Host Poly(A) Polymerases PAPD5 and PAPD7 Provide Two Layers of Protection That Ensure the Integrity and Stability of Hepatitis B Virus RNA.

Noncanonical poly(A) polymerases PAPD5 and PAPD7 (PAPD5/7) stabilize hepatitis B virus (HBV) RNA via the interaction with the viral posttranscriptional regulatory element (PRE), representing new antiviral targets to control HBV RNA metabolism, hepatitis B surface antigen (HBsAg) production, and viral replication. Inhibitors targeting these proteins are being developed as antiviral therapies; therefore, it is important to understand how PAPD5/7 coordinate to stabilize HBV RNA. Here, we utilized a potent small-molecule AB-452 as a chemical probe, along with genetic analyses to dissect the individual roles of PAPD5/7 in HBV RNA stability. AB-452 inhibits PAPD5/7 enzymatic activities and reduces HBsAg both in vitro (50% effective concentration [EC50] ranged from 1.4 to 6.8 nM) and in vivo by 0.94 log10. Our genetic studies demonstrate that the stem-loop alpha sequence within PRE is essential for both maintaining HBV poly(A) tail integrity and determining sensitivity toward the inhibitory effect of AB-452. Although neither single knockout (KO) of PAPD5 nor PAPD7 reduces HBsAg RNA and protein production, PAPD5 KO does impair poly(A) tail integrity and confers partial resistance to AB-452. In contrast, PAPD7 KO did not result in any measurable changes within the HBV poly(A) tails, but cells with both PAPD5 and PAPD7 KO show reduced HBsAg production and conferred complete resistance to AB-452 treatment. Our results indicate that PAPD5 plays a dominant role in stabilizing viral RNA by protecting the integrity of its poly(A) tail, while PAPD7 serves as a second line of protection. These findings inform PAPD5-targeted therapeutic strategies and open avenues for further investigating PAPD5/7 in HBV replication. IMPORTANCE Chronic hepatitis B affects more than 250 million patients and is a major public health concern worldwide. HBsAg plays a central role in maintaining HBV persistence, and as such, therapies that aim at reducing HBsAg through destabilizing or degrading HBV RNA have been extensively investigated. Besides directly degrading HBV transcripts through antisense oligonucleotides or RNA silencing technologies, small-molecule compounds targeting host factors such as the noncanonical poly(A) polymerase PAPD5 and PAPD7 have been reported to interfere with HBV RNA metabolism. Herein, our antiviral and genetic studies using relevant HBV infection and replication models further characterize the interplays between the cis element within the viral sequence and the trans elements from the host factors. PAPD5/7-targeting inhibitors, with oral bioavailability, thus represent an opportunity to reduce HBsAg through destabilizing HBV RNA.

Posttranscriptional modulation of TERC by PAPD5 inhibition rescues hematopoietic development in dyskeratosis congenita.

Reduced levels of TERC, the telomerase RNA component, cause dyskeratosis congenita (DC) in patients harboring mutations in TERC, PARN, NOP10, NHP2, NAF1, or DKC1. Inhibition of the noncanonical poly(A) polymerase PAPD5, or the exosome RNA degradation complex, partially restores TERC levels in immortalized DKC1 mutant cells, but it remains unknown if modulation of posttranscriptional processing of TERC could improve hematopoietic output in DC. We used human embryonic stem cells (hESCs) with a common dyskerin mutation (DKC1_A353V), which have defective telomere maintenance and reduced definitive hematopoietic potential, to understand the effects of reducing EXOSC3 activity, or silencing PAPD5-mediated oligoadenylation, on hematopoietic progenitor specification and function in DC. Reduction of EXOSC3 or PAPD5 levels in DKC1 mutant hESCs led to functional improvements in TERC levels and telomerase activity, with concomitant telomere elongation and reduced levels of DNA damage signaling. Interestingly, the silencing of PAPD5, but not EXOSC3, significantly restored definitive hematopoietic potential in DKC1 mutant cells. Mechanistically, we show that PAPD5 inhibition is sustained in differentiated CD34+ cells, with a concomitant increase in mature, functional, forms of TERC, indicating that regulation of PAPD5 is a potential strategy to reverse hematologic dysfunction in DC patients.

Synthesis of Trifluoromethyl-α,ß-unsaturated Lactones and Pyrazolinones and Discovery of Influenza Virus Polymerase Inhibitors.

To explore the potential biological activities of trifluoromethyl heterocycles, we recently developed a synthetic approach to access a series of α-trifluoromethyl-α,ß-unsaturated lactones and trifluoromethyl pyrazolinones. The compounds were tested for their antimicrobial activity, and we found that some compounds had anti-influenza viral activity. The ß-aryl-α-trifluoromethyl α,ß-unsaturated lactone derivatives 5 g (5-(4-chlorophenyl)-5-methyl-4-phenyl-3-(trifluoromethyl)furan-2-one), 7 b (4-(4-methoxyphenyl)-3-(trifluoromethyl)spiro[furan-5,1'-indane]-2-one), and the trifluoromethyl pyrazolinone 12 c (1-(6-methoxy-2-naphthyl)-2-(trifluoromethyl)-5,6,7,8-tetrahydropyrazolo[1,2-a]pyridazin-3-one) were found to possess promising inhibitory activity against influenza virus type A, strain A/WSN/33 (H1N1). These three hit compounds were successfully optimized, and we identified that the most potent compound 5 h (5-(4-chlorophenyl)-4-(6-methoxy-2-naphthyl)-5-methyl-3-(trifluoromethyl)furan-2-one) showed inhibitory activity against various types of influenza A and B viruses in the low-micromolar range without showing cytotoxicity. Moreover, 5 h was more effective against the clinical isolate A/California/7/2009 (H1N1pdm) strain than the influenza viral polymerase inhibitor, favipiravir (T-705). We also delineated the structure-activity relationship and obtained mechanistic insight into inhibition of the viral polymerase.

Uridylation by TUT4/7 Restricts Retrotransposition of Human LINE-1s.

LINE-1 retrotransposition is tightly restricted by layers of regulatory control, with epigenetic pathways being the best characterized. Looking at post-transcriptional regulation, we now show that LINE-1 mRNA 3' ends are pervasively uridylated in various human cellular models and in mouse testes. TUT4 and TUT7 uridyltransferases catalyze the modification and function in cooperation with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of the L1-ORF1p retrotransposon protein. Uridylation potently restricts LINE-1 retrotransposition by a multilayer mechanism depending on differential subcellular localization of the uridyltransferases. We propose that uridine residues added by TUT7 in the cytoplasm inhibit initiation of reverse transcription of LINE-1 mRNAs once they are reimported to the nucleus, whereas uridylation by TUT4, which is enriched in cytoplasmic foci, destabilizes mRNAs. These results provide a model for the post-transcriptional restriction of LINE-1, revealing a key physiological role for TUT4/7-mediated uridylation in maintaining genome stability.

Fluorescent Branched RNAs for High-Throughput Analysis of Dbr1 Enzyme Kinetics and Inhibition.

We have developed fluorescent 2',5' branched RNAs (bRNA) that permit real time monitoring of RNA lariat (intron) debranching enzyme (Dbr1) kinetics. These compounds contain fluorescein (FAM) on the 5' arm of the bRNA that is quenched by a dabcyl moiety on the 2' arm. Dbr1-mediated hydrolysis of the 2',5' linkage induces a large increase in fluorescence, providing a convenient assay for Dbr1 hydrolysis. We show that unlabeled bRNAs with non-native 2',5'-phosphodiester linkages, such as phosphoramidate or phosphorothioate, can inhibit Dbr1-mediated debranching with IC50 values in the low nanomolar range. In addition to measuring kinetic parameters of the debranching enzyme, these probes can be used for high throughput screening (HTS) of chemical libraries with the aim of identifying Dbr1 inhibitors, compounds that may be useful in treating neurodegenerative diseases and retroviral infections.

Selective inhibitors of trypanosomal uridylyl transferase RET1 establish druggability of RNA post-transcriptional modifications.

Non-coding RNAs are crucial regulators for a vast array of cellular processes and have been implicated in human disease. These biological processes represent a hitherto untapped resource in our fight against disease. In this work we identify small molecule inhibitors of a non-coding RNA uridylylation pathway. The TUTase family of enzymes is important for modulating non-coding RNA pathways in both human cancer and pathogen systems. We demonstrate that this new class of drug target can be accessed with traditional drug discovery techniques. Using the Trypanosoma brucei TUTase, RET1, we identify TUTase inhibitors and lay the groundwork for the use of this new target class as a therapeutic opportunity for the under-served disease area of African Trypanosomiasis. In a broader sense this work demonstrates the therapeutic potential for targeting RNA post-transcriptional modifications with small molecules in human disease.

Design, Synthesis, and Properties of Phosphoramidate 2',5'-Linked Branched RNA: Toward the Rational Design of Inhibitors of the RNA Lariat Debranching Enzyme.

Two RNA fragments linked by means of a 2',5' phosphodiester bridge (2' hydroxyl of one fragment connected to the 5' hydroxyl of the other) constitute a class of nucleic acids known as 2'-5' branched RNAs (bRNAs). In this report we show that bRNA analogues containing 2'-5' phosphoramidate linkages (bN-RNAs) inhibit the lariat debranching enzyme, a 2',5'-phosphodiesterase that has recently been implicated in neurodegenerative diseases associated with aging. bN-RNAs were efficiently generated using automated solid-phase synthesis and suitably protected branchpoint building blocks. Two orthogonally removable groups, namely the 4-monomethoxytrityl (MMTr) group and the fluorenylmethyl-oxycarbonyl (Fmoc) groups, were evaluated as protecting groups of the 2' amino functionality. The 2'-N-Fmoc methodology was found to successfully produce bN-RNAs on solid-phase oligonucleotide synthesis. The synthesized bN-RNAs resisted hydrolysis by the lariat debranching enzyme (Dbr1) and, in addition, were shown to attenuate the Dbr1-mediated hydrolysis of native bRNA.

Selective Suppression of the Splicing-Mediated MicroRNA Pathway by the Terminal Uridyltransferase Tailor.

Several terminal uridyltransferases (TUTases) are known to modulate small RNA biogenesis and/or function via diverse mechanisms. Here, we demonstrate that Drosophila splicing-derived pre-miRNAs (mirtrons) are efficiently modified by the previously uncharacterized TUTase, Tailor. Tailor is necessary and sufficient for mirtron hairpin uridylation, and this modification inhibits mirtron biogenesis. Genome-wide analyses demonstrate that mirtrons are dominant Tailor substrates, and three features contribute to substrate specificity. First, reprogramming experiments show Tailor preferentially identifies splicing-derived miRNAs. Second, in vitro tests indicate Tailor prefers substrate hairpins over mature miRNAs. Third, Tailor exhibits sequence preference for 3'-terminal AG, a defining mirtron characteristic. Our work supports the notion that Tailor preferentially suppresses biogenesis of mirtrons, an evolutionarily adventitious pre-miRNA substrate class. Moreover, we detect preferential activity of Tailor on 3'-G canonical pre-miRNAs, and specific depletion of such loci from the pool of conserved miRNAs. Thus, Tailor activity may have had collateral impact on shaping populations of canonical miRNAs.

Uridylation of RNA Hairpins by Tailor Confines the Emergence of MicroRNAs in Drosophila.

Uridylation of RNA species represents an emerging theme in post-transcriptional gene regulation. In the microRNA pathway, such modifications regulate small RNA biogenesis and stability in plants, worms, and mammals. Here, we report Tailor, an uridylyltransferase that is required for the majority of 3' end modifications of microRNAs in Drosophila and predominantly targets precursor hairpins. Uridylation modulates the characteristic two-nucleotide 3' overhang of microRNA hairpins, which regulates processing by Dicer-1 and destabilizes RNA hairpins. Tailor preferentially uridylates mirtron hairpins, thereby impeding the production of non-canonical microRNAs. Mirtron selectivity is explained by primary sequence specificity of Tailor, selecting substrates ending with a 3' guanosine. In contrast to mirtrons, conserved Drosophila precursor microRNAs are significantly depleted in 3' guanosine, thereby escaping regulatory uridylation. Our data support the hypothesis that evolutionary adaptation to Tailor-directed uridylation shapes the nucleotide composition of precursor microRNA 3' ends. Hence, hairpin uridylation may serve as a barrier for the de novo creation of microRNAs in Drosophila.

Identification of small molecule inhibitors of Zcchc11 TUTase activity.

The RNA-binding protein Lin28 regulates the expression of the let-7 family of microRNAs (miRNAs) during early embryonic development. Lin28 recruits the 3' terminal uridylyl transferase (TUTase) Zcchc11 (TUT4) and/or Zcchc6 (TUT7) to precursor let-7 RNA (pre-let-7) to selectively block let-7 biogenesis. Uridylated pre-let-7 is targeted for decay by the downstream exonuclease Dis3l2 thereby preventing processing to mature let-7. Activation of this oncogenic pathway via up-regulation of Lin28 expression promotes cellular transformation, drives tumorigenesis in mouse models, and is frequently observed in a wide variety of cancer. Recent proof-of-principle experiments showed that Zcchc11 knockdown inhibits the tumorigenicity of Lin28-expressing human cancer cells and established this enzyme as a possible new therapeutic target for human malignancies. However, there are currently no known pharmacological agents capable of targeting this novel enzyme. In this study we developed and applied a sensitive biochemical assay that monitors Zcchc11 activity. Using this assay we performed an automated high-throughput screen of ∼ 15,000 chemicals to identify putative TUTase inhibitors. Several of these small molecules were validated as specific inhibitors of Zcchc11 activity. Our results demonstrate the feasibility of screening for TUTase inhibitors and present a relatively simple platform that can be exploited for future drug discovery efforts aimed at restoring let-7 expression in cancer.

Selective microRNA uridylation by Zcchc6 (TUT7) and Zcchc11 (TUT4).

Recent small RNA sequencing data has uncovered 3' end modification of mature microRNAs (miRNAs). This non-templated nucleotide addition can impact miRNA gene regulatory networks through the control of miRNA stability or by interfering with the repression of target mRNAs. The miRNA modifying enzymes responsible for this regulation remain largely uncharacterized. Here we describe the ability for two related terminal uridyl transferases (TUTases), Zcchc6 (TUT7) and Zcchc11 (TUT4), to 3' mono-uridylate a specific subset of miRNAs involved in cell differentiation and Homeobox (Hox) gene control. Zcchc6/11 selectively uridylates these miRNAs in vitro, and we biochemically define a bipartite sequence motif that is necessary and sufficient to confer Zcchc6/11 catalyzed uridylation. Depletion of these TUTases in cultured cells causes the selective loss of 3' mono-uridylation of many of the same miRNAs. Upon TUTase-dependent loss of uridylation, we observe a concomitant increase in non-templated 3' mono-adenylation. Furthermore, TUTase inhibition in Zebrafish embryos causes developmental defects and aberrant Hox expression. Our results uncover the molecular basis for selective miRNA mono-uridylation by Zcchc6/11, highlight the precise control of different 3' miRNA modifications in cells and have implications for miRNA and Hox gene regulation during development.

Computer-aided discovery of Trypanosoma brucei RNA-editing terminal uridylyl transferase 2 inhibitors.

Human African trypanosomiasis (HAT) is a major health problem in sub-Saharan Africa caused by Trypanosoma brucei infection. Current HAT drugs are difficult to administer and not effective against all parasite species at different stages of the disease which indicates an unmet pharmaceutical need. TbRET2 is an indispensable enzyme for the parasite and is targeted here using a computational approach that combines molecular dynamics simulations and virtual screening. The compounds prioritized are then tested in T. brucei via Alamar blue cell viability assays. This work identified 20 drug-like compounds which are candidates for further testing in the drug discovery process.

Meclizine inhibits mitochondrial respiration through direct targeting of cytosolic phosphoethanolamine metabolism.

We recently identified meclizine, an over-the-counter drug, as an inhibitor of mitochondrial respiration. Curiously, meclizine blunted respiration in intact cells but not in isolated mitochondria, suggesting an unorthodox mechanism. Using a metabolic profiling approach, we now show that treatment with meclizine leads to a sharp elevation of cellular phosphoethanolamine, an intermediate in the ethanolamine branch of the Kennedy pathway of phosphatidylethanolamine biosynthesis. Metabolic labeling and in vitro enzyme assays confirmed direct inhibition of the cytosolic enzyme CTP:phosphoethanolamine cytidylyltransferase (PCYT2). Inhibition of PCYT2 by meclizine led to rapid accumulation of its substrate, phosphoethanolamine, which is itself an inhibitor of mitochondrial respiration. Our work identifies the first pharmacologic inhibitor of the Kennedy pathway, demonstrates that its biosynthetic intermediate is an endogenous inhibitor of respiration, and provides key mechanistic insights that may facilitate repurposing meclizine for disorders of energy metabolism.

The ins and outs of phosphatidylethanolamine synthesis in Trypanosoma brucei.

Phospholipids are not only major building blocks of biological membranes but fulfill a wide range of critical functions that are often widely unrecognized. In this review, we focus on phosphatidylethanolamine, a major glycerophospholipid class in eukaryotes and bacteria, which is involved in many unexpected biological processes. We describe (i) the ins, i.e. the substrate sources and biochemical reactions involved in phosphatidylethanolamine synthesis, and (ii) the outs, i.e. the different roles of phosphatidylethanolamine and its involvement in various cellular events. We discuss how the protozoan parasite, Trypanosoma brucei, has contributed and may contribute in the future as eukaryotic model organism to our understanding of phosphatidylethanolamine homeostasis. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.

Inhibition of RNA lariat debranching enzyme suppresses TDP-43 toxicity in ALS disease models.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease primarily affecting motor neurons. Mutations in the gene encoding TDP-43 cause some forms of the disease, and cytoplasmic TDP-43 aggregates accumulate in degenerating neurons of most individuals with ALS. Thus, strategies aimed at targeting the toxicity of cytoplasmic TDP-43 aggregates may be effective. Here, we report results from two genome-wide loss-of-function TDP-43 toxicity suppressor screens in yeast. The strongest suppressor of TDP-43 toxicity was deletion of DBR1, which encodes an RNA lariat debranching enzyme. We show that, in the absence of Dbr1 enzymatic activity, intronic lariats accumulate in the cytoplasm and likely act as decoys to sequester TDP-43, preventing it from interfering with essential cellular RNAs and RNA-binding proteins. Knockdown of Dbr1 in a human neuronal cell line or in primary rat neurons is also sufficient to rescue TDP-43 toxicity. Our findings provide insight into TDP-43-mediated cytotoxicity and suggest that decreasing Dbr1 activity could be a potential therapeutic approach for ALS.

Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7).

The pluripotency factor Lin28 recruits a 3' terminal uridylyl transferase (TUTase) to selectively block let-7 microRNA biogenesis in undifferentiated cells. Zcchc11 (TUTase4/TUT4) was previously identified as an enzyme responsible for Lin28-mediated pre-let-7 uridylation and control of let-7 expression. Here we investigate the protein and RNA determinants for this interaction. Biochemical dissection and reconstitution assays reveal the TUTase domains necessary and sufficient for Lin28-enhanced pre-let-7 uridylation. A single C2H2-type zinc finger domain of Zcchc11 was found to be responsible for the functional interaction with Lin28. We identify Zcchc6 (TUTase7) as an alternative TUTase that functions with Lin28 in vitro, and accordingly, we find Zcchc11 and Zcchc6 redundantly control let-7 biogenesis in embryonic stem cells. Our study indicates that Lin28 uses two different TUTases to control let-7 expression and has important implications for stem cell biology as well as cancer.

RET1-catalyzed uridylylation shapes the mitochondrial transcriptome in Trypanosoma brucei.

RNA uridylylation is critical for the expression of the mitochondrial genome in trypanosomes. Short U tails are added to guide RNAs and rRNAs, while long A/U heteropolymers mark 3' ends of most mRNAs. Three divergent mitochondrial terminal uridylyl transferases (TUTases) are known: RET1 catalyzes guide RNA (gRNA) uridylylation, RET2 executes U insertion mRNA editing, and MEAT1 associates with the editosome-like complex. However, the activities responsible for 3' uridylylation of rRNAs and mRNAs, and the roles of these modifications, are unclear. To dissect the functions of mitochondrial TUTases, we investigated the effects of their repression and overexpression on abundance, processing, 3'-end status, and in vivo stability of major mitochondrially encoded RNA classes. We show that RET1 adds U tails to gRNAs, rRNAs, and select mRNAs and contributes U's into A/U heteropolymers. Furthermore, RET1's TUTase activity is required for the nucleolytic processing of gRNA, rRNA, and mRNA precursors. The U tail's presence does not affect the stability of gRNAs and rRNAs, while transcript-specific uridylylation triggers 3' to 5' mRNA decay. We propose that the minicircle-encoded antisense transcripts, which are stabilized by RET1-catalyzed uridylylation, may direct a nucleolytic cleavage of multicistronic precursors.

Novel TUTase associates with an editosome-like complex in mitochondria of Trypanosoma brucei.

Expression of mitochondrial genomes in Kinetoplastida protists requires massive uracil insertion/deletion mRNA editing. The cascade of editing reactions is accomplished by a multiprotein complex, the 20S editosome, and is directed by trans-acting guide RNAs. Two distinct RNA terminal uridylyl transferases (TUTases), RNA Editing TUTase 1 (RET1) and RNA Editing TUTase 2 (RET2), catalyze 3' uridylylation of guide RNAs and U-insertions into the mRNAs, respectively. RET1 is also involved in mitochondrial mRNA turnover and participates in numerous heterogeneous complexes; RET2 is an integral part of the 20S editosome, in which it forms a U-insertion subcomplex with zinc finger protein MP81 and RNA editing ligase REL2. Here we report the identification of a third mitochondrial TUTase from Trypanosoma brucei. The mitochondrial editosome-like complex associated TUTase (MEAT1) interacts with a 20S editosome-like particle, effectively substituting the U-insertion subcomplex. MEAT1 and RET2 are mutually exclusive in their respective complexes, which otherwise share several components. Similarly to RET2, MEAT1 is exclusively U-specific in vitro and is active on gapped double-stranded RNA resembling editing substrates. However, MEAT1 does not require a 5' phosphate group on the 3' mRNA cleavage fragment produced by editing endonucleases. The functional RNAi complementation experiments showed that MEAT1 is essential for viability of bloodstream and insect parasite forms. The growth inhibition phenotype in the latter can be rescued by coexpressing an RNAi-resistant gene with double-stranded RNA targeting the endogenous transcript. However, preliminary RNA analysis revealed no gross effects on RNA editing in MEAT1-depleted cells and indicated its possible role in regulating the mitochondrial RNA stability.

DBR1 siRNA inhibition of HIV-1 replication.

BACKGROUND: HIV-1 and all retroviruses are related to retroelements of simpler organisms such as the yeast Ty elements. Recent work has suggested that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate in cDNA synthesis. The putative genomic RNA lariat intermediate is formed by a 2'-5' phosphodiester bond, like that found in pre-mRNA intron lariats and it facilitates the minus-strand template switch during cDNA synthesis. We hypothesized that HIV-1 might also form a genomic RNA lariat and therefore that siRNA-mediated inhibition of expression of the human RNA lariat de-branching enzyme (DBR1) expression would specifically inhibit HIV-1 replication. RESULTS: We designed three short interfering RNA (siRNA) molecules targeting DBR1, which were capable of reducing DBR1 mRNA expression by 80% and did not significantly affect cell viability. We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knockdown led to decreases in viral cDNA and protein production. These effects could be reversed by cotransfection of a DBR1 cDNA indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression. CONCLUSION: These data suggest that DBR1 function may be needed to debranch a putative HIV-1 genomic RNA lariat prior to completion of reverse transcription.

Molecular cloning and functional analysis of a novel cadmium-responsive proto-oncogene.

The molecular mechanisms potentially responsible for cell transformation and tumorigenesis induced by cadmium, a human carcinogen, were investigated by differential gene expression analysis of BALB/c-3T3 cells transformed with cadmium chloride (CdCl(2)). Differential display analysis of gene expression revealed consistent overexpression of mouse translation initiation factor 3 (TIF3; GenBank accession number AF271072) in the cells transformed with CdCl(2) when compared with nontransformed cells. The predicted protein encoded by TIF3 cDNA exhibited 99% similarity to human eukaryotic initiation factor 3 p36 protein. A M(r) 36,000 protein was detected in cells transfected with an expression vector containing TIF3 cDNA. Transfection of NIH3T3 cells with an expression vector containing TIF3 cDNA resulted in overexpression of the encoded protein, and this was associated with cell transformation, as evidenced by the appearance of transformed foci exhibiting anchorage-independent growth on soft agar and tumorigenic potential in nude mice. Expression of the antisense RNA against TIF3 mRNA resulted in significant reversal of oncogenic potential of the CdCl(2)-transformed BALB/c-3T3 cells. Taken together, these findings demonstrate for the first time that the cell transformation and tumorigenesis induced by CdCl(2) are due, at least in part, to the overexpression of TIF3, a novel cadmium-responsive proto-oncogene.