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
Cis-regulatory elements control differential HuB mRNA stability in the soma and germ cells.(A) 3′UTR sequence of the B deletion reporter. Base substitutions were made in 10 nucleotide blocks to create the B substitution mutants Bs1–Bs14. Each 10 nucleotide block was substituted with the sequence GAGGATCCGA. (B) Expression of EGFP and mCherry protein in embryos injected with the indicated Bs substitution mutant reporters. 200 pg of each reporter was injected. All EGFP images were taken at 2000 ms, and all mCherry images were taken at 150 ms. Higher magnification images of mCherry expression in the germ cells are shown as insets. (C) Whole-mount in situ hybridisation using an antisense probe directed against the mCherry coding sequence. Higher magnification images of germ cells are shown as insets.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3113899&req=5

pone-0020773-g004: Cis-regulatory elements control differential HuB mRNA stability in the soma and germ cells.(A) 3′UTR sequence of the B deletion reporter. Base substitutions were made in 10 nucleotide blocks to create the B substitution mutants Bs1–Bs14. Each 10 nucleotide block was substituted with the sequence GAGGATCCGA. (B) Expression of EGFP and mCherry protein in embryos injected with the indicated Bs substitution mutant reporters. 200 pg of each reporter was injected. All EGFP images were taken at 2000 ms, and all mCherry images were taken at 150 ms. Higher magnification images of mCherry expression in the germ cells are shown as insets. (C) Whole-mount in situ hybridisation using an antisense probe directed against the mCherry coding sequence. Higher magnification images of germ cells are shown as insets.

Mentions: To further identify the sequence elements within the B region that are important for the post-transcriptional regulation of the HuB 3′UTR, a substitution mutagenesis approach was taken. Sequential 10-nucleotide blocks of the B region were substituted with the sequence GAGGAUCCGA. In total, 14 mutants were made, termed Bs1 to Bs14 as indicated (Fig. 4A). The Bs substitution mutant RNAs were each injected into 1-cell stage embryos and compared to the activity of the wild type B reporter. Several substitution mutations caused an increase in mCherry expression (Fig. 4B) and RNA stability (Fig. 4C) in the somatic tissue, implying that these substitutions must be disrupting sequence elements which are normally important for somatic instability of the HuB 3′UTR. Most notably, these were Bs2, Bs4, Bs7 and Bs8 (Fig. 4B). Bs2 and Bs4 share an almost identical sequence motif (UCUUUGUGU and UCUUUAUGU respectively) which possibly represent multiple binding sites for the same microRNA or RNA-binding protein. Bs7 and Bs8 do not share this exact sequence motif, but Bs8 is clearly U-rich like Bs2 and Bs4, and may also share a common binding site. Alternatively, given that Bs7 and Bs8 are directly adjacent to each other, they may possibly encompass a single binding site for a destabilising factor that may or may not be the same as that interacting with Bs2 and Bs4. MicroRNA miR-430 is known to promote the deadenylation and clearance of other germ cell specific mRNAs, such as nanos, TDRD7, and dazl, in the somatic tissue of zebrafish [6], [8], however a canonical miR-430 binding site is not present in the HuB 3′UTR. No known zebrafish microRNAs [21], [22] were found to have seed sequence homology to any of the destabilising elements identified, and therefore the mechanism of HuB somatic instability remains unclear.


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)

Cis-regulatory elements control differential HuB mRNA stability in the soma and germ cells.(A) 3′UTR sequence of the B deletion reporter. Base substitutions were made in 10 nucleotide blocks to create the B substitution mutants Bs1–Bs14. Each 10 nucleotide block was substituted with the sequence GAGGATCCGA. (B) Expression of EGFP and mCherry protein in embryos injected with the indicated Bs substitution mutant reporters. 200 pg of each reporter was injected. All EGFP images were taken at 2000 ms, and all mCherry images were taken at 150 ms. Higher magnification images of mCherry expression in the germ cells are shown as insets. (C) Whole-mount in situ hybridisation using an antisense probe directed against the mCherry coding sequence. Higher magnification images of germ cells are shown as insets.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020773-g004: Cis-regulatory elements control differential HuB mRNA stability in the soma and germ cells.(A) 3′UTR sequence of the B deletion reporter. Base substitutions were made in 10 nucleotide blocks to create the B substitution mutants Bs1–Bs14. Each 10 nucleotide block was substituted with the sequence GAGGATCCGA. (B) Expression of EGFP and mCherry protein in embryos injected with the indicated Bs substitution mutant reporters. 200 pg of each reporter was injected. All EGFP images were taken at 2000 ms, and all mCherry images were taken at 150 ms. Higher magnification images of mCherry expression in the germ cells are shown as insets. (C) Whole-mount in situ hybridisation using an antisense probe directed against the mCherry coding sequence. Higher magnification images of germ cells are shown as insets.
Mentions: To further identify the sequence elements within the B region that are important for the post-transcriptional regulation of the HuB 3′UTR, a substitution mutagenesis approach was taken. Sequential 10-nucleotide blocks of the B region were substituted with the sequence GAGGAUCCGA. In total, 14 mutants were made, termed Bs1 to Bs14 as indicated (Fig. 4A). The Bs substitution mutant RNAs were each injected into 1-cell stage embryos and compared to the activity of the wild type B reporter. Several substitution mutations caused an increase in mCherry expression (Fig. 4B) and RNA stability (Fig. 4C) in the somatic tissue, implying that these substitutions must be disrupting sequence elements which are normally important for somatic instability of the HuB 3′UTR. Most notably, these were Bs2, Bs4, Bs7 and Bs8 (Fig. 4B). Bs2 and Bs4 share an almost identical sequence motif (UCUUUGUGU and UCUUUAUGU respectively) which possibly represent multiple binding sites for the same microRNA or RNA-binding protein. Bs7 and Bs8 do not share this exact sequence motif, but Bs8 is clearly U-rich like Bs2 and Bs4, and may also share a common binding site. Alternatively, given that Bs7 and Bs8 are directly adjacent to each other, they may possibly encompass a single binding site for a destabilising factor that may or may not be the same as that interacting with Bs2 and Bs4. MicroRNA miR-430 is known to promote the deadenylation and clearance of other germ cell specific mRNAs, such as nanos, TDRD7, and dazl, in the somatic tissue of zebrafish [6], [8], however a canonical miR-430 binding site is not present in the HuB 3′UTR. No known zebrafish microRNAs [21], [22] were found to have seed sequence homology to any of the destabilising elements identified, and therefore the mechanism of HuB somatic instability remains unclear.

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