The presence of polyriboadenylic acid sequences in calf lens messenger RNA

J. Mol. Biol. (1974) 82, 15-25

The Presence of Polyriboadenylic Acid Sequences in Calf Lens Messenger RNA GENE C.LAVERS,JOHN H. CRENAND ABRAHAMSPECTOR Department of Ophthalmology, College of Pl~yGcians & Surgeons Columbia University, New York, N.Y. 10032, U.X.A. (Received 13 August 1973) The presence of poly(rA) sequences in lens RNA has been demonstrated by the isolation of RNase A and T,-resistant fragments of approximately 50 nucleotide residues. These poly(rA)-rich sequences, obtained from lenses incubated for six hours in organ culture with [3H]adenosine, are located at the 3’ term& of mRNA as determined by 3’ exoribonuclease digestion. Limited digestion of the [3H]adenosine-labeled mRNA with the enzyme led to the abolition of binding to poly(rU)filters and a concomitant loss of template activity with avian myeloblastosis virus RNA-dependent DNA polymerase. Furthermore, after incubation of lenses in organ culture with 3’-deoxyadenosine, the isolated polysomal RNA was unable to function as a template in an avian myeloblastosis virus RNA-dependent DNA polymerase-catalyzed reaction system.

1. Introduction The presence of poly(rA) sequences in eukaryotic mRNAs has been demonstrated in a number of systems (Darnell et al., 1971b; Edmonds et al., 1971; Lee et aZ., 1971; Greenberg & Perry, 1972; Sheldon et al., 1972a). Such segments from 50 to 200 nucleotides in length (Edmonds & Caramela, 1969; Lim & Canellakis, 1970; Darnell et aE., 1971a; Edmonds et al., 1971; Mendecki, et al., 1972; Pemberton & Baglioni, 1972) have been located at the 3’ termini by chemical (Burr & Lingrel, 1971; Nakazato et al., 1973) and enzymatic (Molloy et al., 1972; Sheldon et al., 1972b) means. However, the role of poly(rA) in eukaryotic mRNA remains uncertain. Recent findings suggest that poly(rA) is added to heterogeneous nuclear RNA as a post-transcriptional modification (Darnell et al., 1971a; Edmonds et al., 1971; Mendecki et al., 1972) to the 3’ termini and also appears at the 3’ termini of newly synthesized mRNA (Sheldon et al., 19723; Molloy & Darnell, 1973; Nakazato et al., 1973). In addition, it has been suggested that poly(rA) sequences may be involved in selecting those heterogeneous nuclear RNA molecules which become mRNA (Darnell et al., 1971b; Edmonds et al., 1971; Lee et al., 1971) and its subsequent transport from the nucleus to the cytoplasm (Darnell et al., 1971a; Lee et a.?.,1971), in the initiation of DNA synthesis from RNA (Lai & Duesberg, 1972), in the synthesis of polylysine (Green & Cartas, 1972), and in the interaction with specific binding proteins (Kwan & Brawerman, 1972 ; Blobel, 1973). Such sequences may also be involved in possible roles in cell differentiation (Sarkar et aE., 1973; Slater et al., 1973). The poly(rA)-containing mRNAs of globin (Kaoian et al., 1972; Ross et al., 1972; Verma et al., 1972), immunoglobin (Aviv et al., 1973), lens (Chen et al., 1973a), and 15

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vacoinia (Zassenhaus & Kates, 1972) have been shown to act as templates with AM virust RNA-dependent DNA polymerase (reverse transcriptase) in the presence of oligo(dT) primer. However, the direct involvement of poly(rA) in the reverse transcriptional process of these natural mRNA templates has yet to be elucidated. The lens offers several features which make it an interesting system for the study of mRNA structure and function. The encapsulated lens contains a single layer of epithelial cells which transform into fiber cells and subsequently, with pyknosis of the nucleus, into lens fibers (van Heyningen, 1962). It has been demonstrated that protein synthesis persists in the anuclear region of the lens and that stable mRNA is present in this region (Spector & Kinoshita, 1965; Reeder & Bell, 1967). The tissue is composed primarily of the structural proteins alpha, beta and gamma crystallins. In the calf lens, approximately one half of the total protein synthesis is directed toward the production of a single macromolecule, alpha crystallin (Spector & Kinoshita, 1964; Spector et al., 1968). Lens mRNA has been isolated by zonal centrifugation (Berns et al., 1971; Chen et al., 1973a) and by afhnity chromatography (Chen et al., 19736). It has been shown that the 10 S (Chen et al., 1973a) and 14 8 (Berns et al., 1973a; Chen et al., 1973a) mRNAs isolated by zonal centrifugation and the mRNA isolated from affinity chromatography (Chen et al., 19736) code primarily for the synthesis of complementary DNA with AM virus RNA-dependent DNA polymerase. In this report it is demonstrated that 50-nucleotide poly(rA) sequences are present in lens mRNA at the 3’ termini and that such sequences are most likely required for reverse transcription activity.

2. Materials and Methods (a) lMaterial8 Panoreatic RNase, crystallized 5 times, and ribonucleases T1 and Te were purchased from Calbiochem. The 3’-exoribonuclease from Ehrlich ascites tumor cell nuclei, prepared as described by Lazarus & Sporn (1967), was a generous gift from Dr R. P. Perry, Institute for Cancer Research, Philadelphia, Pa. d-[3H]TTP (spec. act. 1800 cts/min/pmol) was obtained from New England Nuclear, while dATP, dCTP and dGTP, actinomycin D and 3’-deoxyadenosine (Cordycepin) were obtained from Sigma Chemical Company. were obtained from Collaborative Research, Inc., Oligo(d‘J3~~ - 18 and oligo(dT)-celloluse Waltham, Mass. AM virus RNA-dependent DNA polymerase, prepared as described et al., 1971), was kindly provided by Drs S. Spiegelman and D. Kaoian of the (Kacian Institute of Cancer Research, Columbia University, New York, N.Y. [3H]adenosine (spec. act. 10 Ci/mmol), [3H]uridine (spec. act. 50 Ci/mmol) were obtained from New England Nuclear, Boston, Mass. Poly(rU), 3H-labeled poly(rA) (spec. act. 50 Ci/mmol), yeast tRNA and 16 S + 23 S rRNA mixture from EscAerichicG ooli were purchased from Miles Laboratories. Standard 32P-labeled oligonucleotide markers of known sequence were obtained from homoohromatograms of RNase T1 digests of RNA transcribed from simian virus 40 DNA. These standards were the generous gift of Dr S. M. Weissman of Yale University, New Haven, Conn. (b) Lens organ culture, polyribosome isolation and RNA extraction Calf lenses, excised from the eyes of approximately 3 to 4-month old animals were incubated as described by Merola et al. (1960). Cultures were preincubated 30 min before addition of [3H]adenosine or [3H]uridine. In cultures treated with 3’-deoxyadenosine (25 pg/ml). addition of 3H-labeled precursors was delayed for 45 min. Incubations were t Abbreviation

used : AM virus, avian myoblastosis

virus.

POLY(rA)

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LENS

mRNA

17

terminated by removal of the incubation medium and gentle washing of the lenses with ice cold 0.9% NaCl and immediately homogenized. Lens polyribosomes were prepared from calf lenses as described previously (Chen et al., 1973a). The polyribosomes, dissolved in 0.05 M-Tris.HCl, pH 9.0, containing 8 mMmercaptoethanol, were extracted with chloroform : phenol (l/l, v/v) containing 0.1% S-hydroxyquinoline, 0.5% sodium dodecyl sulfate and saturated with 0.05 M-Tris.HCl buffer, pH 9.0. The aqueous phase was adjusted to 0.3 M-LiCl, precipitated by addition of 3 vol. of lOOo/o ethanol and washed with 75% ethanol. The RNA was then dissolved in water and stored frozen at - 70°C.

(c) Poly(rU)$lter

biding

assay and affinity chromatography

on oligo(dT)-celldose

Poly(rU)-filters were prepared by irradiating Whatman GFjC glass filters containing 150 pg poly(rU) with a 30 W Sylvania germicidal lamp and used as described previously (Sheldon et al., 1972a). was prepared by exhaustive washing with water followed by equiliOligo(dT)-cellulose bration with 0.01 M-Tris*HCl buffer, pH 7.5, containing 0.5 M-NaCl to remove unbound in the high salt affinity ligand. RNA samples of approximately 30 A,,, units, dissolved buffer, were applied to 3 cm x 1 cm columns and unbound RNA was eluted and designated as peak A. After washing with at least 10 column volumes of high salt buffer, the bound mRNA fraction was released with water and collected as peak B. The RNA from pooled fractions was adjusted to 0.3 M-Lick and precipitated and washed twice with 75% ethanol. (d) RNase digestions Ribonuclease digestions were carried out for 20 min (Table 1) and 30 min (Fig. 2) at 37°C in O&ml assays containing 10 mrvr-Tris*HCl, pH 7.5, in the presence of poly(rA) (0.02 mg/ml) and 200 miw-KCl. Pancreatic RNase A (6 pg/ml), RNase T, (6 pg/ml) or RNase T, (10 units/ml) were present as specified. The 3’-exoribonuclease was obtained from Ehrlich ascites tumor cell nuclei as described by Lazarus & Sporn (1967). This enzyme preferentially digests poly(rA) sequences bearing a free 3’-OH group. Enzyme kinetics were determined from the hydrolysis of 3H-labeled poly(rA) under standard conditions (Lazarus & Sporn, 1967). Lens mRNA, after exoribonuclease digestion, was extracted with chloroform:phenol as described above. Subsequently, it was used either directly for a&nity binding assays or it was precipitated, as described above, and dissolved in water before assaying template activity with AM virus RNA-dependent DNA polymerase. (e) RNA

gel electrophoresis

RNA samples were electrophoresed in 10% polyacrylamide gels in 0.0033 M-Tris containing 0.03 M-diethylbarbituric acid, pH 8.5 (Richards et aE., 1965), in the presence of 7 M-urea. RNase A and Tr-resistant fragments were dissolved in running buffer containing 10% sucrose, 5 ~1 of O*l”h bromphenol blue and/or 5 ~1 of 9.3% pyronin Y and reference 32P-labeled oligonucleotide markers of 15 and 29 nucleomarker yeast tRNA. Standard tides, each of known sequence, were also included in the appropriate gels for calibration. (f) RNA-dependent

DNA polymerase assay

AM virus RNA-dependent DNA polymerase reactions were done as described previously (Kacian et al., 1972). The basic assay mixture of 100 ~1 contained the following in pmol: Tris.HCl (pH 8.3), 5.0; MgCl,, 0.6; dATP, dCTP, dGTP, each O-02; d-[3H]TTP (spec. act. 1800 cts/min/pmol), 0.004; KCI, 5.0; oligo(dT), 0.4 pg; template RNA, 3 pg; and an appropriate enzyme dilution. The following additional components were used in the appropriate assays as indicated in Table 4: RNase A, 10 pg; RNase T,, 5 units.

(g) Analysis The centrifugation 30% sucrose gradient SW41 rotor (Spinco) yeast tRNA and 16 S

2

of peak B mRNA

of the B peak mRNA fraction was carried out in a linear 15% to in 0.05 M-Tris*HCl, pH 7.4. Sedimentation was carried out in an at 38,000 revs/min for 20 h at 4°C. Standard reference markers of and 23 S E. coli rRNAs were run in a parallel gradient.

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3. Results Calf lenses were incubated in organ culture for six hours in the presence of either [3H]adenosine or [3H]uridine, and the labeled polysomal RNA fraction was extracted with phenol: chloroform, pH 9.0. The extent of the radioactivity retained on poly(rU)-fiberglass filters after digestion with ribonuclease is shown in Table 1. In all cases the same quantity of RNA was tested. Under the conditions used, poly(rA)clusters are not degraded by RNase A (Beers, 1960) or RNase T,. Approximately 12% of the radioactivity was retained on the filters (Table l(a)). Inclusion of RNase T,, which digests poly(rA)-clusters, reduced the binding of the [3H]adenosine-labeled fragments to an insignificant level. In contrast,, digestion of [3H]uridine-labebd polysomal RNA with RNases A and T, alone abolished the retention of radioactivity to the affinity filter (Table l(b)). TABLE 1

Eflect of ribormclease digestion on the afinity binding of [3H]a,denosine or [3H]uridine-labeled RNAs to poly(rU)-j%ers RNA

Treatment

Ctslmin bound (%)

(4 [3H]adenosine

polysomal

RNA

None RNwe

RNase (A + ‘JJ, + T,) poly(rU)

100 12 0.8 0

None RN&se (A + !I?,)

100 0

None RNase (A + ‘I’,) RN&se (A + T, + Ts)

100 98

(A + T,)

(b) [3H]uridine

sH-labeled

polysomal (cl poly(rA)

RNA

poly(rU)

0.4 0

Ribonuclease digestions and poly(rU)-filter binding assays were carried out as described in Materials and Methods. Hybridizations of poly(rU) to either the lens mRNA or poly(rA) were done by inoubating the polymers at 50°C for 30 min in O-05 M-Tris buffer, pH 7.5, and gradual cooling before adjusting to 0.01 x-Tris buffer, pH 7.5, containing 0.12 M-N&I, before poly(rU)filter binding.

In control experiments, 3H-labeled poly(rA) was exposed to the RNases under digestion conditions. As shown in Table l(c), RNases A and T, caused essentially no reduction in binding of 3H-labeled poly(rA) to the poly(rU)-filter, whereas the addition of RNase T, eliminated virtually all specific binding. It is interesting to note that when the polysomal RNA was preincubated with poly(rU), all retention of either the labeled RNA or 3H-labeled poly(rA) was abolished (Table l(a) and (0)). These results suggested the presence of poly(rA)-rich clusters in the polysomal RNA. similar

POLY(rA)

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0.3

0.2 z 5: 2

01

0 Fraction

no.

FIG. 1. Comparison of oligo(dT)-cellulose fractionation of polyribosomal RNA isolated from lenses incubated with and without 3’-deoxyadenosine. [aH]adenosine-labeled polyribosomal RNA was obtained from incubations for 6 h of 2 groups of 90 calf lenses; one group was incubated in the presence of 3’-deoxyadenosine. Two identical columns (3 cm x 1 cm) of oligo(dT)-cellulose were used. Approximately 10 column vohnnes of high salt buffer were used to wash the column (-/I----) after elution of the unbound RNA (peak A). Bound mRNA, peak B, was released with water. One-ml fractions were collected ) and [3H]adenosine cts/min --+-a--) determined. (a) Without 3’and &O ( deoxyadenosine; (b) with 3’-deoxyadenosine.

In order to obtain larger quantities of poly(rA)-rich messenger, preparative aflinity chromatography with oligo(dT)-cellulose was used. A typical fractionation of the [3H]adenosine-labeled RNA isolated from organ cultures incubated for six hours is shown in Figure l(a). Approximately 9% (9520 cts/min) of the radioactivity as well as 3% of the A,,, (O-43 unit) absorbance applied to the column was consistently recovered in the B peak fraction. Since 3’-deoxyadenosine (Cordycepin) has been shown to inhibit the incorporation of adenosine into poly(rA)-clusters of mRNAs (Penman et al., 1970; Adesnik et al., 1972), it was of interest to determine its effect upon C3H]adenosine uptake into lens mRNA. Simultaneous incubations with 3’deoxyadenosine were done under conditions identical to those used to obtain the data illustrated in Figure 1(a). The results of the 3’-deoxyadenosine experiment are shown in Figure l(b). Of the 101,150 cts/min applied to the column only 470 cts/min were detected in the B peak region. It should be noted that the total polysomal RNA recovered from both experiments was essentially the same. This indicates that bulk RNA synthesis appeared to be unaffected by the six-hour incubation with 3’-deoxyadenosine. Consequently, this finding suggests that 3’- deoxyadenosine

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almost completely suppressed the appearance of poly(rA)-containing mRNA in polyribosomes. These results are in general agreement with those obtained by others (Penman et al., 1970; Adesnik et al., 1972; Mendecki et al., 1972; Jelinek et al., 1973). Since these results suggested the presence of poly(rA)-clusters in lens mRNA, it was of interest to determine the size of these unique nucleotide regions. Digestions of [3H]adenosine-labeled lens mRNA were carried out with RNases A and T, under conditions which would preserve poly(rA)-rich clusters. Such RNase-resistant fragments were separated from the remaining digestion products on poly(rU)fiberglass filters and then eluted with water. The eluted RNase-resistant fragments Standard were electrophoresed in 10% polyacrylamide gels containing 7 M-urea. markers of tRNA and 32P-labeled RNA of 15 and 29 nucleotides were run simultaneously. The profile of the [3H]adenosine-labeled fragments is shown in Figure 2(a). One sharp symmetrical peak of radioactivity was found containing 96% of the material applied to the gel, suggesting an homogeneously sized population. Based upon the calibration of the gels, the fragments appear to be approximately 50 residues in length (Fig. 2(b)).

_ ciel slice no.

FIG. 2. Polyacrylamide gel electrophoresis of RNase A and T, resistant fragments of polysomal RNA. sH-labeled mRNA (peak B in Fig. l(a)) was digested with both RNase A and T, for 30 min. The ribonuclease-resistant RNA fragments were electrophoresed in 10% gels containing 7 ~-urea. (a) Profile of [sH]adenosine-labeled fragments (gel slices, 0.78 mm each). (b) Calibration of gels with tRNA and eZP-labeled oligonucleotide markers of 80, 29 and 15 residues, respectively, (-O-O--); mobility of [3H]adenosine-labeled fragments ( l ).

In order to determine the location of the poly(rA) sequences in the peak B mRNA fraction, a 3’-exoribonuclease from Ehrlich ascites tumor cell nuclei was used. The enzyme is relatively specific for poly(rA) sequences with a 3’-OH terminus (Lazarus & Sporn, 1967), as demonstrated for the poly(rA) segments at the 3’ termini of mammalian mRNAs (Sheldon eEal., 1972; Molloy et al., 1972). It sequentially cleaves nucleotides from the 3’ to the 5’ end of RNA. Table 2 shows that, as a function of digestion time with the enzyme, the amount of [3H]adenosine-labeled mRNA bound to the poly(rU)-filter was progressively reduced. In 30 minutes, about 92% of the affinity.binding was eliminated from the filter. Studies on the kinetics of digestion of synthetic 3H-labeled poly(rA) revealed that approximately 30 minutes were necessary

POLY(rA)

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TABLET Effect of 3’.exoribowdease on the binding of [3H]adenosine-labeled lens mRNA to poly(rU)-jiberglass Jilters Cts/min bound (%)

Treatment

100

None 3’-exoribonuclease

0 10 30 60

min min min min

100 67 8 0

[3H]adenosine-labeled mRNA (5 pg/assay) was digested with a 3’-exoribonuclease under the conditions described previously (Lazarus & Sporn, 1967). Portions were removed from the digestion solution at the indicated times and adjusted to 0.01 M-Tris, pH 9.0, and extracted with phenol : chloroform (1: 1, v/v) containing 0.1 y0 %hydroxyquinoline and 0.5 y0 sodium dodecyl sulfate. The aqueous phase was adjusted to 0.12 ~-Kc1 and applied to poly(rU)-filters as described (Sheldon et al., 1972).

to hydrolize 50 residues of [3H]AMP from the polymer based upon the assay of digested (trichloroacetic acid-soluble) and undigested (trichloroacetic acid-insoluble) RNA fractions. This result supports the size determination from the gel, indicating a 50-residue poly(rA)-rich fragment is present at the 3’ terminal of the mRNA. Previous work has suggested that the B peak RNA contains other components besides 10 S and 14 S mRNAs (Chen et al., 1973b). Therefore, B peak material was further fractionated through a 15% to 30% sucrose gradient. The profile obtained from such an experiment is shown in Figure 3. The 10 S and 14 S components represent about 50% of the total material. Translation and reverse transcription assays on all pooled peaks indicated that only the 10 S and 14 S components gave

0.4 - 23 S 1

16s 1

4s 1

FIG. 3. Sucrose centrifugation profile of peak B mRNA. Peak B mRNA, obtained from oligo(dT)-chromatography of polysomal RNA es described in Materids and Methods, was fractionated on 15% to 30% sucrose gradients. Absorbance at 260 nm, arrows indicate standard markers of tRNA and two rRNAs. Bars delineate the (-----I; fractions pooled for subsequent assays.

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3

Effect of 3’deoxyadenosine and ribonuclease on the template activity of lens mRNA with AM virus RNA-dependent DNA polymerase

RNA

Reverse transcriptase activity (%)

Conditions

(a) Polysomal

RNA

(b) Oligo(dT)-purified

None 3’-deoxyadenosine mRNA

100 1.4

None RN&se (A + T,) 3’-exo-RNase

Poly(rU)

100 30 0 5 20 30 60

min min min

1.6 100 78

min min

52

min

16 0 0

3’-Deoxyadenosine treatment of cultures, ribonuclease A and T1 digestions and assays for reverse transcript&se activity are described in Materials and Methods. The 3’-exoribonuclease digestions were as described in Table 2 except that the RNA extracts were adjusted to O-3 M-Lick, precipitated and washed with 75% ethanol and dissolved in water for reverse transoriptase assays.

significant levels of both activities. Since all active mRNA templates with AM virus reverse transoriptase contain poly(rA)-rich clusters (Kacian et al., 1972; Ross et al., 1972; Verma et al., 1972; Zassenhaus & Kates, 1972; Aviv et al., 1973; Chen et al., 1973a), the transcriptional activity observed with these two RNA species lends further support to the idea that they contain poly(rA) sequences. This observation was further substantiated by the demonstration that no reverse transoriptase template activity was found in polysomal RNA obtained from lenses incubated in the presence of 3’-deoxyadenosine (Table 3(a)). To explore further this poly(rA) dependency, peak B mRNA was incubated with 3’-exoribonuclease for varying periods of time. After phenol : chloroform extraction, the RNA samples were assayed with AM virus reverse transcriptase. It can be seen from Table 3(b) that after 30 minutes, 84% of the template activity was lost and at 60 minutes all activity was abolished. It should also be noted that preincubation of peak B RNA with poly(rU) resulted in a total loss of template activity.

4. Discussion The presence of a population of 50-nucleotide sequences of poly(rA) at the 3’ termini of lens mRNA is of particular interest. Eukaryotic mRNAs have been found to contain such sequences in the range of 150 to 250 residues (Darnell et al., 1971a; Edmonds et al., 1971; Mendecki et al., 1972; Pemberton & Baglioni, 1972). In contrast, mRNAs containing poly(rA) of about 50 residues have been reported in HeLa cell mitochondria (Perlman et al., 1973), Saccharomyces cerevisiae yeast (McLaughlin

POLY(rA)

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IN CALF LENS mRNA

23

et al,, 1973; Reed & Wintersberger, 1973) and Xenopus laevis embryos (Crippa et al., 1973). It has been shown that HeLa mRNR poly(rA)-tracts decrease in size from 200 to less than 100 nucleotides after export of newly synthesized mRNA from the nucleus to the cytoplasm (Sheiness & Darnell, 1973). Similarly, X. Zaevis poly(rA) varied in size as a function of incubation time from more than 100 nucleotides after only 30 minutes to less than 50 residues after four hours (Crippa et al., 1973). Whether the homogeneous poly(rA) fragments isolated from lens polysomal mRNA after a six-hour incubation represent previously shortened poly(rA) fragments is not clear and requires further investigation with different incubation times. The lens mRNA population studied was obtained from a preparation containing both anuolear lens fibers as well as nuclear lens fiber cells. Consequently, the results suggest no difference in poly(rA) size within the two regions. While the biological role(s) of poly(rA) in heterogeneous nuclear RNA and mRNA is not understood, the biochemical requirement of poly(rA) for AM virus RNAdependent DNA polymerase template activity is strongly implicated. The removal of the 3’ poly(rA) sequences by a controlled digestion with a 3’-exoribonuclease abolished all oligo(dT)-stimulated incorporation of d-[3H]TMP into CClaCOOHprecipitable material as a linear function with respect to time. Furthermore, poly(rA)deficient polysomal RNA, obtained from 3’-deoxyadenosine treated lenses in organ cultures, also exhibited no template activity with AM virus reverse transcriptase. These data suggest a direct involvement of poly(rA) tracts of lens mRNA in the AM virus reverse transcriptase-catalyzed reaction. It is of interest that essentially all template activity with reverse transcriptase is absent in polyribosomal RNA isolated from 3’-deoxyadenosine-treated lenses. In addition, approximately 80% of the A,,, units and 95% of the [3H]adenosine radioactivity were no longer present in the peak B fractions of such RNA preparations. Similar losses in [3H]adenosine incorporation were also observed with mRNA isolated from HeLa cells (Adesnick et al., 1972) and mouse sarcoma 180 ascites cells (Mendecki et al., 1972) treated with 3’-deoxyadenosine during 30-minute labeling periods. The observations obtained with the lens peak B component indicate that pre-existing mRNA and/or 3’-deoxyadenosine-resistant poly(rA) in the lens has been markedly altered during the six-hour incubation. It is of interest that the peak B RNA isolated from control lens preparations contains about 50% non-mRNA components (Fig. 3 and Chen et al., 19733). At present it is not known whether the peak B material isolated from deoxyadenosine-treated lenses is composed of such non-mRNA components and/or altered mRNA. Recently, Lodish et al. (1973) found that a short internal oligo(rA) segment is present in the mRNA isolated from the eukaryotic cellular slime mold Dictyostelim discoideum. Similar short internal oligo(rA)-clusters without 3’-OH groups have also been observed (Nakazato et al., 1973). It has been suggested that the lens 14 S mRNA, which codes for a 19,500 molecular weight subunit of alpha crystallin, may be bicistronie (Berns et al., 19733). It is possible that oligo(rA) may be present as an intercistronic spacer in the 14 S mRNA. However, the abolition of poly(rU)-filter binding, together with the elimination of oligo(dT)-primed reverse transcriptase activity of lens mRNAs by limited digestion with 3’-exoribonuclease implies that such a possibility is unlikely. Nevertheless, such oligo(rA) segments may be masked in the secondary structure of the mRNA or through binding with mRNA-associated protein(s) (Kwan & Brawerman, 1972; Blobel, 1973). The size homogeneity of the

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lens poly(rA) fragments indicated that both 10 S and 14 S mRNAs probably contain the same small poly(rA) nucleotide segments at their 3’ termini; consequently, the difference between these two molecular forms (10 S and 14 S) can not be attributable to their poly(rA) size. We thank Drs S. Spiegelman and D. Kaoian of Columbia University for kindly making available the avian myeloblastosis virus RNA-dependent DNA polymerase. The authors are also indebted to Dr R. P. Perry of the Institute for Cancer Research, Philadelphia, Pa., for a generous gift of 3’-exoribonuclease: and Dr S. M. Weissman of Yale University for his kindness in providing the 32P-labeled oligonucleotides. This work was supported by grants from the National Eye Institiute, National Institutes of Health. One of us (G.C.L.) is a Postdoctoral Fellow of the National Eye Institute, National Institutes of Health.

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