Belak ZR et al. (2018), Conserved RNA binding activity of a Yin-Yang 1 ...

ECB-ART-46353

Sci Rep 2018 May 23;81:8061. doi: 10.1038/s41598-018-26264-0.

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Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus.

Belak ZR , Ovsenek N , Eskiw CH .

Abstract
Yin-Yang 1 (YY1) is a highly conserved transcription factor possessing RNA-binding activity. A putative YY1 homologue was previously identified in the developmental model organism Strongylocentrotus purpuratus (the purple sea urchin) by genomic sequencing. We identified a high degree of sequence similarity with YY1 homologues of vertebrate origin which shared 100% protein sequence identity over the DNA- and RNA-binding zinc-finger region with high similarity in the N-terminal transcriptional activation domain. SpYY1 demonstrated identical DNA- and RNA-binding characteristics between Xenopus laevis and S. purpuratus indicating that it maintains similar functional and biochemical properties across widely divergent deuterostome species. SpYY1 binds to the consensus YY1 DNA element, and also to U-rich RNA sequences. Although we detected SpYY1 RNA-binding activity in ova lysates and observed cytoplasmic localization, SpYY1 was not associated with maternal mRNA in ova. SpYY1 expressed in Xenopus oocytes was excluded from the nucleus and associated with maternally expressed cytoplasmic mRNA molecules. These data demonstrate the existence of an YY1 homologue in S. purpuratus with similar structural and biochemical features to those of the well-studied vertebrate YY1; however, the data reveal major differences in the biological role of YY1 in the regulation of maternally expressed mRNA in the two species.

PubMed ID: 29795182
PMC ID: PMC5966398
Article link: Sci Rep

Species referenced: Echinodermata
Genes referenced: LOC100887844 LOC115918456 LOC115919910 LOC583082 LOC590297 LOC594261 yy1
Antibodies: LOC754126 Ab3 potekp1 Ab2 yy1 Ab1

Article Images: [+] show captions

	Figure 1. Multiple Sequence Alignment of SpYY1. Results of multiple sequence alignment of YY1 homologues from a diverse set of species using ClustalX version 2.1 software.
	Figure 2. Phylogenetic Comparison of YY1 from Assorted Metazoan Species. A phylogenetic tree comparing YY1 homologues from a diverse set of species was constructed using the drawgram program from the PHYLIP version 3.69 software package using the default parameters.
	Figure 3. Expression of Strongylocentrotus purpuratus YY1 in E. coli. The S. purpuratus YY1 coding sequence was cloned into the bacterial expression vector pRsetB to yield pRSetB-SpYY1. Both empty vector (pRsetB) and the construct pRsetB-SpYY1 were then transformed into BL21(DE3)pLysS E. coli and expression of exogenous protein was induced by addition of IPTG. The expressed SpYY1 protein was then purified using immobilized metal affinity chromatography according to previously described procedures. Bacterial lysates (pRsetB, pRsetB-SpYY1; above each panel) as well as the purified protein preparation (Purified SpYY1; above each panel) were analyzed by SDS-PAGE and Western blotting. (A) SDS-PAGE gel stained with Coomassie brilliant blue. (B) Western blot of bacterial lysates and purified protein probed with anti-Xenopus YY1 antibody. Positions of molecular mass markers are indicated to the left of each panel.
	Figure 4. DNA and RNA binding activity of recombinant SpYY1 and SpYY1 in S. purpuratus ovum lysates. Bacterially expressed and purified S. purpuratus YY1 (250 nM) was combined with 10 nM radiolabelled YY1 DNA consensus probe (A, upper panel) or 10 nM end-labelled U(20) RNA probe (B, lower panel) and various competitor oligonucleotides as indicated, and analyzed by EMSA. For analysis of DNA binding activity in ova, either untreated (−RNase) or RNase treated (+RNase) lysates of S. purpuratus ova were analyzed in EMSA reactions containing 10 nM radiolabelled YY1 DNA consensus probe and various competitor oligonucleotides as indicated above the panel (C). Competitors are indicated above the panel: SpYY1, YY1 protein only, no competitor; +YY1C, 100 nM (10X excess) unlabelled YY1 DNA consensus oligonucleotide; +YY1M, 100 nM (10X excess) unlabelled YY1 DNA mutant consensus oligonucleotide; +U(20), 100 nM (10X excess) unlabelled U(20) RNA; +C(20), 100 nM (10X excess) unlabelled C(20) RNA. The positions of YY1/DNA and YY1/RNA complexes and unbound RNA oligonucleotide (free probe, FP) are indicated to the left of the panels.
	Figure 5. RNA binding activity of YY1 in S. purpuratus ovum lysates. Either untreated (−RNase) or RNase treated (+RNase) lysates of S. purpuratus ova were analyzed in EMSA reactions containing 10 nM radiolabelled poly-U(20) RNA probe and various competitor oligonucleotides as indicated above the panel. Competitors were: +YY1C, 100 nM (10X excess) unlabelled YY1 DNA consensus oligonucleotide; +YY1M, 100 nM (10X excess) unlabelled YY1 DNA mutant consensus oligonucleotide; +U(20), 100 nM (10X excess) unlabelled U(20) RNA; +C(20), 100 nM (10X excess) unlabelled C(20) RNA. The positions of the YY1/RNA (YY1) and non-specific (NS) complexes, as well as free probe (FP) are indicated to the left of the panel.
	Figure 6. Isolation of Messenger Ribonucleoprotein Particles from S. purpuratus ova. Lysates of S. purpuratus ova were either untreated (−RNase) or treated with RNase A/T1 (+RNase) and applied to oligo-dT cellulose columns and the bound proteins analyzed by SDS-PAGE and visualized by staining with Coomassie brilliant blue (upper panel) or subjected to Western blotting with anti-Xenopus YY1 antibody (lower panel). Fractions are indicated above the panel, L, load-on; U, unbound fraction; B, bound fraction. Positions of molecular mass markers and the YY1 band found upon Western blotting are indicated to the left of the panel.
	Figure 7. Nucleocytoplasmic Distribution of SpYY1 in S. purpuratus ova. Ova of S. purpuratus were fractionated into nuclear and cytoplasmic extracts using previously described methods. Whole-cell (W) as well as nuclear (N) and cytoplasmic (C) fractions were analyzed by SDS-PAGE followed by Western blotting. Antibodies used are indicated to the left of the panel, αYY1, Rabbit anti-Xenopus YY1; αH3, Goat anti-Histone H3 antibody, nuclear marker; αActin, Goat anti-Beta-Actin antibody, cytoplasmic marker.
	Figure 8. SpYY1 associates with mRNA and mRNPs in Xenopus oocytes. HA-SpYY1 was expressed in Xenopus oocytes via nuclear microinjection of a CMV-promoter-driven plasmid construct. Following 16 h of expression, oocytes were subjected to analysis. (A) HA-SpYY1-expressing oocyte lysates prepared in the presence of RNase inhibitor (−RNase, upper panel) or treated with RNaseA/T1 mixture (+RNase, lower panel) were subjected to oligo-dT cellulose chromatography, and aliquots of the load-on lysate (L), unbound fraction (U), and the bound fraction (B) were analyzed by Western blotting using an anti-HA antibody. (B) HA-SpYY1-expressing oocytes were lysed to produce whole-cell (W) lysates, or manually enucleated to isolate nuclear (N) and cytoplasmic (C) fractions as indicated above the panels and analyzed by Western blotting with anti-HA, anti-IκB (cytoplasmic marker), or anti-PCNA (nuclear marker) antibodies as indicated to the left of the panels.

References [+] :

Atchison, Function of YY1 in Long-Distance DNA Interactions. 2014, Pubmed