Abstract
Members of the tristetraprolin family of CCCH tandem zinc finger proteins bind to AU-rich elements in certain cellular mRNAs, leading to their deadenylation and destabilization. Studies in knock-out mice demonstrated roles for three of the family members, tristetraprolin, ZFP36L1, and ZFP36L2, in inflammation, chorioallantoic fusion, and early embryonic development, respectively. However, little is known about a recently discovered placenta-specific tristetraprolin family member, ZFP36L3. Tristetraprolin, ZFP36L1, and ZFP36L2 have been shown to shuttle between the nucleus and cytoplasm, using typical hydrophobic amino acid-rich nuclear export sequences, and nuclear localization sequences located within the tandem zinc finger domain. In contrast, we previously showed that green fluorescent protein-labeled ZFP36L3, expressed in HEK 293 cells, remained cytosolic, even in the presence of the nuclear export blocker leptomycin B. We show here that the conserved tandem zinc finger domain contains an active nuclear localization signal. However, the sequence corresponding to the nuclear export signal in the other family members was nonfunctional, and thus did not contribute to the cytosolic localization. The unique C-terminal repeat domain could override the activity of the nuclear localization sequence, preventing the import of ZFP36L3 into the nucleus. Immunostaining of mouse placenta demonstrated that ZFP36L3 was located only in the cytoplasm of trophoblast cells. Thus, in contrast to the other mammalian members of this protein family, ZFP36L3 is a "full-time" cytosolic protein, rather than a nucleocytoplasmic shuttling protein. The significance of this difference in subcellular localization to the physiology of placental trophoblast cells, where ZFP36L3 is selectively expressed, remains to be determined.
Highlights
Members of the tristetraprolin family of CCCH tandem zinc finger proteins bind to AU-rich elements in certain cellular mRNAs, leading to their deadenylation and destabilization
Identification of a Functional Nuclear Localization Sequence (NLS) within the tandem zinc finger (TZF) Domain of L3—Because nuclear localization sequences (NLS) appear to exist within the TZF domains of TTP, L1, and L2, and because TTP family members from a given animal species exhibit great sequence similarity within their TZF domains (Fig. 2, A and B), we investigated whether there was a functional NLS within the TZF domain of L3
Further work is underway to determine the biochemical difference between the two species, but it does not seem to be due to alternative translational initiation, premature truncation, or Through examination and modification of predicted subcellular localization domains, we have identified the basis for the apparent cytosolic restriction of the L3 protein
Summary
Members of the tristetraprolin family of CCCH tandem zinc finger proteins bind to AU-rich elements in certain cellular mRNAs, leading to their deadenylation and destabilization. In contrast to the other mammalian members of this protein family, ZFP36L3 is a “full-time” cytosolic protein, rather than a nucleocytoplasmic shuttling protein The significance of this difference in subcellular localization to the physiology of placental trophoblast cells, where ZFP36L3 is selectively expressed, remains to be determined. L3 was found to bind a tumor necrosis factor (TNF)-based RNA probe in a cell-free system and to promote decay of an ARE-containing RNA target in intact cell transfection experiments (14) This new family member presumably retains the general biological functions of TTP, L1, and L2, i.e. the ability to down-regulate certain ARE-containing mRNAs; this concept remains to be proven in a physiological setting. L3, which is cytoplasmic at steady state when expressed as a GFP fusion protein in transfected HEK 293 cells, does not appear to shuttle sequence; SV40, simian virus 40; 293 cells, human embryonic kidney 293 cells; LMB, leptomycin B; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence; E, embryonic day; GFP, green fluorescent protein; NLS, nuclear localization sequence; NES, nuclear export sequence
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