

Affiliation: Discipline of Biochemistry, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia.

ABSTRACT
The ability of germ cells to carry out a gene regulatory program distinct from the surrounding somatic tissue, and their capacity to specify an entire new organism has made them a focus of many studies that seek to understand how specific regulatory mechanisms, particularly post-transcriptional mechanisms, contribute to cell fate. In zebrafish, germ cells are specified through the inheritance of cytoplasmic determinants, termed the germ plasm, which contains a number of maternal mRNAs and proteins. Investigation of several of these messages has revealed that the restricted localisation of these mRNAs to the germ plasm and subsequent germ cells is due to cis-acting sequence elements present in their 3'UTRs. Here we show that a member of the Hu family of RNA-binding proteins, HuB, is maternally provided in the zebrafish embryo and exhibits germ cell specific expression during embryogenesis. Restriction of HuB mRNA to the germ cells is dependent on a number of sequence elements in its 3'UTR, which act to degrade the mRNA in the soma and stabilise it in the germ cells. In addition, we show that the germ cell specific RNA-binding protein DAZL is able to promote HuB mRNA stability and translation in germ cells, and further demonstrate that these activities require a 30 nucleotide element in the 3'UTR. Our study suggests that DAZL specifically binds the HuB 3'UTR and protects the message from degradation and/or enhances HuB translation, leading to the germ cell specific expression of HuB protein.

Show MeSH
DAZL protein is able to stabilise the HuB mRNA.(A) Expression of EGFP and mCherry protein in embryos injected with 150 pg each of EGFP and mCherry-B reporter RNAs, either alone (labelled B), or with ubiquitous overexpression of HA-tagged DAZL, DND, HuB (400 amol RNA) or VASA (200 amol RNA) as labelled. All EGFP images were taken at 2000 ms and all mCherry images were taken at 500 ms. The fold change in the somatic expression of mCherry from the B reporter-protein combination compared to the B reporter alone is indicated as an inset number in white. (B) mCherry-BΔs91011 reporter RNA +/− overexpression of DAZL. (C) Whole-mount in situ hybridisation using an antisense probe directed against the mCherry coding sequence. Embryos at 9 hpf are shown in the left panels (sagittal view with dorsal side to the right), and embryos at 24 hpf are shown in the right panels with germ cells shown as insets.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3113899&req=5

pone-0020773-g005: DAZL protein is able to stabilise the HuB mRNA.(A) Expression of EGFP and mCherry protein in embryos injected with 150 pg each of EGFP and mCherry-B reporter RNAs, either alone (labelled B), or with ubiquitous overexpression of HA-tagged DAZL, DND, HuB (400 amol RNA) or VASA (200 amol RNA) as labelled. All EGFP images were taken at 2000 ms and all mCherry images were taken at 500 ms. The fold change in the somatic expression of mCherry from the B reporter-protein combination compared to the B reporter alone is indicated as an inset number in white. (B) mCherry-BΔs91011 reporter RNA +/− overexpression of DAZL. (C) Whole-mount in situ hybridisation using an antisense probe directed against the mCherry coding sequence. Embryos at 9 hpf are shown in the left panels (sagittal view with dorsal side to the right), and embryos at 24 hpf are shown in the right panels with germ cells shown as insets.

Mentions: In order to identify the factor responsible for stabilising HuB mRNA specifically in the germ cells, a set of candidate germ cell specific RNA-binding proteins were overexpressed in the somatic tissues of the zebrafish embryo to see whether this misexpression of a germ cell specific RNA-binding protein would lead to a subsequent increase in the somatic expression of the mCherry-B reporter RNA. The western blot of all HA-tagged overexpressed proteins confirmed that all were expressed and were of the expected size (Fig. S3). Fig. 5A shows that overexpression of DND, HuB and VASA caused a modest 1.1 to 1.5 fold increase in mCherry expression in the somatic tissue when compared to the B reporter alone. However, overexpression of DAZL caused a marked increase in somatic expression of mCherry, with a 3.4 fold increase of mCherry protein compared to the B reporter alone. A DAZL RNA-binding mutant containing a phenylalanine to alanine substitution at amino acid position 91 (DAZL F91A) [24], was tested in the same experiment and found to be unable to cause any increase in mCherry expression (Fig. 5A). DAZL overexpression was also tested in the context of the BΔs91011 reporter, and was found to be unable to lead to an increase in mCherry expression (Fig. 5B). Whole-mount in situ hybridisation demonstrates an increase in mCherry-B reporter RNA levels in the somatic tissue of 9 hpf embryos upon DAZL overexpression, which strongly suggests that DAZL acts to stabilise the mCherry-B reporter RNA (Fig. 5C). This increase in reporter RNA levels was less apparent at 24 hpf, however there was still a marked increase in reporter RNA levels in the germ cells when DAZL was overexpressed.


HuB (elavl2) mRNA is restricted to the germ cells by post-transcriptional mechanisms including stabilisation of the message by DAZL.

Wiszniak SE, Dredge BK, Jensen KB - PLoS ONE (2011)

DAZL protein is able to stabilise the HuB mRNA.(A) Expression of EGFP and mCherry protein in embryos injected with 150 pg each of EGFP and mCherry-B reporter RNAs, either alone (labelled B), or with ubiquitous overexpression of HA-tagged DAZL, DND, HuB (400 amol RNA) or VASA (200 amol RNA) as labelled. All EGFP images were taken at 2000 ms and all mCherry images were taken at 500 ms. The fold change in the somatic expression of mCherry from the B reporter-protein combination compared to the B reporter alone is indicated as an inset number in white. (B) mCherry-BΔs91011 reporter RNA +/− overexpression of DAZL. (C) Whole-mount in situ hybridisation using an antisense probe directed against the mCherry coding sequence. Embryos at 9 hpf are shown in the left panels (sagittal view with dorsal side to the right), and embryos at 24 hpf are shown in the right panels with germ cells shown as insets.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3113899&req=5

pone-0020773-g005: DAZL protein is able to stabilise the HuB mRNA.(A) Expression of EGFP and mCherry protein in embryos injected with 150 pg each of EGFP and mCherry-B reporter RNAs, either alone (labelled B), or with ubiquitous overexpression of HA-tagged DAZL, DND, HuB (400 amol RNA) or VASA (200 amol RNA) as labelled. All EGFP images were taken at 2000 ms and all mCherry images were taken at 500 ms. The fold change in the somatic expression of mCherry from the B reporter-protein combination compared to the B reporter alone is indicated as an inset number in white. (B) mCherry-BΔs91011 reporter RNA +/− overexpression of DAZL. (C) Whole-mount in situ hybridisation using an antisense probe directed against the mCherry coding sequence. Embryos at 9 hpf are shown in the left panels (sagittal view with dorsal side to the right), and embryos at 24 hpf are shown in the right panels with germ cells shown as insets.
Mentions: In order to identify the factor responsible for stabilising HuB mRNA specifically in the germ cells, a set of candidate germ cell specific RNA-binding proteins were overexpressed in the somatic tissues of the zebrafish embryo to see whether this misexpression of a germ cell specific RNA-binding protein would lead to a subsequent increase in the somatic expression of the mCherry-B reporter RNA. The western blot of all HA-tagged overexpressed proteins confirmed that all were expressed and were of the expected size (Fig. S3). Fig. 5A shows that overexpression of DND, HuB and VASA caused a modest 1.1 to 1.5 fold increase in mCherry expression in the somatic tissue when compared to the B reporter alone. However, overexpression of DAZL caused a marked increase in somatic expression of mCherry, with a 3.4 fold increase of mCherry protein compared to the B reporter alone. A DAZL RNA-binding mutant containing a phenylalanine to alanine substitution at amino acid position 91 (DAZL F91A) [24], was tested in the same experiment and found to be unable to cause any increase in mCherry expression (Fig. 5A). DAZL overexpression was also tested in the context of the BΔs91011 reporter, and was found to be unable to lead to an increase in mCherry expression (Fig. 5B). Whole-mount in situ hybridisation demonstrates an increase in mCherry-B reporter RNA levels in the somatic tissue of 9 hpf embryos upon DAZL overexpression, which strongly suggests that DAZL acts to stabilise the mCherry-B reporter RNA (Fig. 5C). This increase in reporter RNA levels was less apparent at 24 hpf, however there was still a marked increase in reporter RNA levels in the germ cells when DAZL was overexpressed.

Bottom Line: Restriction of HuB mRNA to the germ cells is dependent on a number of sequence elements in its 3'UTR, which act to degrade the mRNA in the soma and stabilise it in the germ cells.In addition, we show that the germ cell specific RNA-binding protein DAZL is able to promote HuB mRNA stability and translation in germ cells, and further demonstrate that these activities require a 30 nucleotide element in the 3'UTR.Our study suggests that DAZL specifically binds the HuB 3'UTR and protects the message from degradation and/or enhances HuB translation, leading to the germ cell specific expression of HuB protein.

View Article: PubMed Central - PubMed

Affiliation: Discipline of Biochemistry, School of Molecular and Biomedical Science, University of Adelaide, Adelaide, Australia.

ABSTRACT
The ability of germ cells to carry out a gene regulatory program distinct from the surrounding somatic tissue, and their capacity to specify an entire new organism has made them a focus of many studies that seek to understand how specific regulatory mechanisms, particularly post-transcriptional mechanisms, contribute to cell fate. In zebrafish, germ cells are specified through the inheritance of cytoplasmic determinants, termed the germ plasm, which contains a number of maternal mRNAs and proteins. Investigation of several of these messages has revealed that the restricted localisation of these mRNAs to the germ plasm and subsequent germ cells is due to cis-acting sequence elements present in their 3'UTRs. Here we show that a member of the Hu family of RNA-binding proteins, HuB, is maternally provided in the zebrafish embryo and exhibits germ cell specific expression during embryogenesis. Restriction of HuB mRNA to the germ cells is dependent on a number of sequence elements in its 3'UTR, which act to degrade the mRNA in the soma and stabilise it in the germ cells. In addition, we show that the germ cell specific RNA-binding protein DAZL is able to promote HuB mRNA stability and translation in germ cells, and further demonstrate that these activities require a 30 nucleotide element in the 3'UTR. Our study suggests that DAZL specifically binds the HuB 3'UTR and protects the message from degradation and/or enhances HuB translation, leading to the germ cell specific expression of HuB protein.

Show MeSH