Addition of polyadenylic acid to RNA by ATP: Polynucleotidylexotransferase partially purified from HeLa cells

Pergamon Preys

Life Sciences Vol . 14, pp . .437-445, 197.4. Printed in Great Britain

ADDITIdä OF POLYADBRYLIC ACID TO Râib,BY ATP:POLYBQCLEOTIDYLmWTBAIiSFSRA3S PARTIALLY PQRIF~ :~FBAlf~l1" CBt.LS Rusty liens aad~ Gàry~Stair Departaent of"BYochesdYtry~: IInivarsity of Florid* Gainesville, Florida 32ôî0' (Received 310ctober 1973 ; in Snal form 13 December 1973) An ensyaa that catalyses the addition of polyadenylic acid to several RäAs vas partially resolved by salt precipitation and DSAB-callniose chrcsatography from the cytoplaenic coupoaeat of HnLa calls. ATF sad nanganesa are required for the sequential addition of AIiP >toieties to lengthen RBA priaers. The presence of aadogenous prissy and endonuclaolytic activity associated with the en:ysa ara indicated. To further elucidate the role of polyadanylic sequences found on the 3' hydrozyl termini of hetaogeneous nuclear RBA (HaRBA) and nRNA of HeLa cells (1-5) se yell as in nucleic acids of other eukaryotes (ô-9) va have isolated an ATP :polyauclaotidylezotraasferase froze the cytoplaenic canpartaant of HeLa cells.

Thn ea:ysa vas partially purified utilising the procedure developed

with as analogous easyne isolated from corn seedlings (10-11) .

Although the

properties of the Hel.a enayne are similar to the ~ysbe first isolated free calf thymus by Fd~onds sad Abrass (12) and subsequently purified by Winters and Bdsanda (13) sad Taiapalia et al . (14) and' the ensyse purified free rat liver by Ylemparar (15) th~ resolution of thn aaays~e from HeLa calls rill permit coacanitaat is vitro as yell ae is vivo study of the procasaiag of nuclear transcripts in hu~sa cello.

Preliminary findings are presented bare and a

detailed report will be published alserhsre.

Tha asasitivity of thin ensyne

to aavaal zifampicia dsrivatives has been reported (16) . Bnsrs Preparation The an:yma vu isolated froc the soluble proteins o! a pwt~ribosonal atiperoataat fraction from 8 litas of azponwtially proving HeLa 83 cells at 437

438

5

x

HrLa ATP : Polynacleotidylemtrsasternse

10 5 calls per ml .

Vol . 14, No. 3

The cells ware grown in suspension culture in Eagle's

miaiaal essential msdiua supplemented with 3 .Sx (each) of calf and fetal-calf serum (17) .

Calls were harvested by centrifugation, rasuspended and washed

twice is 100 ml Earle's balanced salt solution .

Cells ware rnsuspeadad in 100

ml 10 mM NaCl, 10 ~ Tris (pH 7 .4), 1 .3 mti MgC12 and lysed with a loose fitting Dounce homogeaizer leaving the bulk of nuclei and cLtochondria intact .

Püto-

chondria, nuclei and debris were removed by centrifugation at 27,000 % g for 15 min and the supernatant centrifuged at 105,000 Z g for 90 min to remove ribosames .

The post-riboeooal supernatant was adjusted to 40Z saturation with

(HH4) 2304 (pH 8) and the precipitate, resuspended in 2 .5 mM Trie (pH 8.0), mM 2-mercaptoethaaol, Sx glycerol (G~i) was clarified by raceatrifugation . The salt was adjusted by passage through Sephadez G50 equilibrated and eluted with GEli .

The excluded volume was adsorbed to a colvan of DEAS-cellulose

(25 ~ i .d . R 37 mm) previously equilibrated with GBti and the eaotransferase was eluted with 50 mM (DlH4)250 4 in GEK.

THE AMP incorporating activity preci-

pitated from the high speed supernatant with (NH4) 2504 was enhanced 2-fold by desalting and as additional 150-fold by DEAS-cellulose chromatography with over 90x recovery of added activity (TABLE I) .

The

A280/A260

ratio of the desalted

protein was unaltered by adsorption and elution from DEAE-cellulose (1 .15) and indicated 1 to 2x nucleic acid . Reguireneats for A1~ Incorporation ATP,

the required riboaucleotide precueor was incorporated moat rapidly at

1 .17 di (TABLE II) .

At 2.31 oK ATP (2-fold decrease in pracusor specific

activity) the rate of incorporation was 1/4 that at 1 .17 ml_I indicating inhibi tion by higher nucleotide levels .

Addition of 0.96 mli CTP or 1 a!! DTP to

1.17 mèI ATP significantly reduced [l4 C]Alb incorporation suggesting that these unlabeled nucleotides were either incorporated in lieu of labeled Ate or were inhibitory .

The inability of the HeLa ezotraasferase to incorporate signifi-

cant levels of radioactivity from [14 C]CTP or [l4C]DTP rhea tested at specific

HeLa ATP: Polynacleotidylemtraaeferaae

Vol. 14, No : 3

43g

TABLE I Partial Besolution of Esotransferase fromm Post-+mitochondrial Supernatants of HeLa Cells Fraction

Protein

Activity

Specific Activity

(mg)

(unite)

(unite/mg)

(NH4)2504 , ppt. at 40Z satd .(40 ASP)

190

31 .2

0 .16

Sephadez G50, ezcluded (40 ASP G50)

201

70 .9

0.34

1 .41

66 .2

46 .94

DSAE-cellulose, pooled

Reaction mutates (0 .1 al) containing 7 umolee Tris HC1 (pH 8 .8), 1 uncle dithiothreitol, 0.12 uncles [8-1 C]ATP (1 .45 mCi/mole), 10 ug bovine seras albumin, 0.41 A260 E. cola tRNA, 0 .1 moles MnC12 and either 0.9 mg 40 ASP, 0.8 mg 40 ASP G50 or 14 .1 ug DEAE-pool protein were incubated at 30 ° . Aliquote were delivered to filter paper disks after 30, 60 and 90 min incubation and acid-insoluble radioactivity determined as described previously (10) . A unit is equivalent to 1 mole AMP incorporated per min .

activities 5 to 9-fold that of [14 C]ATP or at specific activities and concentrations comparable with ATP ; with or without 1.14 mM ATP (to permit utilization of ATP) indicated that other nucleotides were incorporated at no more than one per 135 AMP's (TABLE II) . Significant incorporation of ATP was detected is the absence of added RNA, hasever, E. cola tRNA stimulated ANP incorporation pore than 10-fold (TABLE III) .

IInlike the ezotranaferase from corn seedlings (18), the HeLa enzyme was

not stimulated by single stranded, abort, 3' hydrazyl terminated deozyoligomers (TABLE III, Part A) .

Neither addition of

(NH4) 2504 nor replacement of manganese

with magnesium increased HaLa enzyme activity on deozyoligomera .

All HaLa ANAs

ranging in size distribution from 4S to 28S also stimulated A1~IP incorporation (TABLE III, Part B) .

Assuming their average molecular weights were 2 .5 % 104,

6.5 % 10 5 and 1 .7 % 106 for tBPA, 18S and 28S BNA, respectively, then the average moles of ANP added per sole of prianr wan 2 .3, 85 .3 and 183 .6, respectively .

Although all priaere were assayed at rate limiting concentrations the

size distribution among the molecules in each class was relatively wide

440

HeLa ATF: Polyaacleotidyleaotcmafaraae

Vol. 14, No . 3

TABL$ II Specificity of HeLa Bzotraasfarasa for Various Nucleotides Added Nucleotide Labeled

ATP

Incorporated Nucleotide

Sp . Act .

Conc .

DnLbeled

Conc .

(e~Ci/dole)

(a~)

29 .0

0 .03

-

-

292

(~

(pmoles)

ATP

0.79

1 .17

-

-

13,372

ATP

0.38

2 .31

-

-

4,150

ATP

0.79

1 .17

CTP

0.96

1,937

0 .03

DTP

1.0

320

1 .17

DTP

1.0

4,009

0 .01

-

-

0.5

0.97

-

-

34 .1

0.01

ATP

1.14

0.6

0.97

ATP

1.14

21 .8

0.004

-

-

0.3

1 .00

-

-

34 .8

0.004

ATP

1 .14

0 .3

1.00

ATp

1.14

29 .3

ATP ATP CTP CTP CTP CTP DTP DTP DTP UTP

29 .0 0.79 149 1.58 149 1.58 270 1.22 270 1 .22

Total nucleotide incorporated into acid-insoluble material in 90 min pas detarnined .in reaction >liztur a as described is TASLB I but containiag the indicated roacsatrationa of 1~-labeled and ualabnlad miclaotidas sad 7 .87 Ug pooled DßAS anaysn . (fractions froc n 5 to 20x sucrose gradient) .

The 4S fraction sas polydisperead

on SDS-polyacrylaaida gal electrophoresis pith such of the A~60 material migrating more eloaly and thus .larger than tHNA .

Since traces of nndonuclaase

activity aara .datactad in .the -emyyas!pr4paration, a fear nicks in the longer primer molecules gaaarating alaäitioaal 3'hydrozyl teraini could draaatically alter the cai=aslatad priser efficiency .

Therefore, the eatiaate of relative

Vol . 14, No . 3

HeLA : ATP': Polyaacleoddylesotrawfecase

441

TABLE III Specificity of Hala Szotransferasa for Vuious Nucleic Acids Priser

Put A

Part B

Asount Added

~ Incorporated

(Ug)

(pswlas)

None

344

E . coli tBNA

21

4842

Deozyoliga~ar

22

400

E . coli tBNA

2 .1

2840

HeLa 48 BNA

2 .2

1910

HeLa 18S Bäs

2 .1

2689

HeLa 28S BNS

1 .9

2093

Poly(A)

3 .6

6422

Poly(D)

4 .0

2715

Poly(C)

8 .6

487

The total AMP incorporated into acid-insoluble saterial in 90 sin vas detersined in reaction sutures as described in TABLE I but containing, in Put A, the indicated miclaic acid and 5 .9 Vg DEAhrcallulosa aatys~n sad in Part 8, rate lisiting concentrations of the micleic acids indicated and 6.26 ug pooled DEAE as:yee . The HeLa BNAe vara prapued by SDS-phaaol attraction of post-nuclear supernatants of Hala cells a~ resolved by 5 to 20Z sucrose deasity gradient centrifugation . Deotyoligosinr vas prapued by Baited DNAase I digestion of calf thyuus DNA and Sephadez G100 gel filtration as described previously (18) .

prising efficiencq of tRNAs or rBNAs is tenuous.

Aesiming that the homopolys~era

yarn of equivalent eisn distribution than a preferred bindiag of ansyr to poly(A) or preferred addition of A!0' to a tersinal adenosins was indicated by the higher activity of poly (A) as caspared to poly (II) or the inaction poly(Ç) . In the corn ssotranaferass reaction (11), the initial rate of addition of AMP to tBNAs lacking a tarsinal adenosine ie only 1/4 that of tANA suggesting that prissrs with a texsinal adaaosina ars .preferentially elongated. The HeLa sntyan required sanganass and activity vas sisal at 1 nü; activity vas batialy datectabL with 5 sill sanganesa .

B1th suboptisal levels of

HeLa ATP: Polynncleotidylemt~snsferese

442

soo'

e

A

uo i2o ~Ip"ATI011 TeIE 4~1

Vol. 14, No . 3

roo

â0 ws

FIG. Accumulation and electrophoretic swbility of product froa tRNA-primed ATP: polymicleotidylazotransfarasa reaction . A reaction mature (1 .0 al) co taming 70 ynoles Trie HC1 (PH 8 .8), 10 u oles dithiothreitol, 1 .27 bolas [8-l~C]ATP (3 .07 uCi/cola), 2.05 A260 H. soli tBPA, 1 ysole MnC12 and 0 .63 ng pooled DBAB~ cellulose aaayme vas incubated at 30 ° . Aliquots ( .025 mil) vere delivered to filter paper disks at i~icatad tisn, acid-insoluble radioactivity datarmiaed as described previously (10) and the total nmolas A?SP incorporated in the reaction ai:tures plotted in panel A. Additional aliquots (0 .2 ss) vara removed at indicated tisa, made to 45 SDS, 0.02 K SDTA and filtered through Saphada: G25 equilibrated previously with 0.36 Tris, 0 .01 K BATA, 0 .3 K PaH2P04 (pH 7 .4) . Aliquots of the ezcludad vohaa of each pôoled eluatéware resolved by SDS-polyacrylaaida gel (7 .55) electroplwresis.(5 va/gel, 70 >tin) ae described by Loaning (19) . After scanning at 260 m (solid traciag) each gal vas sliced, each elite dried oa a filter paper disk and counted by sciatil Lntion apectrauatry (10) (bar graph) . Electroplwretic migration vas frca left to right . The emigration of 0.08 A260 B. soli tSIIA uwd as prier is indicated in panel B .

K

VoL 14, No. 3

HaLa ATP : Polymcleotidylemtcaaaferase

.443

sanganesa (0 .4 mti , sagaaeius (5 mli, etimulated Al~ incorporation 2-fold . Snsyme free of (HH4) 280 4 (ezcluded fros Snphadez G25) xas atinulated 2-iold by 10 ~ (HH4)2SO4. Product Accuaulation The rate of AMP incorporation xith HaLa azotransferase, analogous to the corn easyoe (11), xaa slox initially, increased to a rapid rata xithin 20 to 60 sin and continued for sots thaw 3 h (Fig ., Panel A) until the priest xas ezbaueted (not sham here) . To desoastrate the sequaatial addition of A!!P moieties to the 3'hydroryl terniaus of priser tBHA, aliquots of the reaction mizture vets reooved at 30 sec, 60, 120 aad 180 oin during Incubation at 30' ; the uaiacorporated ATP vas reooved and the

A260

absorbing a~ acid-insoluble radioactive osteriale xern

rewlved by gel electrophoresis (19)x.

After 30 sec incubatioa the priser

tSNA as xell as the SDS vets detected on the gel scans at slices 31 and 42, respectively (Fig . c.f . panels B and 30 sec) .

An additional peak of A2~

oaterial, migrating to slice 40, vas derived from the ensyme .

liote a lox but

significant level of radioactivity vas detected is the additional peak suggesting that this oaterial vas eadogeaeoua primer (sae TABLE. III, Part A) like that associated xith the enayme isolated frog calf thyous nuclei (20) . Further resolution of the HeLa ensyoe by glycerol gradient centrifugation reduced the acid-insoluble radioactivity accumulated upon incubation of the easyme xithout added prises to 1/40 that accumulated in 90 oia xhan saturated xith tBHA .

After 60 min incubation, the peak of

A260

material at slice 30 vas

broadened sad skeyed toward the slayer sigrating oateriale xith acid-insoluble product diaCributed .undar the shoulder incubation the proportion of

A260

(Fig ., panel 60 oin) .

After 120 min

material migrating as a ahouldgr increased

as did the acid~inaoluble radioactivity associated xith It .

After 180 min

incubation, in +dditioa . to .furthar accumulation .of,sloxly oigrating radioactivity aawciatad xith the shoulder of

A260

material around slice 20, the

asount of acid-insoluble radioactivity migrating xith or faster than the prises

444

HeLa ATP : Polynacleotidylezottaasferase

tßNA increased .

The increased migration of labeled material suggested that the

Vol . 14, No . 3

product was cleaved to smaller oligomere late in the r®action .

It should be

mentioned that the absorbante found in slices 1 to 15 in the 30 sec and 60 min samples disappeared from the 120 min sample and was much reduced in the 1g0 min sample .

Therefore, the development of some of the shoulder of absorbents on

the tRNA peak could represent products derived from the larger material . Since a portion of the primer tRNA increased in size concomitant to the accretion of acid-insoluble radioactivity, since AMP vas incorporated to the ezclusion of UMp or CMP, and since the requirements and characteristics of the HeLa msyme are analogous to those of the corn ezotransferaae where the product vas identified (10,11) ae poly(A), ve conclude that the addition product catalyzed by HeLa enzyme to RIiA is polyadmylic acid .

These praliainary

ezperiments with an enzyme isolated foru a post-ribosomal supernatant suggest that HeLa ATP :polynucleotidyleaotranafsrase may be responsible for the addition of poly(A) sequences to mRäAs after the mRHAs are processed from nuclear transcripts and traasported across the nuclear membrane .

Such an interpretation

moat be only speculative since ve have not ruled out the presence of ezotrans(erase in nuclei as shown in calf thymus (12,20,21) or in mitochondria (22) but recovered in the cytoplasmic compartment of HeLa, rat liver (15), calf thymus (14) or corn cells (10) after its leakage through mambranas or release from lyasd organelles .

Alternatively, the cytoplasmic ezatransferaee may be in

transit from its site of synthesis in the cytoplasm to its functional cellular compartmentalization in the m~cleus . Acknovlad~en ts We wish to acknowledge the technical mpertise of Mrs . Cladia Alvares is ezecutiag these ezpariments .

Thin investigation vas supported is part by the

United States Atomic Easrgy Cassission :

Report Ro . ORO-3982-21'aad Grmt DRG

1138 from the Demon &myon Memorial load for Cancer Rassarch .

Vol . 14, No. 3

MS

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