Characterization of DeltaN-Zfp36l2 mutant associated with arrest of early embryonic development and female infertility

Abstract

The zinc finger protein 36-like 2, Zfp36l2, has been implicated in female mouse infertility, because an amino-terminal truncation mutation (ΔN-Zfp36l2) leads to two-cell stage arrest of embryos derived from the homozygous mutant female gamete. Zfp36l2 is a member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins that can bind to transcripts containing AU-rich elements (ARE), resulting in deadenylation and destabilization of these transcripts. I show here that the mouse Zfp36l2 is composed of two exons and a single intron, encoding a polypeptide of 484 amino acids. I observed that ΔN-Zfp36l2 protein is similar to both wild-type Zfp36l2 and TTP (Zfp36) in that it shuttles between the cytoplasm and nucleus, binds to RNAs containing AREs, and promotes deadenylation of a model ARE transcript in a cell-based co-transfection assay. Surprisingly, in contrast to TTP, Zfp36l2 mRNA and protein were rapidly down-regulated upon LPS exposure in bone marrow-derived macrophages. The ΔN-Zfp36l2 protein was substantially more resistant to stimulus-induced down-regulation than the WT. I postulate that the embryonic arrest linked to the ΔN-Zfp36l2 truncation might be related to its resistance to stimulus-induced down-regulation.

FIGURE 1.

A, schematic structure of the mouse and human ZFP36L2 genes. Panel I, mouse Zfp36l2 gene is composed of two exons encoding a polypeptide of 484 amino acids (aa), in which 17 amino acids are contributed by the first exon (blue rectangle) and 467 amino acids by the second exon (pink rectangle). The single intron (∼400 bp) is spliced in the WT transcript (angled lines). The mouse transcript has five typical ATTTA elements (vertical bars) and a single poly(A) signal sequence AATAAA (small rectangle) at the 3′-untranslated region (3′-UTR). Panel II, ΔN-Zfp36l2 transcript contains the second exon fused with part of the single intron (horizontal line at the 5′-UTR). The only in-frame ATG, located at the beginning of exon 2, is the translation start site resulting in a 455-amino acid polypeptide, lacking 29 amino-terminal amino acids, shown as ΔN (line with double-headed arrows). Panel III, human gene, ZFP36L2, is also composed of two exons, encoding a polypeptide of 494 amino acids. The human 3′-UTR has four ATTTA elements (vertical bars) and one poly(A) signal sequence (small rectangle). B, amino acid sequences composing the full-length of mouse Zfp36l2 aligned with human and Xenopus (XC3H-3) ortholog sequences. The tandem CCCH zinc finger domain (line with dots) that characterizes this small family of proteins is located at the central part of the linear sequence. The second exon and its only methionine are indicated by a blue and red arrow, respectively. The blue lines illustrate the localization of each peptide sequence used to raise different antisera against the mouse protein.

FIGURE 2.

Wild-type (WT) and ΔN-Zfp36l2 (Mut) mouse transcripts. A, total RNA (10 μg per lane) was isolated from mouse embryonic fibroblasts from WT and mutant mice, subjected to Northern blot analysis, and probed with exon 1, exon 2, and 3′-UTR probes. The upper band corresponds to Zfp36l2 and the lower band to the ΔN-Zfp36l2 transcript (arrows). B, cytosolic RNA (10 μg per lane) was extracted from mouse embryonic fibroblasts, subjected to Northern blot analysis, and probed with an intron and Neo probes. The new ΔN-transcript hybridized with an intron probe (arrow, left panel) and is not fused with the Neo mRNA (arrow, right panel). C, Zfp36l2 mRNA is ubiquitously expressed in mouse tissues. Total cellular RNA was isolated from the indicated tissues of adult C57BL6 mice and subjected to Northern blot analysis. Each lane contains 10 μg of total RNA probed with an exon 1 probe. The Zfp36l2 transcript migrates as a single band of ∼3.6 kb, and molecular sizes of the 28 S and 18 S ribosomal subunits are shown. The agarose gel was stained with acridine orange showing similar RNA loading in all lanes. D, Zfp36l2 expression in oocytes. Total RNA was extracted from mouse denuded oocytes, in three independent experiments, and compared with primary bone marrow macrophages. The samples were subjected to reverse transcription (RT-PCR) and real time PCR to quantify Zfp36l2 expression in oocytes relatively to macrophages; Gapdh was used as housekeeping gene.

FIGURE 3.

Immunoreactivity and specificity of each antiserum for Zfp36l2. Western blot analysis using protein extracts from HEK 293 cells transfected with empty vector (lane 1) or each member of mTTP family as follows: full-length Zfp36l2 (lane 2), ΔN-Zfp36l2 starting at the second methionine (lane 3), TTP (lane 4), and Zfp36l1 (lane 5) are shown in A–D. Protein extracts were prepared as described under “Material and Methods.” A, N1-Zfp36l2-AS recognizes only full-length Zfp36l2 (lane 2) but not ΔN-, TTP, or Zfp36l1, lanes 3–5, respectively. B, C2-Zfp36l2-AS detects both versions of Zfp36l2 protein (lanes 2 and 3) and Zfp36l1 (lane 5). C, C1-Zfp36l2-AS recognized different versions of Zfp36l2 (lanes 2 and 3) but not TTP or Zfp36l1 (lanes 4 and 5, respectively). D, preimmune serum does not immunoreact with any CCCH family member. E, Western blot analysis using protein extracts from BMM of WT and mutant (Mut) mice were prepared as described under “Material and Methods.” N1-Zfp36l2-AS recognized the WT endogenous protein but not the mutant present in the ΔN-Zfp36l2 mouse (arrow, left panel). C2-Zfp36l2-AS immunoreacted with WT and ΔN-Zfp36l2 proteins (right panel), and nonspecific bands are indicated by small arrows. F, N2-Zfp36l2-AS detected an ∼60-kDa band in protein extracts from overexpressed Zfp36l2 (lane 2) but not with the empty vector (lane 1). This band recognition was blocked by the competing peptide (pep) (+) (right panel), and nonspecific bands are indicated by small arrows. G, N2-Zfp36l2-AS immunoprecipitated in vitro translated Zfp36l2. Zfp36l2 translated in vitro, using a rabbit reticulocyte system, and labeled with ³⁵S was immunoprecipitated in a dose-dependent manner (arrow). Lanes 1–4 show products containing the same amount of in vitro translated Zfp36l2 protein incubated either with preimmune serum (lane 1) or with decreasing amounts of the antiserum: 1:50, 1:100, or 1:500, lanes 2–4, respectively. As a negative control (lane 5), in vitro translated protein that did not contain exogenous Zfp36l2 mRNA was subjected to IP with the antiserum in a dilution of 1:100. As a positive control, a small amount of crude protein extract (not IP) containing mRNA of Zfp36l2 was loaded in lane 6. H, N2-Zfp36l2-AS immunoprecipitated Zfp36l2 but not the other two family members, TTP or Zfp36l1. A small amount of total in vitro translated protein (not IP) from the rabbit reticulocyte system containing TTP, Zfp36l1, and Zfp36l2 was loaded in lanes 3, 6, and 9, respectively. A fixed dilution (1:50) of the antiserum (lanes 2, 5, and 8) or preimmune serum (lanes 1, 4, and 7) was used to immunoprecipitate TTP, Zfp36l1, or Zfp36l2, respectively. A negative control, similar to the one in lane 5 of the upper panel, was loaded into lane 10. The numbers on the left side of the figures are numeric values for molecular mass controls expressed in kDa. I, N2-Zfp36l2-AS immunoprecipitated endogenous and overexpressed Zfp36l2 protein. Primary cell lines of mouse embryonic fibroblasts (MEF) and BMM or the HEK 293 cells transfected with Zfp36l2 construct were labeled in vivo with ³²P as described under “Materials and Methods” and subjected to immunoprecipitation with preimmune serum (PI) or N2-Zfp36l2-AS antiserum (AS). N2-Zfp36l2-AS (arrow), but not preimmune serum, was able to immunoprecipitate endogenous (three left panels) and overexpressed Zfp36l2 (right panel). The amount of immunoprecipitated material was reduced by a third of the original amount in the presence of the competing peptide (+), two right panels.

FIGURE 4.

Interaction of Zfp36l2 protein with RNA. RNA electrophoretic mobility shift assays were done incubating protein extracts from HEK 293 cells transfected with mouse Zfp36l2 or empty vector (V) with 2 × 10⁵ cpm of ³²P-labeled mTNFα (1309–1332 bp) ARE probe. RNA-protein complexes were resolved in a 6% acrylamide nondenaturing gel. A, increasing amounts of protein extracts containing overexpressed Zfp36l2 resulted in increased shift of the probe electrophoretic migration (lanes 3–5, arrow) in regard to the original probe migration (lane 1, P). Protein extracts from HEK 293 cells transfected with the empty vector (V) result in the formation of two complexes (C1 and C2, double arrows). B, HEK 293 cells were transfected with Zfp36l2-HA construct. Protein extracts were incubated as described previously, except for the presence of increasing amounts of HA-antibody (HA-Ab). The HA-Ab shifted the migration of the Zfp36l2-TNFα ARE complex (supershift, SS, lanes 4 and 5). C, N2-Zfp36l2-AS was able to shift the Zfp36l2-HA-TNFα ARE complex, in a similar fashion as the HA-Ab (SS, in lane 5). D, immunodepletion of Zfp36l2 from protein extracts of HEK 293 cells overexpressing Zfp36l2-HA abolished the shift of TNFα probe. The Zfp36l2 protein present in the extracts of HEK 293 cells transfected with Zfp36l2-HA was incubated with the HA-Ab (lanes 6 and 7) or with N2-Zfp36l2-AS (lanes 8 and 9 and, then immunoprecipitated using protein A-Sepharose beads. The immunodepleted protein extracts (lanes 6–9) were used in the RNA electrophoretic mobility shift assays. In lanes 6 and 8, the Zfp36l2-immunodepleted extracts were incubated with RNA probe plus HA-Ab, and in lanes 7 and 9 the complexes were incubated with the N2-Zfp36l2-AS and resolved in a 6% acrylamide nondenaturing gel. The ability of these antibodies to shift the protein-TNFα-probe complex was virtually gone in the Zfp36l2-immunodepleted extract (lanes 6–9, SS). Unspecific recognition was observed in the AS (arrowhead). The same protein extracts containing Zfp36l2 before the immunodepletion resulted in a shift (lane 3) or in a supershift in the presence of HA or N2-Zfp36l2-AS (lanes 4 and 5, SS). E, recombinant Zfp36l2 protein shifted a human TNFα ARE probe. Increasing amounts of recombinant Zfp36l2 (5, 10, and 15 μl, lanes 2–4, respectively) from a WGS containing Zfp36l2 mRNA shifted a ³²P-labeled human TNFα (1331–1389 bp) ARE probe (upper arrow). As a negative control, 15 μl of wheat germ system that did not contain exogenous Zfp36l2 mRNA was incubated with TNF ARE probe, and this resulted in no shift (lane 1). Unspecific complexes, C1 and C2, were present in lanes 1–4 (lower arrows). Note that the intensity of C1 and C2 complexes varies according to the volume of WGS used in reaction. F, expression of recombinant Zfp36l2 protein from WGS. Recombinant proteins using WGS with or without exogenous Zfp36l2 mRNA were labeled with ³⁵S, loaded in a 10% SDS-PAGE, and exposed to film. As a negative control, in vitro translated protein that did not contain exogenous Zfp36l2 mRNA was loaded in lane 1. Increasing amounts of recombinant Zfp36l2 protein from WGS were loaded in lanes 2–4, respectively.

FIGURE 5.

RNA functional assays of Zfp36l2 and ΔN-Zfp36l2. A, in a gel shift assay, protein extracts from HEK 293 cells transfected with GFP (vector) or Zfp36l2-GFP or ΔN-Zfp36l2-GFP (respectively, lanes 2–3) were incubated with a ³²P-mTNFα-ARE probe for 20 min at room temperature. The products were separated on an 8% nondenaturing polyacrylamide gel followed by autoradiography. Both protein extracts containing Zfp36l2-GFP or ΔN-Zfp36l2-GFP formed complexes with the labeled RNA probe, shifting the migration position of the free probe (lane 4, P) to an upper region (arrow). B, cell-free deadenylation of polyadenylated ARE-containing probes. Protein extracts of HEK 293 cells transfected with different constructs, as indicated in the figure, were incubated with ³²P-labeled ARE-A50 RNA probe at 37 °C in the presence (+) or absence (−) of EDTA to inhibit cellular exonucleases for 60 min. The probes were then purified and subjected to electrophoresis on urea-polyacrylamide gels, followed by autoradiography. The arrows indicate the migration position of the polyadenylated probe (ARE-A₅₀), deadenylated product of the probe (ARE), and empty vector (V), upper, middle and lower arrows, respectively. C, dose-response effect of WT and ΔN-Zfp36l2. A fixed amount of 1 μg of CMV.mTNFα was co-transfected into HEK 293 cells with increasing amounts of the expression vector Zfp36l2-GFP (left panel) or ΔN-Zfp36l2-GFP (right panel). Each condition was adjusted to a final concentration of 3 μg of transfected DNA by addition of vector alone. Twenty four hours after the removal of the transfection mixture, total cellular RNA was harvested. Each lane contains 10 μg of total RNA subjected to Northern blot assay and hybridized sequentially with Zfp36l2, TNFα and cyclophilin ³²P-labeled probes. The upper arrow indicates Zfp36l2 fusion constructs; the double arrows indicate the two species of TNFα mRNA, and the lower arrow indicates the cyclophilin transcript used to normalize the samples. D, Western blot analysis and protein quantification; 25 μg of protein extracts from HEK 293 cells co-transfected with 50 ng of BS+ (vector), GFP, Zfp36l2-GFP, or ΔN-Zfp36l2-GFP constructs in the presence of 1 μg of CMV.mTNFα were probed with a GFP-antibody. The bands were quantified and plotted in a bar graph.

FIGURE 6.

Expression of Zfp36l2-GFP and ΔN-Zfp36l2-GFP. HEK 293 cells were transfected with GFP or GFP fusion plasmids, and samples were simultaneously processed for Western blotting and confocal microscopic analysis. A, Western blots containing 25 μg per lane of cytosolic protein extracts from transfected HEK 293 cells were probed with an anti-GFP antibody. Each lane corresponds, respectively, to cells transfected with empty vector (lane 1), GFP (lane 2), Zfp36l2-GFP (lane 3), and ΔN-Zfp36l2-GFP (lane 4). Note the similar levels of expression of the WT and ΔN-Zfp36l2 fusion proteins. B, shows a confocal image of HEK 293 cells expressing GFP alone, and the other panels show WT-Zfp36l2-GFP (C and E) and ΔN-Zfp36l2-GFP (D and F). Bars represent 10 μm.

FIGURE 7.

LPS induced decay of Zfp36l2 transcript. A, BMM from WT and ΔN-Zfp36l2 mice were stimulated with LPS (1 μg/ml) in a time course. Total cellular RNA was isolated at each time point. Ten μg of RNA was loaded per lane, subjected to Northern blot analysis, and probed with Zfp36l2 exon 2 and Gapdh ³²P-labeled probes (left panel). The relative intensity of Zfp36l2 transcript in WT (black squares) and ΔN-Zfp36l2 mice (open squares) was quantified at each time point and corrected by Gapdh using a PhosphorImager. The final values were expressed as a percentage of the maximum value, which occurred at 15 min (right panel). B, BMM from WT (n = 3) and ΔN-Zfp36l2 (n = 3) mice were isolated and stimulated with LPS (1 μg/ml). Total cellular RNA was isolated at each time point, converted to cDNA, and analyzed by real time PCR for Zfp36l2 and TTP expression levels (black and open squares; black and open circles, respectively). Gapdh was used as housekeeping gene. C, LPS induction of two CCCH transcripts in bone marrow-derived macrophages. Data obtained from Northern blot analysis were modeled and plotted graphically.

FIGURE 8.

ΔN-Zfp36l2 is not down-regulated by LPS stimulation in BMM from ΔN-Zfp36l2 mice. A, RAW 264.7 cells were stimulated with LPS (1 μg/ml), and protein extracts were prepared at each time point. Sixty μg of cytosolic protein extracts were loaded per lane in a 10% SDS-PAGE. Western blots were developed using C2-Zfp36l2-AS as described previously. Upper and lower panels show specific and nonspecific (NS) bands (loading control), respectively. B, Western blot of LPS (1 μg/ml)-stimulated bone marrow-derived macrophages from WT and ΔN-Zfp36l2 animals at different time points were probed with C2-Zfp36l2-AS (upper panel) or GAPDH antibody (lower panel). C, Zfp36l2 band at each time point in A and B was quantified using a National Institutes of Health program. The intensity of Zfp36l2 bands was corrected by Gapdh or nonspecific bands and plotted (squares, black circles, and open circles represent the values obtained from RAW, WT, and ΔN-Zfp36l2 cells, respectively). D, Zfp36l2 protein down-regulation was not influenced by cycloheximide (CHX). Western blot of RAW 264.7 cells stimulated with LPS (1 μg/ml) at the indicated time points. The cells were pretreated with vehicle or cycloheximide (50 μm) for 15 min, left and right panel, respectively. E, intensity of Zfp36l2 and nonspecific (NS) bands were quantified and plotted along the time course of LPS stimulation in the presence of vehicle (open squares) and cycloheximide (black squares).