CCCH Tandem Zinc Finger Proteins Research Articles

Overexpression of stress granule protein TZF1 enhances salt stress tolerance by targeting ACA11 mRNA for degradation in Arabidopsis.

Tandem CCCH zinc finger (TZF) proteins play diverse roles in plant growth and stress response. Although as many as 11 TZF proteins have been identified in Arabidopsis, little is known about the mechanism by which TZF proteins select and regulate the target mRNAs. Here, we report that Arabidopsis TZF1 is a bona-fide stress granule protein. Ectopic expression of TZF1 (TZF1 OE), but not an mRNA binding-defective mutant (TZF1H186Y OE), enhances salt stress tolerance in Arabidopsis. RNA-seq analyses of NaCl-treated plants revealed that the down-regulated genes in TZF1 OE plants are enriched for functions in salt and oxidative stress responses. Because many of these down-regulated mRNAs contain AU- and/or U-rich elements (AREs and/or UREs) in their 3'-UTRs, we hypothesized that TZF1-ARE/URE interaction might contribute to the observed gene expression changes. Results from RNA immunoprecipitation-quantitative PCR analysis, gel-shift, and mRNA half-life assays indicate that TZF1 binds and triggers degradation of the autoinhibited Ca2+-ATPase 11 (ACA11) mRNA, which encodes a tonoplast-localized calcium pump that extrudes calcium and dampens signal transduction pathways necessary for salt stress tolerance. Furthermore, this salt stress-tolerance phenotype was recapitulated in aca11 null mutants. Collectively, our findings demonstrate that TZF1 binds and initiates degradation of specific mRNAs to enhance salt stress tolerance.

A Case Study from the Overexpression of OsTZF5, Encoding a CCCH Tandem Zinc Finger Protein, in Rice Plants Across Nineteen Yield Trials

BackgroundDevelopment of transgenic rice overexpressing transcription factors involved in drought response has been previously reported to confer drought tolerance and therefore represents a means of crop improvement. We transformed lowland rice IR64 with OsTZF5, encoding a CCCH-tandem zinc finger protein, under the control of the rice LIP9 stress-inducible promoter and compared the drought response of transgenic lines and nulls to IR64 in successive screenhouse paddy and field trials up to the T6 generation.ResultsCompared to the well-watered conditions, the level of drought stress across experiments varied from a minimum of − 25 to − 75 kPa at a soil depth of 30 cm which reduced biomass by 30–55% and grain yield by 1–92%, presenting a range of drought severities. OsTZF5 transgenic lines showed high yield advantage under drought over IR64 in early generations, which was related to shorter time to flowering, lower shoot biomass and higher harvest index. However, the increases in values for yield and related traits in the transgenics became smaller over successive generations despite continued detection of drought-induced transgene expression as conferred by the LIP9 promoter. The decreased advantage of the transgenics over generations tended to coincide with increased levels of homozygosity. Background cleaning of the transgenic lines as well as introgression of the transgene into an IR64 line containing major-effect drought yield QTLs, which were evaluated starting at the BC3F1 and BC2F3 generation, respectively, did not result in consistently increased yield under drought as compared to the respective checks.ConclusionsAlthough we cannot conclusively explain the genetic factors behind the loss of yield advantage of the transgenics under drought across generations, our results help in distinguishing among potential drought tolerance mechanisms related to effectiveness of the transgenics, since early flowering and harvest index most closely reflected the levels of yield advantage in the transgenics across generations while reduced biomass did not.

OsTZF1, a CCCH-tandem zinc finger protein gene, driven under own promoter produces no pleiotropic effects and confers salt and drought tolerance in rice

ABSTRACT Different abiotic stresses induce OsTZF1, a tandem CCCH-type zinc finger domain gene, in rice. Here, we report that transgenic rice plants overexpressing OsTZF1 under own promoter (POsTZF1:OsTZF1-OX [for overexpression]) transferred to soil showed normal growth similar to vector control plants. The POsTZF1:OsTZF1-OX produced normal leaves without any lesion mimic phenotype and exhibited normal seed setting. The POsTZF1:OsTZF1-OX plants showed significantly increased tolerance to salt and drought stresses and enhanced post stress recovery. Microarray analysis revealed a total of 846 genes up-regulated and 360 genes down-regulated in POsTZF1:OsTZF1-OX salt-treated plants. Microarray analysis of POsTZF1:OsTZF1-OX plants showed the regulation of many abiotic stress tolerance genes. These results suggest that OsTZF1-OX under own promoter show abiotic stress tolerance and produces no pleiotropic effect on phenotype of transgenic rice plant.

Transcriptome-Wide Identification of CCCH-Type Zinc Finger Proteins Family in Pinus massoniana and RR-TZF Proteins in Stress Response.

CCCH-type zinc finger proteins play an important role in multiple biotic and abiotic stresses. More and more reports about CCCH functions in plant development and stress responses have appeared over the past few years, focusing especially on tandem CCCH zinc finger proteins (TZFs). However, this has not been reported in Pinaceae. In this study, we identified 46 CCCH proteins, including 6 plant TZF members in Pinus massoniana, and performed bioinformatic analysis. According to RT-PCR analysis, we revealed the expression patterns of five RR-TZF genes under different abiotic stresses and hormone treatments. Meanwhile, tissue-specific expression analysis suggested that all genes were mainly expressed in needles. Additionally, RR-TZF genes showed transcriptional activation activity in yeast. The results in this study will be beneficial in improving the stress resistance of P. massoniana and facilitating further studies on the biological and molecular functions of CCCH zinc finger proteins.

The role of senescence-associated gene101 (PagSAG101a) in the regulation of secondary xylem formation in poplar.

Wood is produced by the accumulation of secondary xylem via proliferation and differentiation of the cambium cells in woody plants. Identifying the regulators involved in this process remains a challenging task. In this study, we isolated PagSAG101a, the homolog of Arabidopsis thaliana SAG101, from a hybrid poplar (Populus alba × Populus glandulosa) clone 84K and investigated its role in secondary xylem development. PagSAG101a was expressed predominantly in lignified stems and localized in the nucleus. Compared with non-transgenic 84K plants, transgenic plants overexpressing PagSAG101a displayed increased plant height, internode number, stem diameter, xylem width, and secondary cell wall thickness, while opposite phenotypes were observed for PagSAG101a knock-out plants. Transcriptome analyses revealed that differentially expressed genes were enriched for those controlling cambium cell division activity and subsequent secondary cell wall deposition during xylem formation. In addition, the tandem CCCH zinc finger protein PagC3H17, which positively regulates secondary xylem width and secondary wall thickening in poplar, could bind to the promoter of PagSAG101a and mediate the regulation of xylem differentiation. Our results support that PagSAG101a, downstream of PagC3H17, functions in wood development.

A novel CCCH-type zinc finger protein SAW1 activates OsGA20ox3 to regulate gibberellin homeostasis and anther development in rice.

Male sterility is a prerequisite for hybrid seed production. The phytohormone gibberellin (GA) is involved in regulating male reproductive development, but the mechanism underlying GA homeostasis in anther development remains less understood. Here, we report the isolation and characterization of a new positive regulator of GA homeostasis, swollen anther wall 1 (SAW1), for anther development in rice (Oryza sativa L.). Rice plants carrying the recessive mutant allele saw1 produces abnormal anthers with swollen anther wall and aborted pollen. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRIPSR-associated protein 9-mediated knockout of SAW1 in rice generated similar male sterile plants. SAW1 encodes a novel nucleus-localizing CCCH-tandem zinc finger protein, and this protein could directly bind to the promoter region of the GA synthesis gene OsGA20ox3 to induce its anther-specific expression. In the saw1 anther, the significantly decreased OsGA20ox3 expression resulted in lower bioactive GA content, which in turn caused the lower expression of the GA-inducible anther-regulator gene OsGAMYB. Thus, our results disclose the mechanism of the SAW1-GA20ox3-GAMYB pathway in controlling rice anther development, and provide a new target gene for the rapid generation of male sterile lines by genome editing for hybrid breeding.

Masc-induced dosage compensation in silkworm cultured cells.

The Masculinizer (Masc) gene encodes a CCCH‐tandem zinc finger protein that controls both masculinization and dosage compensation in the silkworm Bombyx mori. We previously measured the masculinizing activity of the lepidopteran Masc proteins using B. mori ovary‐derived cell line BmN‐4. Here, we established an RNA‐seq data‐based assay system in which the level of B. mori Masc (BmMasc)‐induced dosage compensation can be estimated in BmN‐4 cells. Using this system, we found that a cysteine residue at position 301, which was shown to be essential for the masculinizing activity of BmMasc, is also required for dosage compensation. We further investigated the relationships between Masc‐induced cell growth inhibition, masculinizing activity, and the level of dosage compensation, using Masc genes from three lepidopteran insects. In summary, we have established a cell‐based system to monitor levels of Masc‐induced dosage compensation.

In vivo masculinizing function of the Ostrinia furnacalis Masculinizer gene

The Masculinizer gene (Masc) encodes a CCCH tandem zinc finger protein essential for masculinization and dosage compensation in the silkworm Bombyx mori. Previously we identified a Masc orthologue from the crambid Ostrinia furnacalis (OfMasc) and observed its masculinizing activity in the B. mori cultured cell line BmN-4. However, the role of OfMasc in masculinization of O. furnacalis has not been assessed. In this study, we unexpectedly discovered that all of the male larvae that escaped from Wolbachia-induced embryonic male-killing by OfMasc cRNA injection expressed the female-type splicing variants of O. furnacalis doublesex (Ofdsx). To clarify the role of OfMasc in the masculinization process in vivo, we established a system to monitor both sex chromosome- and dsx splicing-based sexes from a single O. furnacalis embryo. Using this system, we investigated the effects of OfMasc knockdown in early embryos on Ofdsx splicing and found that depletion of OfMasc mRNA in male embryos induced the production of the female-type splicing variants of Ofdsx. This result indicates that OfMasc is required for masculinization in O. furnacalis, and that the Masc protein possesses masculinizing activity in an insect species that is phylogenetically distant from Bombycidae.

CaC3H14 encoding a tandem CCCH zinc finger protein is directly targeted by CaWRKY40 and positively regulates the response of pepper to inoculation by Ralstonia solanacearum.

Tandem CCCH zinc finger (TZnF) proteins have been implicated in plant defence, but their role in pepper (Capsicum annuum) is unclear. In the present study, the role of CaC3H14, a pepper TZnF protein, in the immune response of pepper plants to Ralstonia solanacearum infection was characterized. When fused to the green fluorescent protein, CaC3H14 was localized exclusively to the nuclei in leaf cells of Nicotiana benthamiana plants transiently overexpressing CaC3H14. Transcript abundance of CaC3H14 was up-regulated by inoculation with R.solanacearum. Virus-induced silencing of CaC3H14 increased the susceptibility of the plants to R.solanacearum and down-regulated the genes associated with the hypersensitive response (HR), specifically HIR1 and salicylic acid (SA)-dependent PR1a. By contrast, silencing resulted in the up-regulation of jasmonic acid (JA)-dependent DEF1 and ethylene (ET) biosynthesis-associated ACO1. Transient overexpression of CaC3H14 in pepper triggered an intensive HR, indicated by cell death and hydrogen peroxide (H2 O2 ) accumulation, up-regulated PR1a and down-regulated DEF1 and ACO1. Ectopic overexpression of CaC3H14 in tobacco plants significantly decreased the susceptibility of tobacco plants to R.solanacearum. It also up-regulated HR-associated HSR515, immunity-associated GST1 and the SA-dependent marker genes NPR1 and PR2, but down-regulated JA-dependent PR1b and ET-dependent EFE26. The CaC3H14 promoter and was bound and its transcription was up-regulated by CaWRKY40. Collectively, these results indicate that CaC3H14 is transcriptionally targeted by CaWRKY40, is a modulator of the antagonistic interaction between SA and JA/ET signalling, and enhances the defence response of pepper plants to infection by R.solanacearum.

A Knock-In Tristetraprolin (TTP) Zinc Finger Point Mutation in Mice: Comparison with Complete TTP Deficiency.

Tristetraprolin (TTP) is a tandem CCCH zinc finger protein that can bind to AU-rich element-containing mRNAs and promote their decay. TTP knockout mice develop a severe inflammatory syndrome, largely due to excess tumor necrosis factor (TNF), whose mRNA is a direct target of TTP binding and destabilization. TTP's RNA binding activity and its ability to promote mRNA decay are lost when one of the zinc-coordinating residues of either zinc finger is mutated. To address several long-standing questions about TTP activity in intact animals, we developed a knock-in mouse with a cysteine-to-arginine mutation within the first zinc finger. Homozygous knock-in mice developed a severe inflammatory syndrome that was essentially identical to that of complete TTP deficiency, suggesting that TTP's critical anti-inflammatory role in mammalian physiology is secondary to its ability to bind RNA. In addition, there was no evidence for a "dominant-negative" effect of the mutant allele in heterozygotes, as suggested by previous experiments. Finally, mRNA decay experiments in mutant macrophages demonstrated that TTP can regulate the stability of its own mRNA, albeit to a minor extent. These studies suggest that RNA binding is an essential first step in the physiological activities of members of this protein family.

An Uncanonical CCCH-Tandem Zinc-Finger Protein Represses Secondary Wall Synthesis and Controls Mechanical Strength in Rice

Secondary walls, which represent the bulk of biomass, have a large impact on plant growth and adaptation to environments. Secondary wall synthesis is switched and regulated by a sophisticated signaling transduction network. However, there is limited understanding of these regulatory pathways. Here, we report that ILA1-interacting protein 4 (IIP4) can repress secondary wall synthesis. IIP4 is a phosphorylation substrate of an Raf-like MAPKKK, but its function is unknown. By generating iip4 mutants and relevant transgenic plants, we found that lesions in IIP4 enhance secondary wall formation. Gene expression and transactivation activity assays revealed that IIP4 negatively regulates the expression of MYB61 and CESAs but does not bind their promoters. IIP4 interacts with NAC29/NAC31, the upstream regulators of secondary wall synthesis, and suppresses the downstream regulatory pathways in plants. Mutagenesis analyses showed that phosphomimic IIP4 proteins translocate from the nucleus to the cytoplasm, which releases interacting NACs and attenuates its repression function. Moreover, we revealed that IIPs are evolutionarily conserved and share unreported CCCH motifs, referred to as uncanonical CCCH-tandem zinc-finger proteins. Collectively, our study provides mechanistic insights into the control of secondary wall synthesis and presents an opportunity for improving relevant agronomic traits in crops.

Selection for a Zinc-Finger Protein Contributes to Seed Oil Increase during Soybean Domestication

Seed oil is a momentous agronomical trait of soybean (Glycine max) targeted by domestication in breeding. Although multiple oil-related genes have been uncovered, knowledge of the regulatory mechanism of seed oil biosynthesis is currently limited. We demonstrate that the seed-preferred gene GmZF351, encoding a tandem CCCH zinc finger protein, is selected during domestication. Further analysis shows that GmZF351 facilitates oil accumulation by directly activating WRINKLED1, BIOTIN CARBOXYL CARRIER PROTEIN2, 3-KETOACYL-ACYL CARRIER PROTEIN SYNTHASE III, DIACYLGLYCEROL O-ACYLTRANSFERASE1, and OLEOSIN2 in transgenic Arabidopsis (Arabidopsis thaliana) seeds. Overexpression of GmZF351 in transgenic soybean also activates lipid biosynthesis genes, thereby accelerating seed oil accumulation. The ZF351 haplotype from the cultivated soybean group and the wild soybean (Glycine soja) subgroup III correlates well with high gene expression level, seed oil contents and promoter activity, suggesting that selection of GmZF351 expression leads to increased seed oil content in cultivated soybean. Our study provides novel insights into the regulatory mechanism for seed oil accumulation, and the manipulation of GmZF351 may have great potential in the improvement of oil production in soybean and other related crops.

Plant Tandem CCCH Zinc Finger Proteins Interact with ABA, Drought, and Stress Response Regulators in Processing-Bodies and Stress Granules.

Although multiple lines of evidence have indicated that Arabidopsis thaliana Tandem CCCH Zinc Finger proteins, AtTZF4, 5 and 6 are involved in ABA, GA and phytochrome mediated seed germination responses, the interacting proteins involved in these processes are unknown. Using yeast two-hybrid screens, we have identified 35 putative AtTZF5 interacting protein partners. Among them, Mediator of ABA-Regulated Dormancy 1 (MARD1) is highly expressed in seeds and involved in ABA signal transduction, while Responsive to Dehydration 21A (RD21A) is a well-documented stress responsive protein. Co-immunoprecipitation (Co-IP) and bimolecular fluorescence complementation (BiFC) assays were used to confirm that AtTZF5 can interact with MARD1 and RD21A in plant cells, and the interaction is mediated through TZF motif. In addition, AtTZF4 and 6 could also interact with MARD1 and RD21A in Y-2-H and BiFC assay, respectively. The protein-protein interactions apparently take place in processing bodies (PBs) and stress granules (SGs), because AtTZF5, MARD1 and RD21A could interact and co-localize with each other and they all can co-localize with the same PB and SG markers in plant cells.

Commentary: Conservation of AtTZF1, AtTZF2, and AtTZF3 homolog gene regulation by salt stress in evolutionarily distant plant species.

Commentary: Conservation of AtTZF1, AtTZF2, and AtTZF3 homolog gene regulation by salt stress in evolutionarily distant plant species.

Deficiency of the placenta- and yolk sac-specific tristetraprolin family member ZFP36L3 identifies likely mRNA targets and an unexpected link to placental iron metabolism.

The ZFP36L3 protein is a rodent-specific, placenta- and yolk sac-specific member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins. These proteins bind to AU-rich elements in target mRNAs, and promote their deadenylation and decay. We addressed the hypotheses that the absence of ZFP36L3 would result in the accumulation of target transcripts in placenta and/or yolk sac, and that some of these would be important for female reproductive physiology and overall fecundity. Mice deficient in ZFP36L3 exhibited decreased neonatal survival rates, but no apparent morphological changes in the placenta or surviving offspring. We found Zfp36l3 to be paternally imprinted, with profound parent-of-origin effects on gene expression. The protein was highly expressed in the syncytiotrophoblast cells of the labyrinth layer of the placenta, and the epithelial cells of the yolk sac. RNA-Seq of placental mRNA from Zfp36l3 knockout (KO) mice revealed many significantly upregulated transcripts, whereas there were few changes in KO yolk sacs. Many of the upregulated placental transcripts exhibited decreased decay rates in differentiated trophoblast stem cells derived from KO blastocysts. Several dozen transcripts were deemed high probability targets of ZFP36L3; these include proteins known to be involved in trophoblast and placenta physiology. Type 1 transferrin receptor mRNA was unexpectedly decreased in KO placentas, despite an increase in its stability in KO stem cells. This receptor is crucial for placental iron uptake, and its decrease was accompanied by decreased iron stores in the KO fetus, suggesting that this intrauterine deficiency might have deleterious consequences in later life.

Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein

We have recently discovered that the Masculinizer (Masc) gene encodes a CCCH tandem zinc finger protein, which controls both masculinization and dosage compensation in the silkworm Bombyx mori. In this study, we attempted to identify functional regions or residues that are required for the masculinizing activity of the Masc protein. We constructed a series of plasmids that expressed the Masc derivatives and transfected them into a B. mori ovary-derived cell line, BmN-4. To assess the masculinizing activity of the Masc derivatives, we investigated the splicing patterns of B. mori doublesex (Bmdsx) and the expression levels of B. mori IGF-II mRNA-binding protein, a splicing regulator of Bmdsx, in Masc cDNA-transfected BmN-4 cells. We found that two zinc finger domains are not required for the masculinizing activity. We also identified that the C-terminal 288 amino acid residues are sufficient for the masculinizing activity of the Masc protein. Further detailed analyses revealed that two cysteine residues, Cys-301 and Cys-304, in the highly conserved region among lepidopteran Masc proteins are essential for the masculinizing activity in BmN-4 cells. Finally, we showed that Masc is a nuclear protein, but its nuclear localization is not tightly associated with the masculinizing activity.

ABA-induced CCCH tandem zinc finger protein OsC3H47 decreases ABA sensitivity and promotes drought tolerance in Oryza sativa

Water deficit causes multiple negative impacts on plants, such as reactive oxygen species (ROS) accumulation, abscisic acid (ABA) induction, stomatal closure, and decreased photosynthesis. Here, we characterized OsC3H47, which belongs to CCCH zinc-finger families, as a drought-stress response gene. It can be strongly induced by NaCl, PEG, ABA, and drought conditions. Overexpression of OsC3H47 significantly enhanced tolerance to drought and salt stresses in rice seedlings, which indicates that OsC3H47 plays important roles in post-stress recovery. However, overexpression of OsC3H47 reduced the ABA sensitivity of rice seedlings. This suggests that OsC3H47 is a newly discovered gene that can control rice drought-stress response, and it may play an important role in ABA feedback and post-transcription processes.

Post‐transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins bind to AU-rich regions in target mRNAs, leading to their deadenylation and decay. Family members in Saccharomyces cerevisiae influence iron metabolism, whereas the single protein expressed in Schizosaccharomyces pombe, Zfs1, regulates cell-cell interactions. In the human pathogen Candida albicans, deep sequencing of mutants lacking the orthologous protein, Zfs1, revealed significant increases (> 1.5-fold) in 156 transcripts. Of these, 113 (72%) contained at least one predicted TTP family member binding site in their 3'UTR, compared with only 3 of 56 (5%) down-regulated transcripts. The zfs1Δ/Δ mutant was resistant to 3-amino-1,2,4-triazole, perhaps because of increased expression of the potential target transcript encoded by HIS3. Sequences of the proteins encoded by the putative Zfs1 targets were highly conserved among other species within the fungal CTG clade, while the predicted Zfs1 binding sites in these mRNAs often 'disappeared' with increasing evolutionary distance from the parental species. C. albicans Zfs1 bound to the ideal mammalian TTP binding site with high affinity, and Zfs1 was associated with target transcripts after co-immunoprecipitation. Thus, the biochemical activities of these proteins in fungi are highly conserved, but Zfs1-like proteins may target different transcripts in each species.

Rapid proteasomal degradation of posttranscriptional regulators of the TIS11/tristetraprolin family is induced by an intrinsically unstructured region independently of ubiquitination.

The TIS11/tristetraprolin (TTP) CCCH tandem zinc finger proteins are major effectors in the destabilization of mRNAs bearing AU-rich elements (ARE) in their 3' untranslated regions. In this report, we demonstrate that the Drosophila melanogaster dTIS11 protein is short-lived due to its rapid ubiquitin-independent degradation by the proteasome. Our data indicate that this mechanism is tightly associated with the intrinsically unstructured, disordered N- and C-terminal domains of the protein. Furthermore, we show that TTP, the mammalian TIS11/TTP protein prototype, shares the same three-dimensional characteristics and is degraded by the same proteolytic pathway as dTIS11, thereby indicating that this mechanism has been conserved across evolution. Finally, we observed a phosphorylation-dependent inhibition of dTIS11 and TTP degradation by the proteasome in vitro, raising the possibility that such modifications directly affect proteasomal recognition for these proteins. As a group, RNA-binding proteins (RNA-BPs) have been described as enriched in intrinsically disordered regions, thus raising the possibility that the mechanism that we uncovered for TIS11/TTP turnover is widespread among other RNA-BPs.

The Drosophila Tis11 Protein and Its Effects on mRNA Expression in Flies

Members of the mammalian tristetraprolin family of CCCH tandem zinc finger proteins can bind to certain AU-rich elements (AREs) in mRNAs, leading to their deadenylation and destabilization. Mammals express three or four members of this family, but Drosophila melanogaster and other insects appear to contain a single gene, Tis11. We found that recombinant Drosophila Tis11 protein could bind to ARE-containing RNA oligonucleotides with low nanomolar affinity. Remarkably, co-expression in mammalian cells with "target" RNAs demonstrated that Tis11 could promote destabilization of ARE-containing mRNAs and that this was partially dependent on a conserved C-terminal sequence resembling the mammalian NOT1 binding domain. Drosophila Tis11 promoted both deadenylation and decay of a target transcript in this heterologous cell system. We used chromosome deletion/duplication and P element insertion to produce two types of Tis11 deficiency in adult flies, both of which were viable and fertile. To address the hypothesis that Tis11 deficiency would lead to the abnormal accumulation of potential target transcripts, we analyzed gene expression in adult flies by deep mRNA sequencing. We identified 69 transcripts from 56 genes that were significantly up-regulated more than 1.5-fold in both types of Tis11-deficient flies. Ten of the up-regulated transcripts encoded probable proteases, but many other functional classes of proteins were represented. Many of the up-regulated transcripts contained potential binding sites for tristetraprolin family member proteins that were conserved in other Drosophila species. Tis11 is thus an ARE-binding, mRNA-destabilizing protein that may play a role in post-transcriptional gene expression in Drosophila and other insects.