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Characterization of Small Nontranslated Polyadenylylated Proc. Natl. Acad. Sci. USA Vol. 93, pp. 2037-2042, March 1996 Biochemistry Characterization of small nontranslated polyadenylylated RNAs in vaccinia virus-infected cells (inhibition of protein synthesis/in vitro transcription/in vitro translation/in vivo polyadenylylation of tRNAs and small nuclear RNAs) CHUNXIA Lu AND ROSTOM BABLANIAN* Department of Microbiology and Immunology, State University of New York, Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203 Communicated by Howard L. Bachrach, U.S. Department ofAgriculture, Greenport, NY, September 28, 1995 (received for review June 22, 1995) ABSTRACT Host protein synthesis is selectively inhibited uninfected, infected-with-VV or infected-with-UV-irradiated in vaccinia virus-infected cells. This inhibition has been associ- VV (9600 ergs/mm2) HeLa spinner cells. Poly(A)+-containing ated with the production of a group of small, nontranslated, mRNA was isolated from total cytoplasmic RNA by oligo(dT)- polyadenylylated RNAs (POLADS) produced during the early cellulose chromatography. POLADS were isolated as de- part of virus infection. The inhibitory function of POLADS is scribed (10). associated with the poly(A) tail of these small RNAs. To deter- Construction of Plasmids That Contain POLADS. The mine the origin of the 5'-ends of POLADS, reverse transcription cDNA of POLADS was obtained by reverse transcription (13) was performed with POLADS isolated from W-infected cells at using a cDNA synthesis kit (United States Biochemical) and 1 hr and 3.5 hr post infection. The cDNAs ofthese POLADS were cloned into pBS or pBluescript KS 11 +/- at the Sma I site. cloned into plasmids (pBS or pBluescript H KS +/-), and their In Vitro Transcription. Constructs were linearized by Xba I nucleotide composition was determined by DNA sequencing. The or Kpn I, and in vitro transcription was done (14) in the results of this investigation show the following: There is no presence of T3 or T7 RNA polymerase, depending on the specific gene encoding for POLADS. The 5' ends ofPOLADS may orientation of the inserted cDNA of POLADS. be derived from either viral or cellular RNAs. Any RNA sequence Measurement of Protein Synthesis in Vitro. In vitro protein including tRNAs, small nuclear RNAs and 5' ends ofmRNAs can synthesis was done in reticulocyte lysate (15) treated with become POLADS if they acquire a poly(A) tail at their 3' ends micrococcal nuclease (16). Twenty-five-microliter reaction during infection. This nonspecific polyadenylylation found in mixtures were programmed with virus or HeLa mRNA, and vaccinia virus-infected cells is probably conducted by vaccinia proteins were labeled with [35S]methionine (7). virus poly(A)+ polymerase. No consensus sequence is found on Determination of the Origin of 5' Ends of POLADS. Cloned the 5' ends of POLADS for polyadenylylation. The 5' ends of POLADS were sequenced with modified T7 polymerase Se- POLADS have no direct role in their inhibitory activity ofprotein quenase (17). The reaction was done on alkaline-denatured synthesis. double-stranded plasmid templates (18), and either a T7 primer or a reverse primer was used in the reactions. The DNA During vaccinia virus (VV) infection, a population of small sequences of the POLADS were compared with the nucleotide nontranslated polyadenylylated RNAs (POLADS) are pro- sequence database provided by the National Center for Bio- duced. The relation between the production of POLADS and technology Information using the BLAST network service. the selective inhibition of cell protein synthesis has been exten- Construction of Artificial POLADS. Poly(C) and rRNAs sively studied (1-10). POLADS selectively inhibit host protein were hydrolyzed with 0.2 M NaOH for 10 min at 37°C and synthesis (3-7), and the inhibitory moiety has been shown to neutralized with HCl (0.2 M). These RNAs were precipitated reside only on their poly(A) tails (5, 6, 10). The longer the poly(A) with 100% ethanol and electrophoresed on a thin (0.4 mm) tails, the greater the inhibitory activity of POLADS (5, 6, 10). 5% acid/urea/polyacrylamide gel. The 80- to 150-nt-long To further characterize these inhibitors, we cloned POLADS RNAs were eluted from the gel in 0.5 M NH4 (OAC), 10 mM and determined their composition. The results in this investiga- EDTA, and 0.1% SDS at 37°C for 18 hr. Polyadenylylation tion demonstrate that during VV infection, both viral or host of these RNAs (rRNA, poly(C), and tRNA) was done in the mRNAs and, in addition, host tRNA and small nuclear RNA are presence of Escherichia coli poly(A) polymerase. In 100-,l polyadenylylated and may serve as POLADS. Polyadenylylation reaction mixture, there were 8 units of poly(A)+ polymerase, of these normally nonpolyadenylated host cell RNAs is probably 100 mM Tris-HCl (pH 8.0), 500 mM NaCl, 20 mM MgCl2, 5 conducted by VV poly(A) polymerase that is released from the mM MnCl2, 2 mM ATP, bovine serum albumin at 1 ,g/IAl virus core into the cytoplasm during replication. Thus, host RNAs and 9 ,ug of RNA. This reaction mixture was incubated at other than mRNAs become polyadenylylated in vivo. 37°C for 15 min, and after the addition of 1 ,ul of 10% SDS the RNAs were precipitated in ethanol. MATERIALS AND METHODS RESULTS Cells and Viruses. Human HeLa spinner cells were grown in Eagle's spinner medium supplemented with 5% newborn calf Origin of 5' Ends of POLADS at 1 hr Postinfection (p.i.). serum (NCS). The cells were infected with WR strain of Infection of HeLa spinner cells with VV at a multiplicity of 500 vaccinia virus and purified according to the method of Joklik particles per cell resulted in the inhibition of host protein (11). synthesis as early as 1 hr p.i. and reached a maximum at 3.5 hr Isolation ofTotal Cytoplasmic RNA and poly(A) Containing p.i. POLADS at these two time points were isolated, cloned, RNA from HeLa Cells or W-Infected HeLa Cells. Cytoplasmic and sequenced. Out of 44 pBSPOLADS sequenced, 18 (40%) RNA was isolated according to Krystosek et al. (12) from Abbreviations: POLADS, polyadenylylated RNAs; VV, vaccinia virus; The publication costs of this article were defrayed in part by page charge p.i., postinfection; snRNA, small nuclear RNA; PAB, poly(A)-binding payment. This article must therefore be hereby marked "advertisement" in protein. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2037 Downloaded by guest on September 26, 2021 2038 Biochemistry: Lu and Bablanian Proc. Natl. Acad. Sci. USA 93 (1996) were from the host, 11 (25%) were from the virus, and 9 had Table 2. Origin of 5' ends of POLADS 1 hr p.i. homologous to no homology to viral or known host sequences and were tRNA genes probably from the host because the complete sequence of VV Homology, is known (38). Three constructs were too short to be compared Clone Start, nt Stop, nt Origin % to any known sequences, and three others had free poly(A) 64-1 30 75 tRNA-Leu gene-(1-75) 100 stretches of 18-26 nt. These data show that at 1 hr p.i., most 86-1 24 of the 5' ends of POLADS are acquired from the host-cell 64 tRNA-Gly gene-(1-68) 100 RNA. Among 18 cellular POLADS, three were from human small nuclear U2 RNA (snRNA) gene (snRNA-POLADS) shown) indicate that there is no consensus sequence at the (Table 1), two were from tRNA (leucine and glycine) genes 5' ends of POLADS. (tRNA-POLADS) (Table 2), and the remaining 13 5' ends The Effect of Cloned POLADS with Host or Viral 5' Ends originated from various mRNAs (mRNA-POLADS) (Table 3). on Protein Synthesis in Reticulocyte Lysate System. It was Out of the 13 mRNA-POLADS 66% of the sequences do not of interest to test POLADS with host or viral 5' ends for possess the eukaryotic polyadenylylation signal (AAUAAA). inhibitory activity of cellular mRNA translation in the POLADS that contain viral 5' ends are shown in Table 4. cell-free system. A clone of a POLADS homologous to a From 11 viral POLADS, two were from the telomere region portion of bovine casein kinase gene and another homolo- of VV genome (7.5 K region and NR2 region, respectively), gous to 5' flank of ORF B13R of the VV genome were the remaining nine were from various regions of the VV transcribed in vitro (14). Fig. 1 shows the inhibitory activity genome. of these two POLADS with similar lengths of poly(A) tails The Source of 5' Ends of POLADS at 3.5 hr p.i. Out of a ("80 nt). The results demonstrate that both types of PO- total of 43 POLADS obtained at 3.5 hr p.i., 26 (60%) were LADS inhibit the translation of HeLa mRNAs to a similar homologous to various VV genes, and 10 (23%) were extent (72% and 78% inhibition, lanes 2 and 5). In contrast, homologous to various sequences of the host genome. Four were not found to be homologous to any known sequences, 14( T and three had short 5' ends and could not be evaluated. These data also show that at a relatively late time, 5' ends of 120 POLADS are still derived from both host and viral tran- I 00 scripts. But, unlike at 1 hr p.i., at 3.5 hr p.i. POLADS with c viral 5' ends were more abundant. 80 - Among 10 cellular POLADS isolated at 3.5 hr, two came -;7U 60 from the U2 snRNA gene similar to the U2 RNA POLADS 40. isolated at 1 hr.
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