A Nuclear Matrix-Associated Factor, SAF-B, Interacts with Specific Isoforms of AUF1/hnRNP D Yukitomo Arao,* , † Reiko Kuriyama,* Fujio Kayama,† and Shigeaki Kato* , ‡ ,1 *Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan; †Department of Health Science, Jichi Medical School, Yakushiji, Minamikawachi-machi, Tochigi 329-0498, Japan; and ‡CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan Received February 8, 2000, and in revised form May 11, 2000 One class of heterogeneous nuclear ribonucleopro- teins (hnRNPs), AUF1/hnRNP D, consists of four iso- form proteins (p45, p42, p40, and p37) which are gen- erated by alternative splicing. The present study was therefore undertaken to clarify any isoform-specific differences in terms of their functions and nucleocy- toplasmic localization. All isoforms primarily local- ized in the nucleus. However, heterokaryon analysis and a study using RNA polymerase II inhibitor re- vealed that p40/p37 exhibited a continuous shuttling between the nucleus and cytoplasm. Constant nuclear retention activity was mapped to the p45/p42-specific sequence at the C-terminal region, which is retained by alternative splicing. Using this domain as a probe, we performed a yeast two-hybrid screening and we found that scaffold attachment factor B (SAF-B), a nu- clear matrix-associated protein, exhibits protein–pro- tein interaction to this region. Colocalization of p45/ p42 and SAF-B was observed as a speckle in the nu- cleus. Interestingly, p45/p42 isoforms appeared to act as a negative regulator in gene expression by forming a complex with SAF-B. Thus, the present study re- vealed that the isoform-specific functions of AUF1/ hnRNP D are defined by intracellular shuttling capac- ity. © 2000 Academic Press Key Words: AUF1; hnRNP D; SAF-B; nuclear reten- tion signal. Gene expression is controlled by transcriptional and posttranscriptional steps. Posttranscriptional modula- tion is known to be regulated at various steps, such as RNA splicing, polyadenylation, cytoplasmic transpor- tation, and mRNA degradation. Recently, it has been elucidated that numerous RNA binding proteins play pivotal roles in these posttranscriptional events (1– 4). One of the most intriguing aspects of heterogeneous nuclear ribonucleoproteins (hnRNPs) 2 is their intracel- lular transportation, which allows them to function in different ways. Although a subset of hnRNPs is pri- marily located in the nucleus, they are shown to con- tinuously shuttle between the nucleus and cytoplasm (hnRNP A1, E, and K) while others are strictly located in the nucleus (hnRNP C and U) (5). Previous reports suggested that such shuttling proteins play distinct roles upon their shift from nucleus to cytoplasm. For instance, hnRNP K was first identified as a transcrip- tion factor regulating some gene expressions, either positively or negatively, in the nucleus (6 –9). Further- more, a novel role of hnRNP K as a translation repres- sor of 15-lox mRNA in the cytosol was recently discov- ered (10). Thus, these findings suggest that nucleocy- toplasmic shuttling proteins exert distinct functions in the nucleus and cytoplasm, though the molecular in- tracellular localization mechanism of hnRNPs remains largely unknown. AUF1/hnRNP D was found as one such hnRNPs. AUF1/hnRNP D was first purified from cytoplasmic extracts of K562 human erythroleukemia cells by mon- itoring the activities of its binding to the 3' untrans- lated region (UTR) of c-myc mRNA, and later it was 1 To whom correspondence should be addressed. Fax: 81-3-5841- 8477. E-mail: uskato@mail.ecc.u-tokyo.ac.jp. 2 Abbreviations used: hnRNP, heterogeneous nuclear ribonucleo- protein; UTR, untranslated region; DMEM, Dulbecco’s modified Ea- gle’s medium; FCS, fetal calf serum; IPTG, isopropyl--thiogalacto- pyranoside; GST, glutathione S-transferase; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; HA, hem agglu- tinin; ACTD, actinomycin D; GFP, green fluorescent protein; PEG, polyethylene glycol; PR, progesterone receptor; S/MAR, scaffold or matrix attachment region; CTD, C-terminal domain; PMSF, phenyl- methylsulfonyl fluoride; SAF-B, scaffold attachment factor B; TRN, transportin. 228 0003-9861/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 380, No. 2, August 15, pp. 228 –236, 2000 doi:10.1006/abbi.2000.1938, available online at http://www.idealibrary.com on