PIPPin, a Putative RNA-Binding Protein Specifically Expressed in the Rat Brain 1 Daniele Castiglia, 2 Maria Scaturro, Tommaso Nastasi, Alessandro Cestelli, Italia Di Liegro 3 Dipartimento di Biologia Cellulare e dello Sviluppo “Alberto Monroy,” viale delle Scienze, Parco d’Orleans, 90128 Palermo, Italy Received November 21, 1995 In looking for genes encoding RNA-binding factors, we prepared an expression library in gt11, by cloning cDNAs corresponding to the polyadenylated fraction of RNA from rat brain at the embryonal day 18. The library was then screened by binding to a radioactive RNA, transcribed in vitro from a cDNA encoding the rat histone variant H3.3. Here we report some findings concerning a cDNA for a protein which contains two putative double stranded RNA-binding domains (dsBD). The corresponding message is specifically expressed in the brain. Moreover, Southern blot analysis showed that the gene is highly conserved from Drosophila melanogaster to man. © 1996 Academic Press, Inc. In the last few years an ever growing number of evidences has pointed to the importance of regulation of mRNA localization, stability and translation, in the control of gene expression, both in development (1–6) and in the maintenance of a differentiated cell phenotype (7–14). Posttran- scriptional control processes are mediated by several RNA-binding proteins (RBPs) (5,9,15–20) and much progress has been made in the identification and characterization of the functions of many of these factors. The cloning of the corresponding cDNAs and the analysis of the amino acid sequences have led to the discovery of different conserved motifs in RBPs (for review, see: 17); in particular, several RBPs have in common one or more copies of a domain able to bind double- stranded RNA (dsRNA) (15,17–18). Here we describe a cDNA which encodes a protein that contains two putative dsRNA-binding domains and is expressed specifically in the rat brain. MATERIALS AND METHODS Cloning of putative RNA-binding proteins. Polyadenylated mDNA from developing rat brain was used as a template for cDNA synthesis. cDNA was then cloned in the gt11 Sfi-Not vector (Promega, USA), according to the manufacturer’s instructions. Screening of the library was done by probing the ability of the proteins (produced by the clones) to bind a [ 32 P]-radiolabeled H3.3 mRNA, transcribed in vitro from the T3 RNA polymerase promoter of a recombinant pGEM EX (Promega, USA) plasmid which contains an H3.3 insert (acc.n. X73683: ref.21). We obtained two positive clones, the inserts of which were subsequently subcloned into the bluescript plasmid vector. One of these clones (called PIPPin cDNA) has been sequenced on both strands by the dideoxynucleotide chain termination method (22), using the Sequenase DNA- sequencing kit (United States Biochemicals, Cleveland, OH). Sequence alignments were done using MacMolly Tetra Program by Gene Soft. Northern analysis. Total RNA was isolated from different rat adult tissues and from rat brains at different ages of development, according to Chomczynsky and Sacchi (23). Electrophoretic separation and Northern analysis of RNA were performed as described elsewhere (21). For standardization, the membranes were also probed with a 1.4 Kb Bam H1 fragment derived from the human ribosomal gene cluster, which specifically hybridizes to the 28S rRNA (24). Southern analysis. 10g of genomic DNA from different sources was digested with restriction enzymes, separated by 0.8% agarose gel electrophoresis and blotted onto a Hybond N + membrane (Amersham Int.). The blot was then hybridized with the [ 32 P]-labeled PIPPin cDNA. The membrane was washed for 20 min in 2XSSC/0.1% SDS at room temperature and for 20 min in 0.5XSSC/0.1% SDS at 55°C. 1 The sequence of PIPPin cDNA has been deposited in the EMBL/GenBank/DDBJ databases (accession no. X89962). 2 Present address: Istituto Dermopatico dell’Immacolata, IDI, via dei Monti di Creta 104, 00167 Roma, Italy. 3 To whom correspondence should be addressed. Fax: +39-91-420897. BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 218, 390–394 (1996) Article No. 0068 390 0006-291X/96 $12.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.