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Originally published In Press as doi:10.1074/jbc.M314071200 on February 9, 2004

J. Biol. Chem., Vol. 279, Issue 19, 20154-20166, May 7, 2004
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Identification of TINO

A NEW EVOLUTIONARILY CONSERVED BCL-2 AU-RICH ELEMENT RNA-BINDING PROTEIN*

Martino Donnini{ddagger}, Andrea Lapucci{ddagger}, Laura Papucci{ddagger}, Ewa Witort{ddagger}, Alain Jacquier||, Gary Brewer**, Angelo Nicolin{ddagger}{ddagger}, Sergio Capaccioli{ddagger}§§, and Nicola Schiavone{ddagger}¶¶

From the {ddagger}Department of Experimental Pathology and Oncology, School of Medicine, University of Florence, 50134 Florence, Italy, the {ddagger}{ddagger}Department of Pharmacology, School of Medicine, University of Milan, 20129 Milan, Italy, the ||Unité de Génétique des Interactions Macromoléculaires URA 2171-CNRS Institut Pasteur, F-75724 Paris, France, and the **Department of Molecular Genetics, Microbiology and Immunology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Received for publication, December 23, 2003 , and in revised form, February 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulation of mRNA stability by regulatory cis-acting AU-rich elements (AREs) and ARE-binding proteins is an important posttranscriptional mechanism of gene expression control. We previously demonstrated that the 3'-untranslated region of BCL-2 mRNA contains an ARE that accounts for rapid BCL-2 down-regulation in response to apoptotic stimuli. We also demonstrated that the BCL-2 ARE core interacts with a number of ARE-binding proteins, one of which is AU-rich factor 1/heterogeneous nuclear ribonucleoprotein D, known for its interaction with mRNA elements of others genes. In an attempt to search for other BCL-2 mRNA-binding proteins, we used the yeast RNA three-hybrid system assay and identified a novel human protein that interacts with BCL-2 ARE. We refer to it as TINO. The predicted protein sequence of TINO reveals two amino-terminal heterogeneous nuclear ribonucleoprotein K homology motifs for nucleic acid binding and a carboxyl-terminal RING domain, endowed with a putative E3 ubiquitin-protein ligase activity. In addition the novel protein is evolutionarily conserved; the two following orthologous proteins have been identified with protein-protein BLAST: posterior end mark-3 (PEM-3) of Ciona savignyi and muscle excess protein-3 (MEX-3) of Caenorhabditis elegans. Upon binding, TINO destabilizes a chimeric reporter construct containing the BCL-2 ARE sequence, revealing a negative regulatory action on BCL-2 gene expression at the posttranscriptional level.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The fate of mRNAs has recently emerged as an important point of regulation of gene expression. mRNA localization and stability and protein translation are closely controlled. In general, these mechanisms are based on the cooperation between cis- and trans-acting elements. Interactions among regulatory factors constitute the messenger ribonucleoprotein, an integrated dynamic platform for RNA-protein and protein-protein interactions acting on the cellular fate of each bound mRNA (1). It is now clear that this fate is determined within the nucleus where pre-mRNAs are "coated" by heterogeneous nuclear ribonuclear proteins (hnRNPs).1 Some of them are able to shuttle between the nucleus and the cytoplasm (nucleocytoplasmic shuttling): in this manner nuclear and cytoplasmic events of mRNA metabolism are interconnected (2). Messenger ribonucleoprotein particle assembly is so integral to gene expression control that it is functionally conceptualized as a posttranscriptional "operon" (3, 4).

One of the more investigated classes of cis-acting factors are the AU-rich elements (ARE). AREs are involved in messenger ribonucleoprotein assembly, and they are responsible for mRNA half-life control (5). AREs were initially found in the 3'-UTR of the mRNAs of early response genes such as c-FOS, c-MYC, and c-JUN, which code for powerful transcriptional activators, and granulocyte-macrophage colony-stimulating factor, interleukin-2, interleukin-3, and interleukin-6, which code for growth factors and cytokines. These mRNAs are finely regulated in response to external stimuli and are subject to rapid turnover (6, 7). AREs are well recognizable as one or more AUUUA motifs in 3'-UTRs of mRNAs often linked in a typical nonameric unit, UUAUUUA(U/A)(U/A). A more recent ARE classification is reported in the ARED data base (8) where more than 800 ARE-harboring mRNAs are classified on the basis of the number of AUUUA motifs they possess.

AREs bind to a growing number of AU-rich element-binding proteins. Among these proteins are AUF1/hnRNP D; ELAV-like (embryonic lethal abnormal vision); and hnRNP A1, A2, and C proteins belonging to the wide family of hnRNPs; these proteins contain the RNA recognition motif as their RNA-interacting domain (9–12). Other classes of RNA-binding proteins participating in ARE decay have been recently identified. Tristetraprolin is the prototype of ARE-binding proteins that possess a characteristic CCCH zinc finger domain (13). Another example is the hnRNP K homology (KH)-type splicing regulatory protein, a KH-type protein first identified as a splicing factor (14). Recently this protein was also implicated together with AUF1/hnRNP D and tristetraprolin proteins in the exosome recruitment on ARE mRNAs (15).

Considering the complexity of proteins that can bind a particular cis-acting element, we have chosen the yeast RNA THS technique to identify other BCL-2 ARE-binding proteins by a library screening assay. The RNA THS allowed us to clone two cDNAs: p40AUF1 isoform cDNA and a novel human gene on which we have focused in this work. Here we report its isolation and characterization. We refer to this protein as pTINO. The novel protein, prevalently localized in the nuclear-perinuclear compartments of the cell, is a novel regulator of BCL-2 gene expression acting at the posttranscriptional level.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
RNA Three-hybrid Library Screening Assay—The yeast strain L40-coat (Mata, ura3–52, leu2–3, 112, his3–200, trp1-1, ade2, LYS2::(LexA-op)-HIS3, LexA-MS2coat (TRP1)) and plasmids pIIIA/MS2-1, pIIIA/MS2-2, and pIIIA/IRE-MS2 were gifts from Dr. M. Wickens (University of Wisconsin). The RNA expression vector that we chose for hybrid RNA transcription was pIIIA/MS2-1. The human BCL-2 ARE cDNA, cloned into the SmaI site of the pIIIA/MS2-1, spans nucleotides 944–1050 of the human BCL-2 mRNA (GenBankTM accession number M14745 [GenBank] ). The resulting plasmid, pIIIA/MS2-B2ARE, was constructed as described previously (16). A derivative of yeast L40-coat containing pIIIA/MS2-B2ARE was transformed with a human placenta MATCH-MAKER cDNA library (no. HL4025AH, Clontech). Double transformants were plated on synthetic medium lacking leucine and histidine. 3 mM 3-aminotriazole (Sigma) was used to select for relatively high levels of HIS3 reporter gene activation. About 1.2 x 107 yeast transformants were screened. After a week, 1,040 white colonies were picked up and assayed according to the described procedures for RNA THS (17). The RNA-dependent false-positive clones were identified at the end of the screening by mating assay using the yeast R40-coat derivative strain (Mat{alpha}, ura3–52, leu2–3, 112, his3–200, trp1-1, ade2, LYS2::(LexA-op)-HIS3, LexA-MS2coat (TRP1)) containing the unrelated RNA 5'IRE-MS23'; the deletion mutant version of BCL-2 ARE, 5'MS2-B2{Delta}ARE3', whose nonameric motif was deleted; and as a positive control the 5'MS2-B2ARE3'. The B2{Delta}ARE cDNA sequence was deleted of the UUAUUUAU motif, ranging from nucleotides 1005 to 1012 of the human BCL-2 mRNA, and cloned in the SmaI site of the pIIIA/MS2-1, giving rise to pIIIA/MS2-B2{Delta}ARE vector.

RNA Three-hybrid System Mapping Assays—14 different and partially overlapping segments of BCL-2 ARE were cloned into the XmaI and SphI sites of the pIIIA/MS2-2. The first 11 segments (from B2ARE1 to B2ARE11) were PCR-amplified using Pfu DNA Polymerase (Stratagene) and pIIIA/MS2-B2ARE plasmid as template or, only for the segment named B2{Delta}ARE, the pIIIA/MS2-B2{Delta}ARE. The primers used were: fFW1, 5'-TCCcccgggTCAGCTATTTACTGCCA-3'; fFW2, 5'-TCCcccgggGCCAAAGGGAAATATCA-3'; fFW3, 5'-TCCcccgggATTTGTTACATTATTAAG-3'; fFW4, 5'-TCCcccgggATTTATTTATTTAAGACAG-3'; fFW5, 5'-TCCcccgggATTTAAGACAGTCCCATC-3'; fRV1, 5'-ACCTgcatgcTAAATGATATTTCCCTT-3'; fRV2, 5'-ACCTgcatgcTAATAATGTAACAAATAA-3'; fRV3, 5'-ACCTgcatgcTCTTAAATAAATAAATCT; fRV4, 5'-ACCTgcatgcCAAAGACAGGAGTTTTGA-3'; and fRV5, 5'-ACCTgcatgcGATTTCCAAAGACAGGAG-3'. The B2ARE1 segment was PCR-amplified with fFW1 and fRV2, B2ARE2 with fFW2 and fRV3, B2ARE3 with fFW3 and fRV4, and B2ARE4 with fFW4 and fRV5. The B2ARE5 segment was PCR-amplified with fFW1 and fRV1 primers, B2ARE6 with fFW2 and fRV2, B2ARE7 with fFW3 and fRV3, B2ARE8 with fFW4 and fRV4, and B2ARE9 with fFW5 and fRV5. B2ARE and B2{Delta}ARE were amplified with fFW1 and fRV5. All 11 B2AREs were checked for their transcription in yeast baits using Northern analysis (data not shown). B2ARE10, B2ARE11, and B2ARE12 segments were synthesized as complementary oligonucleotides with protruding ends forming XmaI and SphI sites. The L40-coat derivative clones, containing different combinations of hybrid RNAs and activation domain-fused proteins, were assayed for HIS3 and lacZ gene activation.

Plasmids—The 107-bp segment of human BCL-2 mRNA located in the 3'-UTR from nucleotides 944–1050 (GenBankTM accession number M14745 [GenBank] , Ref. 16) was PCR-amplified from pBS-SK-H-BCL-2 and inserted into the unique BglII restriction site downstream of the rabbit {beta}-globin cDNA of the pTRE2pur vector (Clontech). The resulting plasmid was pTRE2pur-B2ARE. pTRE2pur vector harboring the predicted ORF of TINO, named pTRE2pur/TINO, was produced as follows. A 1,485-nucleotide-long fragment was PCR-amplified from the pACT2 vector containing the human TINO cDNA with 5'-GGATCCGCCACCATGACCGAGTGCGTC-3' forward and 5'-GATATCTCAATACTGTCGTTGAAGGGC-3' reverse primers and cloned into the BamHI and EcoRV sites of the pTRE2pur. pQE-TriSystem vector (Qiagen) harboring the predicted ORF of TINO, named pQE-TriSystem/TINO, was produced as follows. A 1,485-nucleotide-long fragment was PCR-amplified from the pACT2 vector containing the human TINO cDNA with 5'-CATGCCATGGCAATGACCGAGTGCGTC-3' forward and 5'-CGCCTCGAGATACTGTCGTTGAAGGGC-3' reverse primers and cloned into the NcoI and XhoI sites of the pQE-TriSystem.

Cell Culture and Transfection—Retinal pigment epithelial (RPE) and HeLa cell lines were purchased from American Type Culture Collection (ATCC), and the HeLa Tet-Off cell line was purchased from Clontech. RPE cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium (Euroclone) and Ham's F-12 medium (Euroclone) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 0.1 µg/ml penicillin/streptomycin. HeLa cells were maintained in Dulbecco's modified Eagle's medium (Euroclone) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 0.1 µg/ml penicillin/streptomycin. HeLa Tet-Off cells were maintained in Dulbecco's modified Eagle's medium (Euroclone) supplemented with 10% tetracycline-free fetal calf serum (Clontech), 2 mM glutamine, 50 IU/ml penicillin, 50 µg/ml streptomycin, and 100 µg/ml G418 (Sigma). Recombinant TINO protein was analyzed by Western blot of the protein lysates extracted from HeLa Tet-Off cells transfected with 4 µg of pTRE2pur/TINO (and pTRE2pur as control) or 4 µg of pQE-TriSystem/TINO (and pQE-TriSystem as control) performed in 6-well plates. For RNase protection assay experiments, HeLa Tet-Off cells were transiently transfected in 6-well plates with 50 ng of pQE-TriSystem/TINO or pQE-TriSystem plasmid and 20 ng of pTRE2pur-B2ARE or pTRE2pur as control in the presence of 10 µg of plasmid DNA carrier. SuperFect transfection reagent (Qiagen) was used according to the manufacturer's instructions.

RNA Extraction and RT-PCR—Midlog cells were detached from culture cell dishes, and about 1 x 106 cells were recovered for RNA extractions using an RNeasy minikit (Qiagen). Total RNA was extracted from RPE, SH-SY5Y, K562, NB4, A431, human umbilical vein endothelial, human embryonic kidney 293, HeLa, and Jurkat T cells. 1 µg of total RNA was reverse-transcribed using 0.5 µg of random examers (New England Biolabs) and 1 µl of Improm-IITM reverse transcriptase (Promega) following the manufacturer's recommendation. The reverse-transcribed product was amplified in a 50-µl volume containing 25 pmol of gene-specific primers, 50 mM Tris-HCl, pH 9.0, 1.5 mM MgCl2, 15 mM (NH4)SO4, 0.1% Triton X-100, 2.5% Me2SO, and 0.7–1 units of DyNAzymeTM EXTTM DNA polymerase (Finnzymes). PCR was performed for 34 cycles with annealing at 59 °C using 5'-GTCAACATGACCGAGTGCG-3' forward and 5'-GGATGATGGAGAAGTGTTCGG-3' reverse primers flanking the major intron site within TINO cDNA.

Northern Blotting Analysis—For tissue distribution analysis, a premade Northern blot containing ~2 µg of poly(A) RNA/lane was used (human MTNTM blot, Clontech). Hybridization conditions were performed according to the manufacturer's instructions. The cDNA probe was derived after ApaI restriction enzyme cutting of the novel gene (nucleotides 521–1118 of TINO cDNA). The cDNA fragment was gelpurified and then subjected to the random priming reaction for radioactive labeling using 24 ng of DNA and [{alpha}-32P]dATP (3,000Ci/mmol, Amersham Biosciences) with the DECA Prime II kit (Ambion).

5'- and 3'-Rapid Amplification of cDNA Ends (5'- and 3'-RACE)— The Human Placenta FirstChoiceTM RACE Ready cDNA kit (Ambion) was used for 5'- and 3'-RACE analysis of TINO transcripts. It provides a mixture of two different types of cDNA population for each type of analysis. The cDNAs serves as templates in nested PCRs using adapter sequence-specific primers (provided with the FirstChoiceTM RACE Ready cDNA kit) and gene-specific primers. The sequences of the gene-specific primers used are as follows: 5'GSP1, 5'-ATGTGCGCCTCGATCTCCTC-3' (nucleotides 485–466 of TINO cDNA), and 5'GSP2, 5'-CTGCTGGATGCGCTTGATGG-3' (nucleotides 375–356 of TINO cDNA) for 5'-RACE; 3'GSP1, 5'-ATTCGCGTGGAGACGGAGAC-3' (nucleotides 1411–1430 of TINO cDNA), and 3'GSP2, 5'-GAAGCCGTTTTCTGATTTGACTTTTCTCGCCG-3' (nucleotides 1627–1658 of TINO cDNA) for 3'-RACE. The DyNAzymeTM EXTTM polymerase (Finnzymes) was used in all nested RNA T4 ligation-mediated RACE PCRs according to the manufacturer's instructions.

GENSCAN Analysis—The GENSCAN software is available on the Internet.2 The predicted additional 5'-exon of TINO gene was amplified using the following forward primers: G1, 5'-ATGCCCAGCTCGCTCGGCCA-3'; G2, 5'-TGGCGCTGGACCAGCTGTCG-3'; and G3, 5'-TGGCGACACGGACGAGGA-3'. The reverse primers were: R1, 5'-GCGTGGCGCGGATGATGGAG-3' (nucleotides 232–213 of TINO cDNA); and R2, 5'-TCCCAGTGACCGCGAACAC-3' (nucleotides 439–421 of TINO cDNA). The PCR profile time and temperatures were as follows: initial denaturation at 94 °C for 3 min followed by 35 cycles (94 °C for 30 s, 62 °C for 1 min 30 s, 72 °C for 1 min 30 s) with a final elongation step at 72 °C for 5 min.

Antibodies and Western Blotting Analysis—Rabbit polyclonal anti-pTINO antibody was raised against three synthetic peptides from pTINO. The peptides were the following: pep1, NH2-CKIKALRAKTNTYIK-COOH (amino acids 24–38); pep2, NH2-CKRIQQRTHTYIVTPGRDKE-COOH (amino acids 121–139); and pep3, NH2-CKTPNQGRRPPTATA-COOH (amino acids 199–212). pep2 and pep9 possess an additional residue (in bold) at the N-terminus in order to facilitate the conjunction with the protein carrier. The peptides were synthesized at the PRIMM s.r.l. (Milan, Italy) synthesis facility using the solidphase peptide synthesis method with Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by reverse-phase high pressure liquid chromatography. The amino-terminal amino acid for each peptide was coupled to the keyhole limpet hemocyanin protein, then the three keyhole limpet hemocyanin-coupled peptides were injected into two rabbits, and serum was collected after the second injection. The antisera were purified by affinity column chromatography using CNBr-Sepharose resin (Amersham Biosciences) for each purification, aliquoted, and kept at -20 °C until use. For total protein preparation and Western analysis, cells were collected, washed with ice-cold phosphate-buffered saline, and lysed with radioimmune precipitation assay lysis buffer (100 µl/well; 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 5 µg/ml aprotinin, 5 µg/ml leupeptin, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS). For Western blotting, 25 µg of total proteins were separated by 12.5% SDS-PAGE in 1x Tris-glycine-SDS running buffer (ICN Biomedicals Inc.) and transferred with the Trans-blot SD/semidry transfer cell (BioRad) onto nitrocellulose membranes (Schleicher & Schuell) in 25 mM Tris, 192 mM glycine, 20% methanol, and 0.1% SDS at 0.330 A for 30 min in a cold room. Membranes were blocked with 5% skim milk in PBS-Tween 20 at room temperature for 1 h prior to addition of rabbit polyclonal anti-pTINO antibody (1:1,000) diluted in PBS-Tween 20 overnight at 4 °C or mouse monoclonal anti-His(C-term) antibody (Invitrogen, no. R930-25) (1:5,000) diluted in PBS-Tween 20 with 5% skim milk overnight at 4 °C. After washing, membranes were incubated with peroxidase-conjugated secondary antibodies for 1 h at room temperature (1:10,000, rabbit, Amersham Biosciences; 1:5,000, goat, Sigma) and washed again prior to detection with ECL Plus reagents on ECL Hyperfilm (Amersham Biosciences).

In Vitro Translation—The ORF fragment of TINO gene was PCR-amplified from plasmid pQE-TriSystem/TINO using the following primers: the forward primer 5'-GTTAATACGACTCACTATAGGGAAATAATAGTCAACATGACCGAGTGCGT-3' containing the T7 promoter sequence (underlined) and the reverse primer 5'-CACTTAGTGATGGTGATGGTGATGGTGGTGCTCGA-3' containing the nucleotide sequence for the 8-histidine stretch present in the DNA template. The PCR product was incubated with the TNT-coupled transcription-translation reticulocyte lysate (Promega) in the presence of cold methionine according to the manufacturer's instructions.

Gel Electrophoresis Mobility Shift Assay—Mobility shift assays were performed using a radiolabeled 107-nucleotide-long RNA corresponding to the human BCL-2 mRNA located in the 3'-UTR from nucleotides 944 to 1050 (GenBankTM accession number M14745 [GenBank] ) as described in Lapucci et al. (16). Increasing volumes of reticulocyte lysate product were incubated for 20 min at room temperature with 2 fmol (2 x 104 cpm/fmol) of 32P-labeled BCL-2 ARE riboprobe in the presence of 10 mM Tris (pH 7.5), 100 mM KOAc, 5 mM Mg(OAc)2, 2 mM dithiothreitol, 100 mM spermine, 10% glycerol, 5 units of RNasin (Promega), 50 µg of heparin, and 0.2 µg/µl of yeast tRNA (total volume, 20 µl). Samples were electrophoresed in a 6% acrylamide gel (polyacrylamide:bisacrylamide, 60:1), dried, and analyzed using the CycloneTM Storage Phosphor System (PerkinElmer Life Sciences).

Intracellular Localization—HeLa cells were seeded at very low density (about 10,000 cells/cm2) and grown on glass coverslips (15 x 15 mm) in 12-well plates. Harvested cells were washed two times with 1 ml of cold PBS and fixed for 20 min in 3.7% paraformaldehyde in PBS. Following three washes for 2 min each with PBS, the cells were permeabilized with 1 ml of 0.25% Triton X-100 in PBS for 5 min at room temperature and washed three times for 2 min each in PBS. The following incubations were performed in the dark. Nuclei were stained with Hoechst 33258 (Sigma, no. B2883, blue fluorescence) diluted 1:1,000 in PBS for 30 min at 37 °C. The cells were washed three times for 5 min at room temperature and incubated with 1 ml of blocking buffer (3% bovine serum albumin, 0.1% Triton X-100 in PBS) for 1 h at room temperature. The cells were incubated with primary rabbit polyclonal anti-TINO antibody diluted 1:500 in blocking buffer overnight at 4 °C and the next day washed three times for 15 min each in washing buffer (0.1% Triton X-100 in PBS). The cells were then incubated with secondary fluorescein isothiocyanate-conjugated anti-rabbit antibody (Chemicon, no. AP156F, green fluorescence) diluted 1:800 for 60 min at room temperature. Following a final three washes with 1 ml of washing buffer for 5 min each at room temperature, the samples were dried, mounted onto glass slides, and examined with a Nikon fluorescence microscope using a B-2A filter for fluorescein isothiocyanate and UV-2A filter for Hoechst 33258.

RNase Protection Assay—HeLa Tet-Off cells were seeded in 6-well plates at a density of 1.5 x 105 cells/well 1 day prior to transient transfections. Two days posttransfection cells were treated with 2 µg/ml doxycycline (Clontech), and total RNA was collected at various time points and harvested in lysis solution containing guanidine thiocyanate (Ambion). Lysates were passed through shredder columns (Qiagen) and stored frozen at -20 °C. The plasmids pGAPM, containing a portion of the human glyceraldehyde-3-phosphate dehydrogenase coding region (18), and pBS{Delta}{beta} (19), containing the rabbit {beta}-globin cDNA, were linearized with DdeI and NcoI enzymes, respectively, and used for in vitro run-off transcriptions to produce specific antisense riboprobes. 10 fmol of 32P-labeled riboprobes were generated using T7 (pGAPM) and T3 (pBS{Delta}{beta}) RNA polymerases in the presence of [{alpha}-32P]UTP (800 Ci/mmol, Amersham Biosciences) according to the manufacturer's instructions for the MaxiScript kit (Ambion). Riboprobes were synthesized to yield transcripts with specific activities of 1–2 x 104 cpm/fmol, and they were added (10 fmol each) to the total RNA isolated according to the Direct ProtectTM Lysate RNase protection assay kit (Ambion). Protected RNA fragments were fractionated by denaturing gel electrophoresis, dried, and analyzed using a Cyclone Storage Phosphor System (PerkinElmer Life Sciences). The glyceraldehyde-3-phosphate dehydrogenase band intensities were used to normalize {beta}-globin bands to correct for loading errors. RNA half-lives and first-order decay constants were calculated from plots of percentage of remaining RNA versus time using Prism 3.03 (GraphPad software).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of TINO—To identify trans-acting factors that interact with the BCL-2 ARE, a human placenta cDNA library was screened by the RNA THS. The bait RNA sequence used in the yeast RNA Three-hybrid library screening was the 107-nucleotide-long BCL-2 ARE (107-mer) located in the 3'-UTR of BCL-2 mRNA. For the synthesis of the hybrid RNA, the 107-mer cDNA was cloned downstream of the MS2 sequence in the pIIIA/MS2-1 RNA expression plasmid. The chimeric RNA is 5'MS2-B2ARE3'. Of the initial 1,040 white yeast double transformants recovered from plates of synthetic complete medium lacking Leu and His and containing 3 mM 3-aminotriazole, 11 were selected for sequencing reactions at the end of the screen. Indeed only these clones demonstrated binding specificity for the BCL-2 ARE as verified by yeast mating assays using the bait negative controls 5'IRE-MS23', the unrelated RNA, and 5'MS2-B2{Delta}ARE3', the 107-mer sequence deleted of the UUAUUUAU segment. After sequencing, three different interacting sequence tags (ISTs) were selected for analysis. The first cDNA sequence identified (two ISTs), shared by six independent yeast clones, was the 40-kDa isoform of AUF1 protein, p40AUF1, an ARE-binding protein that we already demonstrated to interact in vitro with the BCL-2 ARE (16). The third IST, shared by five independent yeast clones, encoded a novel protein as determined using nucleotide-nucleotide BLAST. Consequently the presence of a previously uncharacterized human gene was revealed that we named TINO (Fig. 1).



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  		FIG. 1.
In vivo interaction of BCL-2 ARE and TINO by reconstituted RNA THS: verifying binding specificities by mating assay. Two positives cloned by the two library screening assays, p40AUF1 and pTINO, were used to confirm their specificity of interaction with the ARE motif. The hybrid RNAs used as negative controls were 5'MS2-B2{Delta}ARE3' (the mutant RNA deleted of nonamer motif) and 5'IRE-MS23' (the unrelated RNA). The RNA expression vectors were transformed in isogenic R40-coat yeast strain, and the selected transformants were mated with nine different L40-coat yeast clones harboring ISTs. The diploid cells containing the RNA-binding proteins p40 long interspersed nuclear element 1 (LINE-1), hnRNP A/B, fibrillarin, the transcriptional transactivator TWIST, and the unrelated protein hemoglobin are the negative controls. pTINO and p40AUF1 derive from two independent screenings of the library. The diploids (three independent clones for each type of RNA/protein combination-containing yeast) were selected on plates of synthetic complete medium lacking uracil and Leu and replicated on a filter for lacZ reporter gene transactivation assay.

 
A second independent RNA three-hybrid library screening assay with a modified and improved version of the RNA THS was applied.3 Also with this modified RNA THS, p40AUF1 and TINO were recovered; in particular the same IST of the novel gene was cloned. The lacZ reporter gene assay indicated that TINO protein (pTINO) is the IST most specifically interacting with the BCL-2 ARE. Indeed a strong decrease in the transactivation signal was observed when this fusion protein was tested with 5'IRE-MS23' or 5'MS2-B2{Delta}ARE3' RNAs for RNA-protein interactions. Thus we focused on the novel gene TINO (GenBankTM accession number AF458084 [GenBank] ). Recently a related cDNA sequence, KIAA2031 (GenBankTM accession number AB107353 [GenBank] ), was obtained from sequencing projects of large human transcripts with unknown function (20).

TINO Protein Interacts in Vitro with the 107-mer ARE—To confirm the interaction of the novel protein with the 107-mer sequence, a gel electrophoresis mobility shift assay was performed. The pTINO was produced using the reticulocyte lysate system because of the difficulties producing recombinant protein either in bacteria or insect cells probably due to its toxicity. Increasing amounts of in vitro synthesized TINO protein mixture were incubated with the radioactively labeled 107-mer ARE; efficient complex formation was observed with pTINO for which the intensity parallels the volume of the protein synthesis mixture (Fig. 2).



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  		FIG. 2.
Gel shift assay. pTINO was expressed using an in vitro system for coupled transcription and translation synthesis using PCR products as DNA templates. Increasing amounts of in vitro synthesized TINO protein mixture were incubated with the radioactively labeled 107-mer ARE motif, causing efficient RNA-protein complex formation (lanes 1–6). By contrast, no RNA-protein complex formation was obtained when TINO protein mixture was incubated in the presence of an unrelated radioactively labeled RNA probe ({beta}-globin RNA, 340 nucleotides, lanes 8 and 9) or when the in vitro synthesized luciferase protein was incubated with the 107-mer ARE probe (lane 7).

 
TINO and AUF1 Proteins Recognize Different Binding Sites of the 107-mer ARE—Using the RNA THS we identified the site of the BCL-2 ARE bound by pTINO. In agreement with the results of the binding specificity test performed with the mating assay, pTINO recognizes the motifs possessing the nonamer region (B2ARE2, B2ARE3, and B2ARE7); importantly the RNA-protein interaction is lost (B2ARE4) without the adenine stretch just upstream of this element (Fig. 3, A and B). To determine the minimal motif bound by pTINO, we tested B2ARE11 and B2ARE12 segments. We concluded that the 26-nucleotide-long sequence B2ARE11, containing both the adenine stretch and the nonamer, is the binding site of pTINO (Fig. 3, A and B).



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  		FIG. 3.
Mapping assays. The RNA THS was used to determine the binding sites of pTINO, p37AUF1, and p40AUF1 on the BCL-2 ARE. For this goal, 42 L40-coat derivative yeast clones, each containing a different combination of hybrid RNA and RNA-binding protein, were constructed. The upper panel (A) shows the sequences of the ARE segments cloned into the pIIIA/MS2-2 (on the right) and the results of HIS3 and lacZ reporter gene activation for each L40-coat derivative clone (on the left). HIS3 and lacZ genes were tested at the same time. Indeed a single yeast colony for each yeast clone was grown as a circular spot of about 5 mm in diameter for 2 days on synthetic medium lacking uracil, leucine, and histidine and containing 10 mM 3-aminotriazole, selecting for HIS3-activating cells. Afterward the yeast cells were covered with a thin layer of soft agar containing 0.1% SDS, for cellular permeabilization, and 0.04% 5-bromo-4-chloro-3-indolyl-{beta}-D-galactopyranoside (X-gal) as a substrate for the {beta}-galactosidase. The lower panel (B) summarizes the results of RNA THS mapping assays. The binding sites of the RNA-binding proteins pTINO (gray), p37AUF1 (white with fine black outline), and p40AUF1 (white with thick black outline) are indicated by the colored lines subtended along the minimal motifs of BCL-2 ARE recognized by each protein. The pentamer AUUUA and the nonamer AUUUAUUUAUUUA motifs are underlined in each panel; the base substitution 981T->G of the BCL-2 ARE (16) is in red (A).

 
To compare the binding profiles of pTINO and AUF1 on the BCL-2 ARE, we mapped the binding sites of p40AUF1 (amino acids 94–297), the other relevant protein found in the library screening, and p37AUF1 (amino acids 1–257), which recently we disclosed to be able to interact with the BCL-2 ARE in RNA THS (16). p40AUF1 is able to bind the segments within the first 75 nucleotides of the BCL-2 ARE (B2ARE1, B2ARE2, B2ARE3, B2ARE5, B2ARE6, and B2ARE7), which identify a larger RNA recognition sequence (Fig. 3, A and B). We determined that p40AUF1 interacts independently with B2ARE10, B2ARE11, and the B2ARE12 motifs (Fig. 3, A and B). p37AUF1 recognizes the motifs B2ARE1, B2ARE2, B2ARE3, and B2ARE7, showing that its binding site is immediately upstream of the nonamer region. Accordingly we found that p37AUF1 is able to interact with B2ARE10 (Fig. 3, A and B). We conclude that pTINO and AUF1 isoforms display different, and partially overlapping, interaction profiles with the BCL-2 ARE.

Genomic Structure and Analysis of the Human TINO Gene— The cloned TINO cDNA contains 2,132 bp (Fig. 4A) that fully overlap human chromosome 19 within the 19p13.3 band, forming three exons on the corresponding genomic DNA region (Fig. 4B). The predicted first exon is short, containing only 67 bp, and it is separated from the second exon by a 10,541-bp intron with canonical GT-AG splice sites; the second (1,347-bp) and the third (718-bp) exon are only 187 bp apart. These 187 bp constitute a short intron possessing the AT-AG dinucleotides as splicing consensus boundaries for the donor and the acceptor site, respectively. This splicing junction AT-AG is a non-canonical splice site belonging to the AT-AC group controlled by the novel type of U12-based spliceosome (21), whose consensus sequences, /ATATCCTTT for the donor site and YAG/for the acceptor site, are present in the 187-bp boundaries. (Slashes indicate the boundary between intron and exon.) The TINO cDNA sequence completely overlaps with another described human mRNA sequence of unknown function that was recently cloned (KIAA2031, GenBankTM accession number AB107353 [GenBank] ) (20). Thus TINO and KIAA2031 appear related. The short intron between exons 2 and 3, arising from the genomic distribution of TINO sequence, is retained in the KIAA2031 cDNA and consequently may represent an alternative intron. TINO and KIAA2031 mRNAs can be considered as alternatively spliced transcripts. However, this is not the only difference between the two sequences: the 5' end of the KIAA2031 cDNA is longer than that of our cloned DNA; indeed an additional 121 bp are present. These additional 121 bp represent the upstream adjacent region on the genomic sequence of the first exon of 67 bp.



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  		FIG. 4.
A, nucleotide and amino acid sequence of TINO cDNA. Numbers on the left indicate nucleotides, and numbers in parentheses on the right indicate amino acids. The stop codon is indicated by an asterisk. The predicted ORF reveals 490 amino acids. The two KH domains for RNA bindingare underlined; the RING domain is in bold characters and underlined. The polyadenylation signal in the 3'-UTR is in white on a black background. B, genomic structure of the TINO gene. The genomic organization of TINO and KIAA2031 are shown. Introns are shown as lines, and exons are shown as boxes. Coding regions are depicted in gray, and non-coding regions are in white. Numbers above indicate nucleotide positions within the two cDNAs. Splice-pair dinucleotides GT-AG (canonical splice site) and AT-AG (non-canonical splice site) are indicated below. C, modular structures of TINO and KIAA2031 proteins. Shown is a schematic representation of the protein motifs within TINO and KIAA2031 proteins. Numbers above indicate the amino acids of the predicted structural motifs, KH1, KH2, and RING domains. The representation also shows the sequence of the alternative carboxyl-terminal ends of the two proteins.

 
TINO cDNA gives rise to a predicted partial protein of 490 amino acids (Fig. 4C). pTINO is an RNA-binding protein possessing two KH domains within its amino-terminal region with the KH1 domain spanning from amino acid 1 to 69 and the KH2 domain spanning from amino acid 96 to 163. In addition, a carboxyl-terminal RING domain spanning from amino acid 424 to 463 is predicted for which the general function is as an E3 ubiquitin-protein ligase. The sequence of the protein pTINO completely overlaps with the KIAA2031 protein (515 amino acids) except for the carboxyl terminus where an alternative 19 amino acids are present. The two predicted proteins differ starting from amino acid 471 of pTINO or amino acid 511 of KIAA2031 at which 19 amino acids (RVETETPQPGGASALQRQY) for pTINO or 4 amino acids (HIFS) for KIAA2031 are present. Curiously the last 4 amino acids and the stop codon of KIAA2031 are encoded by the 187-bp alternative intron present in the transcript (Fig. 4C). Moreover an additional 40 amino acids from the KIAA2031 cDNA can be predicted at the amino terminus compared with pTINO. KIAA2031 protein is probably a partial product since its initial amino acid is a proline and not a methionine.

A search for homologous proteins recovered the putative ORF of the KIAA2009 sequence (GenBankTM accession number AB095929 [GenBank] ) whose locus lies on human chromosome 15q25.2 (Fig. 5). pTINO may be a prototype of a protein family whose members are the KIAA2009 product and another putative member located on human chromosome 1q22. The latter is predicted because of the high identity score of local alignment obtained with our cDNA sequence and human expressed sequence tags overlapping with that DNA region (GenBankTM accession numbers BE315075 [GenBank] , BG746475 [GenBank] , and BQ947909 [GenBank] ). No functionally characterized homologous sequences were obtained among other mammalian species, but TINO homologies are predicted by automated computational analysis from the available GenBankTM expressed sequence tags in Mus musculus (GenBankTM accession number XP_137153) and in Rattus norvegicus (GenBankTM accession number XP_218846 [GenBank] ) species. More interestingly, the sequence comparison with known sequences from other organisms revealed a high degree of conservation of TINO protein. Two orthologous proteins exist: posterior end mark-3 (PEM-3) from Ciona savignyi (22) and muscle excess-3 (MEX-3) from Caenorhabditis elegans (23). These proteins possess the highest sequence identities concentrated at the level of the two KH domains for the nucleic acid binding with PEM-3 sharing 74% identity and MEX-3 sharing 69% identity (Fig. 5).



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  		FIG. 5.
Protein sequence alignment. The protein sequence comparison among pTINO (Homo sapiens), KIAA2009 (H. sapiens), PEM-3 (C. savignyi, GenBankTM accession number AB001769 [GenBank] ), and MEX-3, isoform a (C. elegans, GenBankTM accession number U67864 [GenBank] ) protein sequences is shown (ClustalW version 1.8). PEM-3 and MEX-3 orthologous proteins possess the highest identity within the two KH domains (the amino acids in bold type). The RING domain is underlined. Identical amino acid residues that are shared are in gray (in light gray if they are shared with pTINO sequence or in dark gray if they are not) and marked with an asterisk. Similar amino acids are marked with colons. Secondary structural features ({beta}-sheets as {beta} and {alpha}-helices as H) are shown above, and they correspond to the boxed amino acids.

 
Expression and Size Determination of the TINO mRNA and Protein—We examined the expression profile of endogenous TINO gene using human Northern blots containing poly(A) RNA from different healthy tissues (Fig. 6A). A predominant {approx}3.1–3.2-kb transcript was detected that was present at different levels in all the human tissues tested including heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. A putative splicing variant was also detected in heart and skeletal muscle tissues with an apparent molecular size of {approx}6.5 kb. These data indicate that most likely TINO and KIAA2031 cDNA are related to partial transcripts.



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  		FIG. 6.
A, expression profile of TINO by Northern analysis. To determine the tissue distribution of TINO mRNA, a premade Northern membrane blotted with 5 µg of poly(A) RNAs from normal human tissues (Clontech) was hybridized with a specific probe. The highest hybridization signal is with the poly(A) RNA extracted from human placenta; two bands are visible in lanes 1 and 6 with the heart and skeletal muscle RNAs. From this analysis the estimated TINO mRNA size is about 3,100–3,200 nucleotides. A putative {approx}6.5-kb splicing variant present in heart and skeletal muscle is marked by arrowheads. B, expression profile of TINO mRNA by qualitative RT-PCR in human cell lines. Total RNA extracted from the human RPE cell line and human transformed cell lines (SH-SY5Y, K562, NB4, A431, human umbilical vein endothelial cells (HUVEC), human embryonic kidney (HEK) 293, HeLa, and Jurkat T) was reverse-transcribed with random primers, and the initial 223 bp of TINO cDNA was amplified with specific primers. C, analysis of recombinant pTINO in HeLa Tet-Off cells. Western blot analysis of recombinant pTINO was carried out with the total protein extracts of transiently transfected HeLa Tet-Off cells using the mouse monoclonal anti-His(C-term) antibody ({alpha}-HIS tag) or the rabbit polyclonal anti-pTINO antibody ({alpha}-pTINO). HeLa Tet-Off cells were transfected with either the pQE-TriSystem/TINO vector containing the TINO ORF linked to the histidine tag (lanes 2 and 5) or doxycycline-sensitive pTRE2pur/TINO containing the TINO ORF alone (lane 4). The corresponding controls (mock) are in lanes 1 and 6 for the first and lane 3 for the second type of vector. The polyclonal antibody recognizes the recombinant pTINO in the range of about 60 kDa. M is the molecular weight marker. D, adsorption experiments. To identify the band(s) corresponding to the endogenous product(s) of TINO, a Western blotting analysis with the rabbit polyclonal anti-pTINO antibody ({alpha}-pTINO) adsorbed with the specific peptides was performed. In particular, the peptides were incubated for 1 h at room temperature with anti-pTINO at a 1:20 (antibody/peptides) molar ratio. Thus, total protein extracts of HeLa Tet-Off cells were immunoblotted with the antibody/peptide mixture (pept) or with the primary antibody alone (-). unsp indicates the unspecific protein product probed by the anti-pTINO antibody; it corresponds to the lower band of the doublet spanning from about 70 to 75 kDa.

 
Also the expression data from different human immortalized (RPE) and neoplastic cell lines (SH-SY5Y (neuroblastoma), K562 (chronic myelogenous leukemia), NB4 (acute promyelocytic leukemia), A431 (epidermoid carcinoma), primary human umbilical vein endothelial cells, human embryonic kidney 293, HeLa (adenocarcinoma), and Jurkat T (acute T cell leukemia)) were obtained. Total RNA was extracted, reverse-transcribed, and amplified with gene-specific primers. The specific band was detected in all the tested cell lines with the exception of K562 cells where a very faint band was detectable (Fig. 6B).

Ectopical expression of the TINO gene was examined by Western blotting analysis. Total protein extracts from transiently transfected HeLa Tet-Off cells were collected and analyzed with the anti-pTINO or the anti-His(C-term) antibody (Fig. 6C). Following transfection of the TINO ORF with the pTRE2pur-based vector, the recombinant protein was immunodetected (Fig. 6C, lane 4). By contrast, no signal was detected in the total protein extract prepared from HeLa Tet-Off cells transfected with the empty vector (mock) (Fig. 6C, lane 3). Recombinant pTINO runs in SDS-PAGE with an apparent molecular mass of about 60 kDa, although the calculated mass from the cDNA sequence is about 50 kDa. The peptide antibody specifically recognized the recombinant protein. As a further proof of the specificity, a band of identical size is detectable in HeLa Tet-Off protein extracts probed with the anti-pTINO or the anti-His(C-term) antibody following transient transfection of the His-tagged TINO ORF (Fig. 6C, lanes 2 and 5). By contrast, neither the His-tagged nor the untagged protein was detectable with the relative antibodies in the protein extract of the corresponding controls (mock) (Fig. 6C, lanes 1 and 6). The slight difference in molecular mass between the recombinant proteins expressed from the tetracycline-sensitive promoter or the constitutive promoter is due to the histidine stretch present in the recombinant protein produced by the second kind of vector. The probing of protein extracts with the anti-pTINO antibody detects three others strong bands, two of which possess an apparent molecular weight of about 70–75 kDa and one of about 39 kDa. However, adsorption experiments with specific peptides allowed identification of an unspecific band (Fig. 6D).

Analysis of 5'- and 3'-UTR Regions of TINO mRNA by RACE Analysis—From Northern Blotting analysis we concluded that the cloned TINO cDNA does not correspond to the full-length mRNA; we then used RNA T4 ligation-mediated RACE for 5' and 3' transcript end cloning. With specific primers for 5' and 3' ends, nested PCRs from human placenta cDNA were performed, and the amplicons were cloned in the pCRII vector (Invitrogen) through TA cloning and then sequenced. By means of 3'-RACE we cloned the 3' end of the mRNA and verified that it is perfectly overlapping with the TINO and KIAA2031 cDNAs (data not shown); this fact suggested that the missing mRNA region of TINO is in the 5' end. Despite many attempts, we were unable to clone the true 5' end of the transcript by RNA T4 ligation-mediated RACE; the cloned products showed deletions of the known extreme 5' end at the ligation point.

Analysis of 5' End of TINO mRNA by GENSCAN—To clone the missing 5' end of TINO cDNA we applied a predictive method that defined the entire gene. The GENSCAN program (24) enabled us to analyze the genomic region that we assumed contained our gene, a 21,942-bp region, from bp 1493021 to bp 1514963 of chromosome 19 (GenBankTM accession number NT_011255 [GenBank] .13). This DNA region flanks the neighboring MBD3 gene (methyl-CpG binding domain protein 3). The predicted peptide completely overlaps the KIAA2031 protein and, except for the 4 last amino acids, pTINO and gives rise to a putative 651-amino acid protein. In addition, the major intron and the predicted poly(A) signal overlap with the structure of our gene (data not shown). However, the amino-terminal region of the predicted protein is 136 amino acids longer than KIAA2031; these additional amino acids correspond to a 408-bp exon, reaching a length of about 3,100 bp when added to the 2,441 bases of the KIAA2031 sequence and to the {approx}200 bp of the poly(A) tail. Thus, to validate this prediction, we designed three forward primers within this region, and we used them for PCR amplifications starting from total RNA extracted from RPE and HeLa cells and reverse-transcribed with random primers (Fig. 7A). In both cell lines, the G2 forward primer alone, paired with the R1 reverse primer encompassing the first splice site junction of the gene, gave rise to PCR products. PCR amplicons can be obtained also with the G2-R2 primers (data not shown). On the other hand, no amplification products were obtained when G1-R1 and G3-R1 primer pairs were used. We focused on the G2-R1 major products, identified as two bands, that we gel purified and sequenced. The sequencing data demonstrate that the TINO cDNA was detected (Fig. 7B). For each sequenced product, the relative sequence in the genomic context and the putative amino-terminal region of TINO/KIAA2031 protein are shown. The major PCR product adds 77 bp to the 5' end of the KIAA2031 transcript. Also the human expressed sequence tag fs06d08.y1 (GenBankTM accession number CD674038 [GenBank] ) contains a 5' end similar to the TINO/KIAA2031 gene; indeed it is almost completely overlapping with this product, and the two sequences share 98% identity over the additional 77 bp.



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  		FIG. 7.
A, GENSCAN analysis. Schematic strategy (on the left) and RT-PCR products (on the right) of analysis of the 5' end of TINO with R1 and R2 reverse primers derived from TINO cDNA and G1, G2, and G3 forward primers designed with GENSCAN software. B, GENSCAN RT-PCRs. Distributions of the Upper and Lower RT-PCR products (capital letters and underlined) on the chromosome 19. Consensus splice sites are in gray, pentameric intronic enhancers are in dark gray, and branch sites are in white on a black background. The first nucleotide (G) of the TINO sequence cloned with the RNA THS is in gray on a black background. The relative protein sequences of the Upper and Lower products with underlined novel predicted amino acids are shown under each genomic context. M is the molecular weight marker.

 
Localization of pTINO—We determined the intracellular localization of endogenous pTINO. Immunofluorescence revealed that pTINO was localized predominantly in the nucleus, where local accumulation in clusters is visible, and also in the perinuclear region. No expression was observed in the nucleoli. In addition, a spotted distribution of the protein is visible in the cytoplasm (Fig. 8, A and B). Nuclear staining faded away when anti-pTINO antibody was preincubated with its peptide antigens. However, the faint cytoplasmic staining remained (data not shown).



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  		FIG. 8.
Immunolocalization of endogenous TINO protein in HeLa cells. Shown is immunocytochemically stained protein pTINO demonstrating a prevalent nuclear and perinuclear localization (B) with fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody (green fluorescence) (A). Nuclear DNA was counterstained with Hoechst 33258 (blue fluorescence).

 
pTINO Influences the Levels of a {beta}-Globin/ARE Chimeric Transcript—We proceeded to study the function of TINO by its transient transfection into HeLa Tet-Off cells. We linked a {beta}-globin reporter gene that is under the transcriptional control of a doxycycline-sensitive promoter (25) to the 107-mer ARE sequence in the pTRE2pur/B2ARE construct. Upon transient transfection, {beta}-globin/ARE mRNA of uniform length is synthesized. Addition of doxycycline blocks transcription of the {beta}-globin/ARE gene. The functional contribution of TINO to ARE-directed mRNA decay was examined by cotransfection of repressible reporter plasmids encoding either the {beta}-globin mRNA linked to the 107-mer ARE sequence or the {beta}-globin mRNA alone. HeLa Tet-Off cells were cotransfected with the pQE-TriSystem alone (mock) or with pQE-TriSystem/TINO (Fig. 9). Upon transient transfection into HeLa Tet-Off cells, reporter mRNA synthesis was allowed for 48 h until addition of doxycycline. RNA levels of reporter constructs were determined by RNase protection assay. Control {beta}-globin mRNA lacking the ARE was intrinsically stable, and TINO did not induce its decay as observed during the 6-h interval (Fig. 9A, quantification in C). {beta}-Globin/ARE mRNA decays faster with a half-life of 6.3 h. When TINO was cotransfected, the half-life of the chimeric RNA was reduced to 2.9 h, indicating an enhanced decay rate (Fig. 9B, quantification in C). We conclude that the BCL-2 ARE destabilizes the {beta}-globin/ARE mRNA, and ectopic expression of TINO acts through the BCL-2 ARE to enhance this effect.



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  		FIG. 9.
mRNA stability analysis with pTINO. HeLa Tet-Off cells were transiently cotransfected with vector only (MOCK) or a vector encoding pTINO (TINO) and a vector producing reporter RNA {beta}-globin (A)or {beta}-globin + ARE (B)of the BCL-2 3'-UTR. The synthesis of the reporter RNAs was inhibited by doxycycline (Dox) 48 h posttransfection, and total RNA was isolated at different time points. Reporter {beta}-globin RNAs were quantified by RNase protection assay. Endogenous glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used for normalization. Half-lives of reporter RNAs with or without TINO were calculated from first-order decay constant (k) (C) (see "Experimental Procedures").

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we describe pTINO, a novel human, evolutionarily conserved protein that interacts with the AU-rich element of BCL-2 mRNA. TINO gene was identified following two serial library screenings of two different versions of the yeast RNA THS. The IST of TINO resulted from analysis of its binding specificity with the BCL-2 ARE sequence at the end of RNA-protein interaction screenings. The RNA THSs were reconstituted by yeast mating assays to validate the binding specificity interaction of the novel cloned protein with the BCL-2 ARE. TINO protein did not transactivate the lacZ reporter gene in the presence of the unrelated 5'IRE-MS23' RNA bait and did not significantly interact with the hybrid RNA 5'MS2-B2{Delta}ARE3' where the BCL-2 ARE is deleted of the nonamer (Fig. 1). In contrast, RNA-protein complex formation both in vivo and in vitro occurs with the wild-type BCL-2 ARE sequence (Figs. 1 and 2). This indicates that the nonamer is needed for RNA-protein complex formation. However, the nonamer is not the only motif of BCL-2 ARE required for the interaction of pTINO. We mapped the binding site of pTINO and disclosed that it must also contain the adenine stretch immediately upstream of the nonamer (Fig. 3, A and B). Although this relatively long sequence (5'-AGAAAAAAAGAUUUAUUUAUUUAAGA-3') is not shared with other ARE-harboring human mRNAs, we cannot exclude the possibility that pTINO could interact with other motifs.

The cloned cDNA is a 2,132-bp segment whose protein product has two KH domains (Fig. 4, A and C). The conserved KH motif, first identified in human hnRNP K, is shared by a wide variety of nucleic acid-binding proteins, many of which take part in the complex network of protein-protein and RNA-protein interactions regulating gene expression in eukaryotes (26). KH domains consist of an {alpha}-{beta} fold with a topology similar to that found in ribosomal proteins (27). Like other RNA binding motifs, KH domains are found in either one or multiple copies; for example hnRNP K and human onconeural ventral antigen-1 possess three copies, fragile X mental retardation protein 1 possesses two copies, and even 14 copies are present in chicken Vigilin. Two KH domains are predicted at the amino terminus of pTINO. At the carboxyl terminus a RING module is predicted. This RING finger is a specialized type of Zn2+ finger domain of 40 amino acids that bind two atoms of zinc, probably involved in mediating protein-protein interactions. Based on the cysteine/histidine pattern, the variant of RING module present in pTINO is the C3HC4 type. The RING domain has been implicated in a range of biological processes. E3 ubiquitin protein ligase activity is ascribed to the RING domain of C-CBL protein, which is able to recruit an E2 ubiquitin-conjugating enzyme and its own substrates (28). It is believed that E3-like activity is a general function of the RING domain.

Evidence of the high degree of interspecies conservation of pTINO is revealed from data base searches using protein-protein BLAST (Fig. 5). pTINO is probably the prototype of a small protein family of KH-containing RNA-binding proteins. Indeed transcripts (KIAA2009) and expressed sequence tags of unknown genes have been cloned and predicted to codify polypeptides similar to pTINO, especially with respect to the KH domains. Two putative other members of the protein family can be found on human chromosomes 15q25.2 and 1q22. Among the ancient bilaterian animals, such as ascidians and nematodes, two orthologous proteins, PEM-3 (C. savignyi) and MEX-3 (C. elegans), respectively, have high homology with pTINO (Fig. 5). PEM-3 and MEX-3 proteins are RNA-binding proteins whose biological relevance appears during embryonic development (22, 23). Many crucial decisions, such as the timing of cell division and cell fate determination, are made in the early phases of embryonic development when little or no transcription occurs, and gene expression often relies on posttranscriptional controls (29). The MEX-3 protein is implicated in anterior-posterior differentiation of C. elegans embryos; in particular mex-3 expression is required for proper muscle development of the worm. We know that MEX-3 is implicated in gene expression control of the pal-1 gene and that this control is dependent on the 3'-UTR region of the pal-1 mRNA. In particular, MEX-3 is a negative regulator of pal-1 gene expression, possibly repressing PAL-1 translation by a direct interaction with the corresponding mRNA region (30). Indeed recombinant MEX-3 protein was demonstrated to bind to the pal-1 3'-UTR in vitro (31). The evolutionarily conserved pTINO interacts with the cis-acting element of the antiapoptotic BCL-2 gene. Apparently there is no relation between the two genes, pal-1 and BCL-2, but the final output of their expression control in muscle progenitor cells could be the same. For example, BCL-2 is expressed in human skeletal muscle cells at an early stage of myogenic differentiation, promoting clonal expansion (32). BCL-2 protein levels decrease along the myogenic pathway from muscle stem cell to myofiber. All these observations suggest that TINO may be a negative regulator of the BCL-2 gene during embryonic development.

MEX-3 acts in the cytoplasm during the early cleavage stages of the embryonic cell and is localized in P-granules (23), which are cytoplasmic structures containing RNAs and RNA-binding proteins playing a role in mRNA processing or packaging (33). By contrast, we observed that endogenous protein is mostly distributed in the nuclear-perinuclear cell compartment (Fig. 8).

Northern blot analysis of the transcript of TINO indicates an apparent size of about 3.1 kb present in all tested poly(A) RNAs extracted from healthy human tissues (Fig. 6A). Placenta shows the stronger hybridization signal, while in the heart and skeletal tissue additional putative {approx}6.5-kb splicing variant is present. However, due to the low level of expression of this product, it can also be either a related transcript or a processing intermediate. The recently cloned KIAA2031 (20) cDNA is located in the same genetic locus as TINO on chromosome 19, suggesting that TINO and KIAA2031 are the same gene. Both sequences completely overlap except for two differences: the KIAA2031 transcript possesses a 187-nucleotide short intron containing the stop codon, which is not present in the TINO transcript. This raises the possibility that the two mRNAs are the result of the alternative splicing known as intron retention (34).

Northern analysis suggested that TINO and KIAA2031 sequences do not correspond to the full-length mRNA, indicated by the size discrepancies between the expected and detected transcripts. To identify the 5' and 3' ends of the mRNA of TINO, we applied two different approaches: PCR-based 5'- and 3'-RACE and the gene prediction program GENSCAN. RACE analysis showed that the missing part of the transcript is the 5' end. The 3'-RACE product was completely overlapping with the terminal 3'-UTR of TINO and KIAA2031. Through the GENSCAN analysis, we detected two RT-PCR products in HeLa and RPE cells (Fig. 7A), extending by 198 and 159 nucleotides the 5' ends of TINO cDNAs. We believe these represent alternative splicing products. Their sequences were aligned within the corresponding genomic region and translated into protein. At the genomic level they form two short introns (Fig. 7B). The splicing consensus boundaries are GC-CG for the longer and GA-CG for the shorter form. These acceptor and donor site pairs are neither canonical nor noncanonical splice sites, nevertheless we cannot exclude the possibility that the transcripts derive from alternative splicing because of the presence of a branch site and pentamer (CTGCC) functioning as intronic splicing enhancer (35). This kind of pentamer accurately identifies human short introns. It represents the major contribution to intron recognition when computational analyses are performed. At the protein level, the difference seems to be functionally sound. Indeed the two products codify proteins differing only at a particular phosphorylation site, whose consensus sequence is RXRXX(S/T), which is the substrate of protein kinase B/AKT. This site, in the form RGRPGT, is present in the longer product (Fig. 7B).

The antibody obtained with a peptide-based method allowed the detection of three major bands in Western blot, possibly corresponding to the endogenous pTINO: a doublet of about 70–75 kDa and one single band of about 39 kDa (Fig. 6C, lanes 3 and 6). The lower band in the upper doublet appears to be unspecific because it is clearly detected even when the antibody is blocked by the specific peptides (Fig. 6D). The size of the specific upper band suggests the presence of an extra sequence with respect to the recombinant pTINO. The extra sequence corresponds to the unknown amino terminus of the novel protein, while the lower band probably derives from proteolytic processing yielding the band of about 39 kDa.

What is the functional significance of the physical association between pTINO and the BCL-2 ARE? To address this question, we used an inducible {beta}-globin reporter gene with the BCL-2 ARE linked to the 3'-UTR. Transient transfection of TINO significantly accelerated (2-fold) mRNA reporter decay when the 107-mer ARE motif was present in the mRNA 3'-UTR (Fig. 9). Since the destabilizing protein AUF1 is another BCL-2 mRNA AU-rich element-binding protein (16), we suppose that pTINO is part of the mechanism of posttranscriptional control of BCL-2 expression and probably acts "cooperatively" with AUF1 (Fig. 3, A and B). In particular, the pooling of the binding sites of TINO and AUF1 over 48 nucleotides of the BCL-2 ARE (where pTINO partially shares its binding site with p40AUF1 and is contiguous to the binding site of p37AUF1, while p37AUF1 completely overlaps the binding site of p40AUF1) suggests a strictly coordinated mode of action that could explain different outputs of BCL-2 expression in basal condition and during apoptosis. The ARE-based decay pathway is a cytoplasmic event, while the subcellular localization of the endogenous pTINO we observed is mostly nuclear and perinuclear (Fig. 8). Accordingly we suspect that the BCL-2 mRNA destabilizing properties can be enhanced by pTINO redistribution to the cytoplasm upon apoptosis. One example of regulated ARE-binding protein is furnished by CUG-binding protein 2, a multifunctional protein containing three non-identical RNA recognition motifs, that is capable of apolipoprotein B and cyclooxygenase-2 AU-rich sequence interaction. Interestingly CUG-binding protein 2 is a highly conserved AU-rich element-binding protein able to relocate from the nucleus to the cytoplasm following apoptotic stimuli and thus able to repress cyclooxygenase-2 mRNA translation (36, 37). Our future research on TINO will examine its functional participation in cellular life and death, both in physiological and pathological conditions, and verify its cellular redistribution and transport pathway.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF458084 [GenBank] .

* This work was supported by grants from the Italian Association for Cancer Research (AIRC), Ministero della Università e Ricerca (MIUR), Ente Cassa di Risparmio di Firenze, and Consiglio Nazionale delle Ricerche/MIUR (Grant Progetto Finalizzato Oncologia, Ministero della Salute) (to S. C.) and by National Institutes of Health Grant CA 52443 (to G. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Recipient of a fellowship from Fullbright and Fondazione Italiana per la Ricerca sul Cancro. Back

§§ To whom correspondence may be addressed. Tel.: 390554282309; Fax: 390554282333; E-mail: sergio{at}unifi.it. ¶¶ To whom correspondence may be addressed. Tel.: 390554282309; Fax: 390554282333; E-mail: nicola{at}unifi.it.

1 The abbreviations used are: hnRNP, heterogeneous nuclear ribonucleoprotein; THS, three-hybrid system; ARE, AU-rich element; KH, hnRNP K homology; AUF1, AU-rich factor 1; IST, interacting sequence tag; PEM-3, posterior end mark-3; MEX-3, muscle excess protein-3; RACE, rapid amplification of cDNA ends; RT, reverse transcription; UTR, untranslated region; ORF, open reading frame; RPE, retinal pigment epithelial; PBS, phosphate-buffered saline; E3, ubiquitin-protein ligase; E2, ubiquitin-conjugating enzyme. Back

2 See genes.mit.edu/GENSCAN.html. Back

3 M. Donnini, A. Lapucci, L. Papucci, E. Witort, A. Jacquier, S. Capaccioli, and N. Schiavone, unpublished results. Back


   ACKNOWLEDGMENTS
 
We are grateful to Matteo Lulli, Francesca Fedeli, Elisabetta Borchi, and Federico Perna for technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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