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Specific Binding of a Hela Cell Nuclear Protein to RNA Sequences in The Proc. Nati. Acad. Sci. USA Vol. 86, pp. 4858-4862, July 1989 Biochemistry Specific binding of a HeLa cell nuclear protein to RNA sequences in the human immunodeficiency virus transactivating region (chemical nuclease imprinting/untranslated RNA binding protein 1) RICHARD GAYNOR*tt, EMMANUEL SOULTANAKIS*t, MICHIo KUWABARA*§, JOSEPH GARCIA*t, AND DAVID S. SIGMAN*§ *Molecular Biology Institute, and tDivision of Hematology-Oncology, Department of Medicine, and §Department of Biological Chemistry, School of Medicine, and tWadsworth Veterans Hospital, University of California, Los Angeles, CA 90024 Communicated by Paul D. Boyer, March 27, 1989 ABSTRACT The transactivator protein, tat, encoded by been shown to be involved in increasing the steady-state level the human immunodeficiency virus is a key regulator of viral of RNA, it has also been reported to increase the translation transcription. Activation by the tat protein requires sequences of RNA (15, 17, 22). The possibility that increased levels of downstream of the transcription initiation site called the trans- RNA are attributable to an antitermination mechanism has activating region (TAR). RNA derived from the TAR is capable also been raised (5, 18-20, 27). In contrast to other transac- offorming a stable stem-oop structure and the maintenance of tivating proteins, such as ElA (28), which activate a number both the stem structure and the loop sequences located between of viral and cellular promoters, tat activation is specific for + 19 and +44 is required for complete in vivo activation by tat. the HIV LTR. It is of interest to identify features ofthe TAR Gel retardation assays with RNA from both wild-type and responsible for this selectivity. mutant TAR constructs generated in viro with SP6 polymerase Since TAR RNA secondary structure appeared to be one indicated specific binding ofHeLa nuclear proteins to the TAR. component required for complete activation by the tat pro- To characterize this RNA-protein interaction, a method of tein, the possibility that a cellular protein may mediate the chemical "imprinting" has been developed using photoacti- interaction of tat with TAR RNA has been explored. The vated uranyl acetate as the nucleolytic agent. This reagent nicks methods used were gel-retardation analysis (29) and a new RNA under physiological conditions at all four nucleotides in a method of examining RNA-protein interactions in which reaction that is independent of sequence and secondary struc- light-activated uranyl acetate is used as the nucleolytic agent. ture. Specific interaction of cellular proteins with TAR RNA This reagent, which was previously shown to cut DNA in a could be detected by enhanced cleavages or imprints surround- sequence-independent reaction (30), cleaves RNA in a pri- ing the loop region. Mutations that either disrupted stem mary and secondary structure-independent manner under base-pairing or extensively changed the primary sequence physiological conditions. Both methods demonstrate specific resulted in alterations in the cleavage pattern ofthe TAR RNA. binding of a HeLa nuclear protein which we designate as Structural features ofthe TAR RNA stem-oop essential for tat untranslated RNA binding protein 1 (URBP-1) to TAR RNA. activation are also required for specific binding ofthe HeLa cell nuclear protein. MATERIALS AND METHODS Plasmid Constructs and Labeling of mRNAs. Wild-type and Gene expression of the human immunodeficiency virus mutant HIV mRNAs shown in Fig. 1 were constructed by (HIV) is dependent on multiple cis-acting sequences in the taking a Pvu II/Xho I fragment (-18 to +80) from pJGFCAT long terminal repeat (LTR) which serve as the binding sites derivatives (10) and inserting them into a pGEM derivative in for cellular proteins that are important in viral gene regulation which the Pst I site was converted into an Xho I site by linker (1-13). In addition, at least three viral proteins-tat, rev, and insertion. Each pGEM construct was digested with HindIII, nef-are also involved in modulating gene expression (14- and RNA synthesis, labeling, and purification were per- 26). One of these HIV-encoded proteins, tat, requires for its formed using the reagents and procedures of the Riboprobe action a region of the HIV LTR extending from -17 to +80, System II (Promega) (29). known as the transactivating region (TAR) (1, 5-10). Gel Retardation Assays and Chemical Footprinting of RNA. TAR sequences may be involved in tat regulation of HIV Gel retardation assays were performed as described by transcription at both the DNA and RNA levels. For example, Leibold and Munro (29). Internally labeled mRNA (50,000 it is the recognition site for cellular DNA binding proteins cpm) was incubated with 1-5 ,ug ofa heparin-agarose column- UBP-1 (12) and UBP-2 (10) (UBP, untranslated binding purified HeLa nuclear extract (31) in 10 ,ul for 20 min (5, 30). protein). Mutagenesis of multiple UBP-1 binding sites in the When included, 1 pmol ofthe indicated unlabeled competitor TAR resulted in decreased activation by the tat protein in mRNA was addedjust prior to the addition oflabeled mRNA. transient gene expression assays with the HIV LTR (9, 10). For chemical cleavage, RNA was 5'-end-labeled (100,000 On the other hand, RNA from the TAR is capable offorming cpm) and incubated as in the gel retardations except 50 mM a stable stem-loop structure (4). Mutagenesis of the TAR NaCl was used instead of 100 mM NaCl and reaction volumes indicated that maintenance ofstem base-pairing between +19 were 100 1.L. Then, 10 Jud of 20 mM uranyl acetate was added and +44 was critical for tat activation (6, 10). However, the followed by exposure to short-wave UV light (7000 p.W/cm2) primary sequence of the loop region between +28 and +34 for 30 min. RNA was extracted, ethanol precipitated, and was also critical for this activation (6, 10). loaded onto a 10% polyacrylamide sequencing gel. Footprint- The mechanism(s) by which the tat protein activates HIV ing lanes were calibrated with partial ribonuclease T1 digests gene expression is not known. Although the tat protein has and autoradiography was performed. The publication costs of this article were defrayed in part by page charge Abbreviations: HIV, human immunodeficiency virus; LTR, long payment. This article must therefore be hereby marked "advertisement" terminal repeat; TAR, transactivating region; UBP, untranslated in accordance with 18 U.S.C. §1734 solely to indicate this fact. binding protein; URBP-1, untranslated RNA binding protein 1. 4858 Biochemistry: Gaynor et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4859 A G G G G C G G G U G U G U G U G C A C A C A C A C A C - G C- G C - G C - G C - G G - C G- C G - C G - C C - C A -U A - U A - U A - U UA U G- C U U G- C U G- C U G- C U G- C U A- U U A- U US U UA a G- C G - C G- C G - C UG - A.C A - U A - U ~OU A .. C- G C - G C - G m-g - G C - G G C - G - G AC A G- A-C-C AC AG -C C G G- C G - C A- U A- U A - U A- U A - U U - A U - A U - A U - A U- A U - A U - A U - A U - A - G - C G C G - C G - C G - C G*U G U CG U G.U U - A U - A U - A U - A U - A C - G C - G C - G C - G C - G U*G U*G U .G U G U G C - G C - G C - G C - G C - G U - A - U- A C C U -A C U U-A C U - A U - A U - A U - A C U - A G - C G - C G - C G - C G - C - G - C G C G - C G - C G - C m GpppG- CACU GpppG- CACU m GpppG- CACU m GpppGC CACU GpppG- CACU +1 +62 +1 +62 +1 +62 +1 +62 +1 +62 WT(+80) [+31/+34] [+19/+22] [+40/+43] 1+19/+22]/[+40/+43] B (; ( (; (; C U G U G C A C A C A C- (; G - C G - C (;-C A- U AJ-UA U A-U C-G U CCA U A-U C U A- U C-C AA-U;^-U A-U A-U C-GC C -(; C - C- G A A' G- C G-C C -C A- A-U A-U U (; - C U'-AU-AG- C U-A U-A C - C C-G C-A-G2G A-U FIG. 1. Secondary structure ofTAR C-G :h-U C-C U-A: mutants. RNA transcribed from the UF C U-A C -G TARwith stem base pairingand the loop region are shown. (A) Wild type and C U GCCCUl mutants [+31/+34], [+19/+22], [+40/ UA U-A +43], and [+19/+22]/[+40/+43] are in- C-CG-C G-C C-C dicated. (B) Wild type and mutants G-C C-C ATAR-sense and ATAR-antisense are m Gppp G G mCppp G G mCppp G C indicated. The sequencefrom +1 to +62 +1 +59 +1 +60 +1 +60 in each construct is shown and the po- sitions ofthe mutations are indicated by WT(ATAR) ATAR-Sense ATAR-Antisense the shaded regions. RESULTS extract (Fig. 2A). This species specifically competed with a Gel-Retardation Analysis of Wild-Type and Mutant TAR 50-fold excess of unlabeled wild-type RNA, but not with a RNAs.
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