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Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains.

Dattoli AA, Hink MA, DuBuc TQ, Teunisse BJ, Goedhart J, Röttinger E, Postma M - Sci Rep (2015)

Bottom Line: Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility.Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility.Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam Science Park 904, NL-1098 XH Amsterdam The Netherlands.

ABSTRACT
SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

No MeSH data available.


Related in: MedlinePlus

A) Phylogenetic tree of related Scratch and Snail zinc-finger proteins. Thistree was based on the alignment of the sequence region that comprised thefive zinc-finger domains (see Methods). C2H2 motif prediction was performedon all sequences using SMART and the proteins were colour coded based on thepredicted first zinc-finger domain. Proteins in black are predicted to havethe first zinc-finger domain, comprising a total of five zinc-fingerdomains. Proteins in red are predicted to have only four zinc-fingerdomains, hence missing the first zinc-finger domain. The first zinc-fingerof NvSNAILB (orange) is weakly recognized (E-value 0.25; seesupplementary dataset 4 for SMART predictions). This analysis shows that thenumber of zinc fingers among snail proteins is variable across species andindicates multiple duplication events, zinc-finger domain loss and geneloss. Phylogenetic analysis. C2H2 zinc-finger domains where predictedusing the Simple Modular Architecture Research; Multiple sequence alignmentwas performed with Muscle 3.8.31 software58 and weresubsequently manually improved (in MacVector 11.0.2). Handling of themultiple sequence alignment was done using jalview 2.7 software59. Tree reconstruction was performed with MR.BAYES360, the consensus tree shown was based on 2,000,000generations. Trichoplax adhaerens (Ta) SNAIL (XP_002108251) has beenraced out from Trichoplax adhaerens coding DNA for this study (see primersequences in supplementary information). B) Proposed evolutionaryhistory of Snail. A proto SNAIL/SCRATCH gene was duplicated early duringevolution (Placozoan-cnidarian-bilaterian) ancestor, giving rise to twogenes, of which one evolved into the Scratch family and the other into theSnail family. A subsequent duplication event gave rise to two snailgenes, of which one lost the first zinc-finger domain and the secondretained the first zinc-finger domain. Nematostella vectensiscomprises two snail genes, SNAILA and SNAILB, of which the latterstill has a partial first zinc-finger domain but was not completely lost.This can indicate that Nematostella vectensis lost the gene with onlyfour zinc-finger domains and underwent another duplication event with thefive zinc-finger domains SNAIL or that the first zinc-finger domain ofNvSNAILB more slowly evolved compared to other species.
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f2: A) Phylogenetic tree of related Scratch and Snail zinc-finger proteins. Thistree was based on the alignment of the sequence region that comprised thefive zinc-finger domains (see Methods). C2H2 motif prediction was performedon all sequences using SMART and the proteins were colour coded based on thepredicted first zinc-finger domain. Proteins in black are predicted to havethe first zinc-finger domain, comprising a total of five zinc-fingerdomains. Proteins in red are predicted to have only four zinc-fingerdomains, hence missing the first zinc-finger domain. The first zinc-fingerof NvSNAILB (orange) is weakly recognized (E-value 0.25; seesupplementary dataset 4 for SMART predictions). This analysis shows that thenumber of zinc fingers among snail proteins is variable across species andindicates multiple duplication events, zinc-finger domain loss and geneloss. Phylogenetic analysis. C2H2 zinc-finger domains where predictedusing the Simple Modular Architecture Research; Multiple sequence alignmentwas performed with Muscle 3.8.31 software58 and weresubsequently manually improved (in MacVector 11.0.2). Handling of themultiple sequence alignment was done using jalview 2.7 software59. Tree reconstruction was performed with MR.BAYES360, the consensus tree shown was based on 2,000,000generations. Trichoplax adhaerens (Ta) SNAIL (XP_002108251) has beenraced out from Trichoplax adhaerens coding DNA for this study (see primersequences in supplementary information). B) Proposed evolutionaryhistory of Snail. A proto SNAIL/SCRATCH gene was duplicated early duringevolution (Placozoan-cnidarian-bilaterian) ancestor, giving rise to twogenes, of which one evolved into the Scratch family and the other into theSnail family. A subsequent duplication event gave rise to two snailgenes, of which one lost the first zinc-finger domain and the secondretained the first zinc-finger domain. Nematostella vectensiscomprises two snail genes, SNAILA and SNAILB, of which the latterstill has a partial first zinc-finger domain but was not completely lost.This can indicate that Nematostella vectensis lost the gene with onlyfour zinc-finger domains and underwent another duplication event with thefive zinc-finger domains SNAIL or that the first zinc-finger domain ofNvSNAILB more slowly evolved compared to other species.

Mentions: To be able to characterize the cellular behaviour of SNAILA and, B, we first madea detailed comparison of structures and sequences of snail orthologs from theanimal kingdom. Snail proteins belong to the Snail superfamily, which includesalso Scratch and Slug proteins. They usually are (with few exceptions) about 270amino acids long; their N-terminus (generally the first 20 amino acids) exhibitshigh variability across species. It usually comprises the N-terminal SNAG domain(Fig. 1A,B), represented by a short sequence of nineamino acids (amino acids 1–9) and also several positively chargedresidues reported to be involved in nuclear localisation2122.The SNAG domain has been reported to be involved in interactions with cofactors,specifically the arginine in 3rd position and the serine in4th seem to be specifically involved in transcriptionalactivity through interaction with cofactors whereas arginine and lysine at the8th and 9th position have been reported alsoto act as a nuclear localisation signal (NLS)12232224. Thealignment shown in Fig. 1 (Fig. 1B)shows the high conservation in the SNAG domain between cnidarians andvertebrates. Both NvSNAILA and NvSNAILB comprise a conserved SNAGdomain and also several positively charged residues at the N-terminal.Furthermore, all proteins consist of one or more short sequences of positivelycharged lysine (K) or arginine (R), which may act as a putative NLS. Forvertebrates such as humans, mice and amphibians some of these overlap with theSNAG domain (R3, K/R8K9 in orange) and others are located in position 15 and 16of the N-terminal of snail proteins (KK or RK shown in pink)2122. Also the cnidarian representatives still have the same putative NLS sequencebut they are found at different positions, for instance K13–14 forNvSNAILA and K10- R13 for NvSNAILB instead of being located atresidue 15–16. Due to the high variability of the N-terminal domain,it is often excluded from phylogenetic analysis11, and rather theC-terminal of the protein comprising the zinc-finger domains (generally startingfrom amino acid 120) is more suitable. Snail family members all possess at leastfour zinc-finger domains (Znf II-V), and often a fifth zinc-finger domain (ZnfI) is found N-terminally to Znf II. The four zinc-finger domains at theC-terminus are reported as essential for DNA binding and also comprise a nuclearlocalisation signal2225. In order to carefully group snailfamily proteins, we constructed a tree based on 69 snail super-family proteinscomprising 49 snail and 20 scratch proteins (Fig. 2A)using the alignment of the region that covers the five zinc–fingerdomains (see supplementary dataset 2,3 for accession numbers and sequencealignment). We used the Simple Modular Architecture Research Tool (SMART,26) to predict the presence of zinc fingers in each protein (seesupplementary information for SMART E-values); the scratch proteins were used asan out-group. From the data obtained, cnidarians are the first animals to haveduplicated snail isoforms and interestingly, the sea anemone Nematostellavectensis has two snail proteins previously stated as having both fivezinc-finger domains: SNAILA and SNAILB (Znf I-V)1127. Finally,vertebrates have at least three SNAIL representatives. In this phylum, among thesnail proteins, fish, mice, frogs and humans have one snail representativecontaining four zinc-finger domains (Znf II-V). Similarly to what has beenpreviously shown11, these results show that animals usually have(at minimum) a single snail gene with four zinc-finger domains and a second genewith five zinc-finger domains. It is likely that the loss of the firstzinc-finger domain occurred in several lineages over time. Both NvSNAILAand B have been previously described as SNAIL proteins containing five zincfingers. However, since loss of the first zinc seems to be occurred severaltimes across the evolution we decided to look more carefully at the sequences ofNvSNAILA, B compared to vertebrates SNAILs respectively possessing 4and 5 zinc finger. As shown in the alignment (Fig. 1C) thefirst zinc-finger domain of NvSNAILB is different compared toNvSNAILA and HsSNAIL 1 and 2 used as vertebrate representativeswith 4 and 5 zinc fingers, respectively and appears to be partial as the secondhistidine is lacking. Together these results indicate that NvSNAILA and Bshow high conservation of the SNAG, putative NLS and zinc finger domainscompared to vertebrates, however despite being often characterized as a fivezinc-finger domains SNAIL, NvSNAILB might actually have lost the firstfinger.


Domain analysis of the Nematostella vectensis SNAIL ortholog reveals unique nucleolar localization that depends on the zinc-finger domains.

Dattoli AA, Hink MA, DuBuc TQ, Teunisse BJ, Goedhart J, Röttinger E, Postma M - Sci Rep (2015)

A) Phylogenetic tree of related Scratch and Snail zinc-finger proteins. Thistree was based on the alignment of the sequence region that comprised thefive zinc-finger domains (see Methods). C2H2 motif prediction was performedon all sequences using SMART and the proteins were colour coded based on thepredicted first zinc-finger domain. Proteins in black are predicted to havethe first zinc-finger domain, comprising a total of five zinc-fingerdomains. Proteins in red are predicted to have only four zinc-fingerdomains, hence missing the first zinc-finger domain. The first zinc-fingerof NvSNAILB (orange) is weakly recognized (E-value 0.25; seesupplementary dataset 4 for SMART predictions). This analysis shows that thenumber of zinc fingers among snail proteins is variable across species andindicates multiple duplication events, zinc-finger domain loss and geneloss. Phylogenetic analysis. C2H2 zinc-finger domains where predictedusing the Simple Modular Architecture Research; Multiple sequence alignmentwas performed with Muscle 3.8.31 software58 and weresubsequently manually improved (in MacVector 11.0.2). Handling of themultiple sequence alignment was done using jalview 2.7 software59. Tree reconstruction was performed with MR.BAYES360, the consensus tree shown was based on 2,000,000generations. Trichoplax adhaerens (Ta) SNAIL (XP_002108251) has beenraced out from Trichoplax adhaerens coding DNA for this study (see primersequences in supplementary information). B) Proposed evolutionaryhistory of Snail. A proto SNAIL/SCRATCH gene was duplicated early duringevolution (Placozoan-cnidarian-bilaterian) ancestor, giving rise to twogenes, of which one evolved into the Scratch family and the other into theSnail family. A subsequent duplication event gave rise to two snailgenes, of which one lost the first zinc-finger domain and the secondretained the first zinc-finger domain. Nematostella vectensiscomprises two snail genes, SNAILA and SNAILB, of which the latterstill has a partial first zinc-finger domain but was not completely lost.This can indicate that Nematostella vectensis lost the gene with onlyfour zinc-finger domains and underwent another duplication event with thefive zinc-finger domains SNAIL or that the first zinc-finger domain ofNvSNAILB more slowly evolved compared to other species.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507178&req=5

f2: A) Phylogenetic tree of related Scratch and Snail zinc-finger proteins. Thistree was based on the alignment of the sequence region that comprised thefive zinc-finger domains (see Methods). C2H2 motif prediction was performedon all sequences using SMART and the proteins were colour coded based on thepredicted first zinc-finger domain. Proteins in black are predicted to havethe first zinc-finger domain, comprising a total of five zinc-fingerdomains. Proteins in red are predicted to have only four zinc-fingerdomains, hence missing the first zinc-finger domain. The first zinc-fingerof NvSNAILB (orange) is weakly recognized (E-value 0.25; seesupplementary dataset 4 for SMART predictions). This analysis shows that thenumber of zinc fingers among snail proteins is variable across species andindicates multiple duplication events, zinc-finger domain loss and geneloss. Phylogenetic analysis. C2H2 zinc-finger domains where predictedusing the Simple Modular Architecture Research; Multiple sequence alignmentwas performed with Muscle 3.8.31 software58 and weresubsequently manually improved (in MacVector 11.0.2). Handling of themultiple sequence alignment was done using jalview 2.7 software59. Tree reconstruction was performed with MR.BAYES360, the consensus tree shown was based on 2,000,000generations. Trichoplax adhaerens (Ta) SNAIL (XP_002108251) has beenraced out from Trichoplax adhaerens coding DNA for this study (see primersequences in supplementary information). B) Proposed evolutionaryhistory of Snail. A proto SNAIL/SCRATCH gene was duplicated early duringevolution (Placozoan-cnidarian-bilaterian) ancestor, giving rise to twogenes, of which one evolved into the Scratch family and the other into theSnail family. A subsequent duplication event gave rise to two snailgenes, of which one lost the first zinc-finger domain and the secondretained the first zinc-finger domain. Nematostella vectensiscomprises two snail genes, SNAILA and SNAILB, of which the latterstill has a partial first zinc-finger domain but was not completely lost.This can indicate that Nematostella vectensis lost the gene with onlyfour zinc-finger domains and underwent another duplication event with thefive zinc-finger domains SNAIL or that the first zinc-finger domain ofNvSNAILB more slowly evolved compared to other species.
Mentions: To be able to characterize the cellular behaviour of SNAILA and, B, we first madea detailed comparison of structures and sequences of snail orthologs from theanimal kingdom. Snail proteins belong to the Snail superfamily, which includesalso Scratch and Slug proteins. They usually are (with few exceptions) about 270amino acids long; their N-terminus (generally the first 20 amino acids) exhibitshigh variability across species. It usually comprises the N-terminal SNAG domain(Fig. 1A,B), represented by a short sequence of nineamino acids (amino acids 1–9) and also several positively chargedresidues reported to be involved in nuclear localisation2122.The SNAG domain has been reported to be involved in interactions with cofactors,specifically the arginine in 3rd position and the serine in4th seem to be specifically involved in transcriptionalactivity through interaction with cofactors whereas arginine and lysine at the8th and 9th position have been reported alsoto act as a nuclear localisation signal (NLS)12232224. Thealignment shown in Fig. 1 (Fig. 1B)shows the high conservation in the SNAG domain between cnidarians andvertebrates. Both NvSNAILA and NvSNAILB comprise a conserved SNAGdomain and also several positively charged residues at the N-terminal.Furthermore, all proteins consist of one or more short sequences of positivelycharged lysine (K) or arginine (R), which may act as a putative NLS. Forvertebrates such as humans, mice and amphibians some of these overlap with theSNAG domain (R3, K/R8K9 in orange) and others are located in position 15 and 16of the N-terminal of snail proteins (KK or RK shown in pink)2122. Also the cnidarian representatives still have the same putative NLS sequencebut they are found at different positions, for instance K13–14 forNvSNAILA and K10- R13 for NvSNAILB instead of being located atresidue 15–16. Due to the high variability of the N-terminal domain,it is often excluded from phylogenetic analysis11, and rather theC-terminal of the protein comprising the zinc-finger domains (generally startingfrom amino acid 120) is more suitable. Snail family members all possess at leastfour zinc-finger domains (Znf II-V), and often a fifth zinc-finger domain (ZnfI) is found N-terminally to Znf II. The four zinc-finger domains at theC-terminus are reported as essential for DNA binding and also comprise a nuclearlocalisation signal2225. In order to carefully group snailfamily proteins, we constructed a tree based on 69 snail super-family proteinscomprising 49 snail and 20 scratch proteins (Fig. 2A)using the alignment of the region that covers the five zinc–fingerdomains (see supplementary dataset 2,3 for accession numbers and sequencealignment). We used the Simple Modular Architecture Research Tool (SMART,26) to predict the presence of zinc fingers in each protein (seesupplementary information for SMART E-values); the scratch proteins were used asan out-group. From the data obtained, cnidarians are the first animals to haveduplicated snail isoforms and interestingly, the sea anemone Nematostellavectensis has two snail proteins previously stated as having both fivezinc-finger domains: SNAILA and SNAILB (Znf I-V)1127. Finally,vertebrates have at least three SNAIL representatives. In this phylum, among thesnail proteins, fish, mice, frogs and humans have one snail representativecontaining four zinc-finger domains (Znf II-V). Similarly to what has beenpreviously shown11, these results show that animals usually have(at minimum) a single snail gene with four zinc-finger domains and a second genewith five zinc-finger domains. It is likely that the loss of the firstzinc-finger domain occurred in several lineages over time. Both NvSNAILAand B have been previously described as SNAIL proteins containing five zincfingers. However, since loss of the first zinc seems to be occurred severaltimes across the evolution we decided to look more carefully at the sequences ofNvSNAILA, B compared to vertebrates SNAILs respectively possessing 4and 5 zinc finger. As shown in the alignment (Fig. 1C) thefirst zinc-finger domain of NvSNAILB is different compared toNvSNAILA and HsSNAIL 1 and 2 used as vertebrate representativeswith 4 and 5 zinc fingers, respectively and appears to be partial as the secondhistidine is lacking. Together these results indicate that NvSNAILA and Bshow high conservation of the SNAG, putative NLS and zinc finger domainscompared to vertebrates, however despite being often characterized as a fivezinc-finger domains SNAIL, NvSNAILB might actually have lost the firstfinger.

Bottom Line: Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility.Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility.Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam Science Park 904, NL-1098 XH Amsterdam The Netherlands.

ABSTRACT
SNAIL transcriptional factors are key regulators during development and disease. They arose early during evolution, and in cnidarians such as Nematostella vectensis, NvSNAILA/B are detected in invaginating tissues during gastrulation. The function of SNAIL proteins is well established in bilaterians but their roles in cnidarians remain unknown. The structure of NvSNAILA and B is similar to the human SNAIL1 and 2, including SNAG and zinc-finger domains. Here, we performed a molecular analysis on localization and mobility of NvSNAILA/B using mammalian cells and Nematostella embryos. NvSNAILA/B display nuclear localization and mobility similar to HsSNAIL1/2. Strikingly, NvSNAILA is highly enriched in the nucleoli and shuttles between the nucleoli and the nucleoplasm. Truncation of the N-terminal SNAG domain, reported to contain Nuclear Localization Signals, markedly reduces nucleolar levels, without effecting nuclear localization or mobility. Truncation of the C-terminal zinc-fingers, involved in DNA binding in higher organisms, significantly affects subcellular localization and mobility. Specifically, the zinc-finger domains are required for nucleolar enrichment of NvSNAILA. Differently from SNAIL transcriptional factors described before, NvSNAILA is specifically enriched in the nucleoli co-localizing with nucleolar markers even after nucleolar disruption. Our findings implicate additional roles for SNAG and zinc-finger domains, suggesting a role for NvSNAILA in the nucleolus.

No MeSH data available.


Related in: MedlinePlus