Tissue and speeies-specific effects of small molecular weight nuclear RNA's on transcription in isolated mammalian nuclei

Departments of' Bior'ogy and C/~epnisfry, University of New Brunswick, Fredericton, A1.B., Canada E3B BE1 Received November 7 , 1988

Liu, We-C., Godbout, R., Jay, E., Yu, K. K.-Y. 2% Krause, M. 0. (1981) Tissue and species- specific effects of small molecular weight nuclear RNBB's on transcription in isolated mammalian nuclei. Cat?. 9. Biockent. 59, 343-352

Small molecular weight nuclear RNA9s (SnRNA's) purified from the 0.35 NaCl extract s f chromatin from human and monkey tissues have been found to stimulate transcription of chromatin dam isolated nuclei in a tissue- and species-specific manner. While SnRNA from normal human cells (WI38 fibroblasts and placenta) stimulates homologous transcription to some extent, it has a greater activity on transcription of heterologous tissue of the same species and no activity on heaerolsgous tissue of a different species (monkey kidney cells). Likewise SnRNA from monkey cells stimulates transcriptio~a sf homologous chromatin but has no eRect s n human cells. Fractionation of the RNA9s in pslyacrylamide gradient slab gels revealed that in d l cases the active RNA was 160-175 nucleotides in length. Our results are compatible with the hypothesis that the active RNA's are involved in the determination and maintenance of tissue differentiation by recognizing promoter or regulator sequences in the DNA and act at the level s f the nucleossme t~ Induce tissue-specific genes.

Liu, W.-C., Godbout, R., Jay, E., Yu, M. K.-Y. & Krause, M. 0. (1981) Tissue and species- specific effects sf small molecular weight nuclear RNA" on transcription in isolated m a m d i m nuclei. Cm. J . Biocbtern. 59, 343-352

Les RNA nuclCaires (SnRNA) de faible poids molkulaire, purifids depuis la chromatine extra& des tissus de l'homrne et du singe avec du 0,35 M NaCl, stirnulent la transcription de Ia chromatine dans des noyaux isolCs et d'une fason spkifique du eissu et de B'espkce. Le SnRNA des cdlules humaines norrnales Qfibroblastes WI38 et placenta) stimule jusqu'h un certain point la transcription homolsgue, mais il exerce une activitC plus grande sur la transcription d9un tissu h6t6rologue de la mCme espkce et une activitk nulle sur un tissu hCt6rologue d'une es@w diR6rente (mllules rdwales de singe). De la m&me fa~on , le SnRNA des cellules de singe stimule la transcription d'une chromatine hsmologue, mais il n9exerce aucun eRet sur les cellules humaines. Le fractisnnement des RNA daams des gels de polyacrylamide rCvkle que dans tous les cas, le RNA actif a une longueur de 160 B 175 nucldotides. Nos rCsuItats sont compatibles avec l'hypoth&se que les RNA actifs sont irnpliques dans %a diternaination et le maintien de la diR6renciation tissuhire en reconnaissant les skquences du promoteur ou du reguhteur dans le DNA et agissent au niveau du nucldosorne pour induire les gknes spCcifiques des tissus.

[Traduit par le journal]

Introdnctio~ sites and rate of elongation of RNA chains. Frac- tionation of the S ~ R N A in polyacrylarnide gradient

We have that an ex- slab gels revealed that the W A was 160- tracted from the chromatin of SV40-transformed 175 nucleotides in length and appeared to act in a human WI3 8 fibroblasts is involved in regulation of tissue- and species-speciec manner ( ). transcription in homologous isolated nuclei as well Much controversy has arisen in the literature as in nuclei of untransformed human and monkey concerning the role of RNA9s in cells (1, 2). Stimulation in normal cell nuclei was chromatin structure and function, starting with reported to involve both an increase in initiation the work of Banner and coworkers (3-5) which

was hater refuted as artifactual. Notwithstanding ABBREVIATIONS: SnRNA, small molecular weight nuclear

RNA; BME, basal medium; DMEM, Dulbecco,s biochemical and metabolic characterization on modified Eagle's medium; PVS, polyvinyl sulfate: TCA, hWNA's has k e n ~ a r ~ e b out in a number of - - - trichloroacet~c acid; A M D , actinornycin D; HMG, hi& laboratories. They were first found in the nuclei of mobility group; heterol., heterologous; homol., homologous. mammalian cells if$-8) but since &en several species

'Address all correspondence to: Dr. M- 0. fiause, De- were found in the cell cytoplasm (9-12) as weU as partment of Biology, University of New Bmnswick, Bag Service No. 45111. Fredericton. N.B.. Canada. ESB 6E1. in " of other organisms ( 3-15) in Tel. (506) 443-4603. adenovims-infected cells ( 16-1 8). Although con-

W8-4018 j81 jQ50343-10%0d .W j O @ 1981 National Research Council of Canada/Conseil national de rxherches daa Canada

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344 CAN. 6 . BIOCHEM. V8L. 53, 1981

centrated in the nucleus some of these RNA's ap- pear to '%shuttle9' between the nucleus and the cyto- plasm 14). The role of these RNA9s in the cell, however, is still obscure.

Our finding of an SnRNA subspecies which can stimulate transcription in a tissue- and species- specific manner has been supported by results from other groups as well. A 4.5%; SnRNA has been re- ported to increase the number of binding sites for WNA polymerase to initiate transcription on calf thymus chromatin (19), while a low molecular weight RNA isolated from 16-day-old chick em- bryonic heart was found to be capable of inducing specific changes in early chick blastoderrn cells simi- lar to those of embryonic heart differentiation (20).

The present communication was aimed at investi- gating the activity of %mRNA9s from different human and monkey tissues as well as to compare and char- acterize the active RNA9s separated in polyacryl- amide gradient slab gels.

Materials and methods Log-phase cultures of WI38 human diploid fibroblasts

were cultivated in BME as previously reported (2). Human p%acenhs were obtained fresh from Dr. Everett Chalmers Hospital, Fredericton and processed within 2 h after de- livery by a modification of a reported procedure (21). The tissue was washed in Eagle's spinner salts and homogenized in a Waring blender, three to four times for 15 s each time at low speed in the cold room. The hornogenate was filtered through 18 layers of cheesecloth, 20% glycerol was added, and the suspension was frozen at -90°C until use.

Vero cells derived from African green monkey kidneys were gown in DMEM supplemented with 10% v/v fetal bovine serum. In all cell types, in vvivo labelling of SnRNA was carried out using [3'P]orthophosphate (New England Nuclear) at a concentration of 30 pCi . mL-I ( I Ci = 37 GBq) in phosphate-free $ME or DMEM for 12-24 h.

Isolafion of nuclei and chromarirs Cultured human cdls were harvested and washed with

Eagle's spinner salt solution, and nuclei were isolated as described (2).

Because of greater fragility vero nuclei were isolated in lysing buffer (0.32 M sucrose, 5 mM MgCL, 0.2 mM CaCL, 5 rnM 2-mercaptoethanoB, and 0. B % Triton X- TOO), washed twice with the same buffer without Triton, and pelleted by centrifugation at 1808 >< g for 10 min. Placenta nuclei were isolated in the same buffer in a Dounce homogenizer with the loose pestle until free of cytoplasmic contamination. Sucrose was added to 1 M and the suspension was layered over a 2 M sucrose cushion and centrifuged at 27 000 x g for 15 min. The nuclear pellet was washed twice with 0.32 M sucrose, 5 mM MgCle, and 5 mM Zmercaptoetha- nol, and pelleted by centrifugation at 1008 >< g for 18 min. For transcription assay all the isolated nuclei were resus- pendkd in 40% glycerol and 50 rnM Tris-HCI pH 8.0, and kept frozen at -90°C until use. For isoIation of SnRNA, PVS was added to all solutions after cell lysis at a con- centration of 25 pg - mL-'.

Chromatin was isolated as previously described (2).

Exgraction o f SnRNA Isolated chromatin was resuspended in 0.35 M NaCl,

20 mM Tris-HCl pH 7.5, and 5 rnM 2-mercaptoethanol at a concentration of f mg DNA. 3 mLsP extraction buffer, and homogenized for 28 min on ice. After centrifugation at 185 008 x g for 2 h to pellet the chromatin, the supernatant was collected, sarcosyl and EDTA were added to 8.25% and 1 waM, respectively, and incubated with 250 pg-mL-' proteinase K for 15 min at 37°C. The reaction was stopped by addition of equal volumes of phenol saturated with buffer (8.35 M NaCl, $0 mM Tris-HCl pH 7.5, 1 mM EDTA, and 0.2% sarcosyl) and chloroforrn-isoamgrl al- cohol (24:B v/v). The organic phase was discarded, the extraction was repeated twice more, and the nucleic acids precipitated with 2.5 volumes of ethanol at -40°C and kept under ethanol until use.

When SnRNA's were to be eased prior to gel fractiona- tion, or as controls, DNase digestion was carried out. The nucleic acid precipitate was dissolved in 8.1 M NaCl, 10 mM Tris-HC1 pH 7.5, and 5 mM MgCL. RNase-free DNase (Boeringer) was added to a concentration of 20 yg-mL-' and the mixture incubated for 15 min at 37°C. The reaction was stopped by addition of proteinase K and sarcosyl as above, and the mixture re-extracted with phenol - chloro- form - isoamyl alcohol and precipitated in the same manner.

Slab nel elecgrar~hsrest sf SnRNA ~ ~ & t r o ~ h o r e ~ j s of S ~ R N A was carried out as described

(2). Samples were dissolved in 100 pL 8.1 M Tris-HC1 pH 8.0, and 1 mM EDTA with xylene cyan01 as dye marker, and layered onto the wells of 26 cm x 48 cm slab gels made up in a 5-15 9% polyacrylarnide gradient (0.5 9% bisacryl- amide and 5-1576 sucrose). Electrophoresis was carried out at 168 V until the maker was about 5 cm from gel bottom (about 48 h) , using 48 rnM Tris-acetate pH 7.0, 1 mM EBTA, and 5 mM NaOAc as tray buffer. With this method DNA contaminants stayed at the top of the gel and only RNA bands migrated through it.

a2P-labelled SnRNA bmds were detected by autoradiog- raphy on Kodak X-OMAT X-ray film. Unllabelled bands were detected under UV after staining the gels with 580 pg L-I ethidium bromide for 20 min.

Recovery of RNA from gel slices was carried out as described (2).

Assay of franscripbion in isolated nuclei Transcription assays were carried out in $0-pL volumes

containing 0.9 pg DNA as freshly thawed nuclei in 50 mM Tris-HC1 pH 8.8; 5 waM MgC12; 10 mM 2-mercaptoetha- nol; 0.5 mM ATP, GTP and CTP; 0.1 mM UTP; 50 pCi ["HIUTP (New England Nuclear, 35 Ci mmol-I) ; 10% glycerol; 0.1 96 bovine serum albumin; and 0.4 U Escherichia cali RNA polymerase purified by the method of Burgess and Jendrisak (22). SnRNAs were dissolved in transcription buffer and added to nuclei at the start of incubation.

Incubations were carried out at 25% for 30 min. Weac- tions were stopped by the addition of 2 mL 10% TCA m d 40 mM Na4P2O7, and precipitation on ice for 1% mine

Precipitates were collected by centrifugation at 8000 x g for 10 min. Pellets were dissolved in 50 mM NaOH to re- lease trapped rH1JUTP and nucleic acids reprecipitated as above, collected on GF-C filters, washed twice with 10 mL ice-cold TCA, washed once with 10 mL 95% ethand, dried, and counted in a Beckman ES 7000 liquid scintillation spectrometer.

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LIU ET Ak. 345

DNA was determined either by UV absorption s s by the indsle procedure (23) and RNA by UV absorption (1 BDso = 33 pg RNA).

SnRNA from normal human cells: WI38 and pla- centa

Experiments were initially set out to test the un- fractionated SnRNA's as to their ability to stimulate homologous and heterologous nuclei in transcription assays in vitro. Crude SnRNA preparations are pro- tein-free since they have been treated with protein- ase K and phenol-extracted three times as detailed in Materials and methods. However, they can be contaminated with traces of DNA; therefore, con- trols using NaBW-treated SnRNA under conditions where all RNA was destroyed allowed for quanti- tation of the SnRNA effect. Moreover, since we ob- tained similar results on the effect of SnRNA using either endogenous or E. eoli RNA polymerase (1) and the latter provided a more efficient system, we decided to make use of the bacterial polymerase to test further properties of the SnRNA.

FIG. I . Titration curves for transcription of WI38 nuclei in the presence and absence of SnRNA from SV40 trans- formed cdls and variable amounts of E. coli RNA polymerase. Q, WI38 control; 0 , with SnRNA at 0.1 : 1 (w/w) proportion to DNA; a d n, with the same propor- tion of SnRNA treated with 0.3 M NaBH for 3 h at 37°C.

placenta nuclei

r

0025 0.05 0.7 0.2 A J ~ Sn RNA added per 7 ~ g DNA

FIG. 2. Dosesesponse curve for activity sf S n W A from difYerent human cdls on stimulating transcription of hornologsus nuclei in the presence of E. coli RNA polymerase at 4.3 pg DNA.U-'. A, SV-WI38 SnRNA; 0 , placenta SnRNA; and W , WI38 SnRNA.

When using the bacterial enzyme it is important to maintain a high DNA to polyermase ratio since, if one saturates the template with the enzyme, the dif- ferential template activities of different kinds of chromatin are minimized or eliminated. A typical titration curve for WI38 nuclei in the presence md absence of SnRNA from SV40-transformed WI38 nuclei is presented in Fig. 1. Note that NaOH treat- ment eliminates +j of the stimulatory activity of SnRNA indicating that + is probably due to con- taminating DNA. In this particular preparation we did not subject the RNA to DNase treatment, hence the effect of DNA contaminants is maximal. How- ever, we found that DNase treatment does not com- pletely eliminate the traces of DNA in the prepara- tion and therefore with each preparation of cmde SnRNA we ran a NaBW-treated control. All sub- sequent assays were run at DNA to RNA polymer- ase ratios of 4-5 pg DNA .U-P.

A comparison of the effect of SnRNA's from placenta, W138 fibroblasts, and SV40-transformed W138 cells on transcription of their homoIogous nuclei using E. esli RNA polymerase is presented in Fig. 2. Since the effect of SV-WI38 SnRNA had

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346 CAN. S. BIBCHEM. VOL. 59, 1981

pog Sn RNA added p r % AQ DNA

FIG. 3. Dose-response curves for activity of SnRNA from human placenta m d 8.8138 cdls on sthulating trans- cription s f homslsgsus ( @, placenta nuclei + placenta SnRNA; and M, WE38 nuclei + WE38 SnRNA) and heteroidsgous nuclei (8, placenta nudei + Wf38 SnRNA; and 8, W138 nuclei + placenta SnRNA) in the presence of Ee cesdi WNA polymerase at 4.3 kg DNA oU-'.

been found to be dose-dependent ( I ) , dose- response curves rather than single-dose assays were compared. It can be seen that SwRNA from normal cells has only a slight stimulatsry effect on their own hsmologous nuclei9 quite different from that of SnRNA from SV40-trans£ ormed WI3 8 cells. If, however, one interchanges placenta md WI38 SnRNA, testing them with keterologous nuclei9 a marked stimulation can be observed-(Rg. 3 ) . The transcriptional activity of placenta nuclei with no SnRNA added is twofold higher than that of WI38. Yet SnRNA from W138 cells does stimulate tran- scription of placenta nuclei almost as much as SnRNA from transformed cells against ksmologsus transformed nuclei while %Psmo%sgous placenta SnRNA has a much lower effect on placenta nuclei. Similarly, SnRNA from placenta cells has much greater stirnulatory activity on W138 than on placenta nuclei. It appears, therefore, that SnRNA from normal cells can activate extra genes in a heterslogous tissue. Perhaps in homologous nuclei the sites sf activation are already nearly saturated with endogenous SnRNA9s.

"1 3 FIG. 4 . Autoradiography sf 32P-labelled SnRNA from

W138 cdls (A) and ethidilum bromide stained ( B ) poly- acrylamide gradient slab gel of SnRNA from human placenta (lane 1) and W138 (lane 2) . Markers 5S and tRNA were in lane 3, Numbers indicate the areas sf the placenta gel which were later extracted and tested for transcription assay. Letters identify the bands according to Penman (15) and between parentheses, those identified by Busch ( 5 ) . The gds were deetrsphoreed at 160 V using 40mM Trisaeetate pH 7.0, 1 mM EBTA, and 5 rnM NaOAe as tray buffer.

Ts make a more meaningful comparison and to eliminate the possible effect of DNA contaminants we needed to-know the subspecies composition of

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TABLE 1. Effect of placenta SanRNA purified by polyacrylarnide gel electrophoresis on transcription sf ho~nolsgous and heterologous WI38 nuclei in vitro with E. coli RNA polymerase1

D N A-dependent [3H]UTP incgsrporabion

(pmol . mg DNA-'. 30 min-I) Apprcax. AMD

SnRNA slice rauclestide Placenta nuclei WH38 nuclei A+SnRNA/CI B+SnRNA/C2 inhibition no.% lengths ( 4 @I (Dl (El % E/D

No addition I 2 3 4 5 6 7 8 9

10 1 1 12

1SnRNA's were extracted from gel slices as described in Materials and methods. Recovered RNA was divided into three identical portions: one tested with placenta nuclei as templates, one with WI3M nuclei as templates, and the last with W138 as template but in the presence of 100 pgstnL-I AMD, SnRNA (20 pg) was applied to the gel with 85-95 % recovery from each band. Values shown were corrected by subtracting picamoles UMP incorporated in the presence of 100 gg.mL-' AMD, therefore represent DNA-dependent RNA synthesis. Results were obtained by averaging data from three separate experi- ments.

'Slice numbers correspond to those depisted in Fig. 4.

placenta SnRNA as compared with normal WI38 and to identify the 'kctive5' RNA9s as to their size categories.

A sample of WI38 and placenta SnRNA frac- tionated in an acrylarnide gradient slab gel and stained with ethidium bromide is shown in Fig. 4B. On panel A an autoradiograph of 3T-labelled SnRNA from WI38 cells is shown since the auto- radiograph shows the bands more clearly than the ethidium bromide stained gel illustrated in panel B. We found that there was no need for DNase treat- ment of the SnRNA prior to gel electrophoresis since all traces of DNA stayed within stacking gel and none penetrated the lower gel. RNase- or alkali- treated preparations resulted in clear gels (2) . The letters identify the RNA bands according to the nomenclature of Penman ( 15) with that of Busch within parentheses 47). As expected, the position of the major bands is very similar in the two human tissues. The RNA's were then recovered from the numbered regions indicated in Fig. 4 and tested for their transcriptional activity with both WI38 and placenta nuclei. The results are presented in Table 1 together with the estimated nucleotide lengths of the RNA's in each region. These estimates were based on migration of yeast ribosomal and tRNA markers as well as on migration of U1 and U2 SnRNA whose size is known since they have been completely sequenced (7). It can be deduced that an WNA fraction 160-4 75 nucleotides in length

(slice no. 41, can activate heterologous W%38 nuclei threefold, twice the stimulation observed with ho- mologous placenta nuclei. Lower molecular weight RNA9s appear slightly inhibitory. Determination of ratios of stirnulatory activities of an RNA subspecies (heterologous-homologous transcription) turned out to be highly reproducible because it eliminates errors due to variable relative amounts sf RNA added in the nuclear transcription assay. Because the amounts of WNA recovered from gel sfices are too low to quantitate, we subdivided each recovered sample into three aliquots, one for homologous and one for heterologous transcription assays, and the third as a control in the presence of 180 pg . mk-l AMD to correct for possible contribution sf the added RNA as a template (2) . The latter turned out to be practically negligible since RNA-directed RNA synthesis requires Mn" as cofactor, and no Mn2+ was present in the transcription buffer.

SnRNA from monkey kidney cells Qvers) Experiments designed to test whether SnRNA's

from two different primate species (human and monkey) had any activity on heterolsgous tsm- scription, utilized unfractionated %nRNA9s from WI38 and vero cells. The results, presented in Table 2, indicate that addition of SnRNA from nonatrans- formed cells to homologous cell nuclei can result in a modest increase of transcription. However, when the same RNA is added to nuclei of a different

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TABLE 2. Activity of unfra~tionated SnRNA extracted from either human WI38 or monkey vero cells on transcription s f hornolsgsans and hetesologous nuclei with E. ccoli RNA polymerase

pnmsl[3H]UTT incorporated (30 min)l Change in transcription (treated/control)

WI38 nuclei Vera nuclei With homologous SnRNA With %leters%sgous SwRNA

No addition 1650 1810 +WH38 SnRNA 2584 1578 +Vero SnRNA 1445 2639

BSnRNA9s were added to nulei at ra concentration of 0.1 :I w/w to DNA.

TABLE 3. Effect of vero SnRNA purified by polyacrylarnide gel electrophoresis on transcription of W138 and vero nuclei ia vibro with E. coli RNA polymerase1

DNA-dependent [3H]UTP incorporation

(prnol .mg DNA-'. 30 min-1) Approx. AMD

SnRNA slice nucleotide W 138 nuclei Vero nuclei A+ SnWNA/C1 B +SnRNA/C2 inhibition no.) lengths (A) '3) 03 bE) (;6 E D

No addition 1 2 3 4 5 6 7 8 9

B 0 11 I 2 13 14 15 1 6

-- -

lSnRNA's were extracted from gel slices as described in Materials and methods. Recovered RNA was divided into three identical portions: one tested with W148 nuclei, one with vero nuclei, and the last with W138 nuclei in the presence of 1069pg.mL-1 AAMD. Values shown were corrected by subtracting picomoles UMP incorporated in the presence of AMD, therefore represent chromatin DNA-dependent RNA synthesis.

'Slice numbers correspond to those depicted in Fig. 5.

species, there appears to be a slight inhibition. These results indicate a discriminatory effect of SwRNA in homoBogous and hetesolsgous chromatin. To test whether the active RNAys from WI38 m d vero cells fall in the same size categories as those found for placenta cells, we fractionated the SnRNA9s from the two primate species by polyacrylamide gradient gel electrophoresis. Figure 5A shows an ethidiurn bromide stained gel obtained with untreated (lane 2) and urea-treated (Bane 4 ) SnRNA from vero cells fractionated in the same gel system as in Fig. 4. Lane 1 shows the position of the markers 5s and tRNA. Figure 5B shows that autoradiograph ob- tained with the same gel with ""P-labelled SnRNA9s. Here also the letters correspond to the nomenclature of Penman (15) with that of Busch within paren- theses (7). A band pattern similar to that observed

in human tissues is apparent. Yet there are a few differences in minor bands particularly in the L region as compared with Fig. 4 and SV-WI38 SnRNA as previously reported (2). In the latter article we observed a shift in the active region of SV-WI38 SnRNA upon urea treatment, with no visible change in band pattern. Untreated RNA showed the highest activity in a region estimated to be 300-450 nucleotides in length while urea- treated SnRNA showed the highest activity in a region of 160-1'75 nucleotides. Here too no diRer- ence in migration is observed as a consequence of urea treatment except for one band below the 5S which disappears into the 5 s band in urea-treated samples, presumably due to loss of secondary stmc- ture. In these gels secondary structure is never eom- pletely eliminated. We did run urea-gradient gels to

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LIU EF AL. 349

% RNA

4 2 3 2 3 FIG. 5. Ethidiurn bromide stained (A) and autoradio-

graph (B) of SnRNA from vero cells fractionated in a polyacrylamide gradient slab gel. Numbers indicate the areas of the gel which were later extracted and tested for transcription assay. Letters identify the bands according to Penman ( 1 5 ) and Buck within parentheses (7) . Lane 1, markers 5S and tRNA; lane 2, untreated SwRNA; and lane 3, urea-treated SnRNA.

minimize effects of secondary structure on migra- tion; however, the activity of SnRNA's recovered from urea gels was noticeably lower (results not shown).

The gels were then sliced as indicated by the numbered regions in Fig. 5 and the respective RNA's were recovered and tested for their activity on transcription of homologous and heterologoui nuclei.

The results of the transcription assay using vero SnRNA's on WI38 and vero nuclei are presented in Table 3. The calculated mean number of nucleo- tides of the RNA's extracted from each numbered region are also presented. While vero SnRNA" ap- pear somewhat inhibitory when tested on WI38 nuclei, they behave distinctly differently when tested on homologous nuclei. Here again a marked stimu- lation is achieved with an SnRNA species ranging in size between 160-4 75 nucleotides. Other dis- criminatory effects of SnRNA on the two kinds of nuclei appear to be rather minor, and possibly are not significant.

The results of the transcription assays using WI38 SnRNA9s on W138 and vero nuclei are presented in Table 4, together with the calculated number of nucleotides for the RNA's extracted from each gel slice. Discriminatory effects of the SnRNA be- tween homologous and heterologous species can be seen primarily in regions 7 and 8. However, in the case of region 7 it appears to be due to inhibition of heterologous nuclei rather than to stimulation of homologous ones. Here again it is region 8, con- taining RNA's ranging in size between 160-475 nucleotides, which has the highest stimulatory activ- ity on nuclei of the same species. In normal human and monkey cells we did not detect activity in the upper regions (above 300 nuclestides) as in the case of SV40-transformed WI38 cells (2). In the case of the human cell SnRNA represented in Fig. 4, we did not even slice the gels beyond the 225 nucleo- tide region since preliminary trials showed no activ- ity in the upper portions of the gel. However more recent experiments indicate that these results are due to nicking of the active RNA around the middle of the molecule. We observed that in normal and SV40-transformed mouse 3T3 cells, the activity shift from the larger to the half size RNA species could be prevented by the use of two or more nuclease inhibitors together with greater speed dur- ing preparation of SnRNA (24). Because we have observed activity shifts in the absence of any ap- parent differences in band pattern, we think it is unlikely that the active molecules can be totally identified with any one of the major bands, although they may comigrate with one of them.

The results presented in this communication indi-

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TABLE 4. EEmt of W138 SnRNA purified by polyacrylamide gel electrophoresis om transcription of W138 and vers nuclei in viiro with E. coli RNA polymerase

-

DNA-dependent [3HIUTP incorporation

(prnsl .mg DNAA' 30 mhrH) Approx. AM19

SnRNA slice nucleotide W138 nuclei Vero nuclei A+SnRNA/CI B+SnRNA/Cs inhibition no, lengths (a> (B) [Dl BE) '7 , c D/E

No addition 5 6 7 8 9

1 0 11 12 13 14 15 16

%nRWA's were extracted from gel slices as described in Materids and methods. Recovered WNA was divided into three identical portions: one tested with WI38 nuclei. one with vers nuclei, and the last with vero nuclei in the presence OF 100pgoanL-"MD. Values shown were corrected by subtracting piconmoles incorporated in the presence of AMD, therefore, represent chromatin DNA-dependent WNA synthesis.

%%.lice numbers csersspond to those depicted in Fig. 5.

cate that a subspecies of SnRNA estimated to be 160-175 nuclestides in length is capable of stimu- lating transcription of isolated nuclei in a tissue- and species-specific manner.

Since the stimulatory activity could be observed with either E. eoli or endogenous RNA polymerase ( 1) it appears likely that this KNA acts at the level of the chromatin template. Although E. coli RNA polymerase may not specifically recognize euka- ryotig: promoters, it has been reported that it is capable of transcribing active regions of the chromatin, presumably because the DNA in those regions is made available for interaction with the enzyme ( 2 5 ) . The greatest drawback in the use of the bacterial enzyme is the known RNA-dependent RNA polymerase activity of the E. csli RNA polymerase (26). However, such drawbzcks can be corrected by eliminating Mnw from the transcrip- tion buffer and using A as control so that one can subtract, from the stimulatory activity observed with any active SnRNA, all those counts which were not inhibited by the drug (2) .

We have found that, at high DNA to polymerase ratio, the kinetics of the transcription assay m d stimulatory properties of the ""active33nnRNA, using isolated nuclei and bacterial WNA polymerase, are very similar to those observed with endogenous en- zyme ( 1 ). Pa&icularly where numerous SnWA9s must be tested and compared, the heterolsgsus sys- tem is considerably more economic since one can scale down the amount of DNA template 2nd hence

SnRNA needed for the assay. Using purified RNA bands extracted from gel

slices we were able to conclude that the stimulatory effect of the active SnaRNA is indeed chromatin DNA-dependent. Contrsls using SnRNA minus chromatin or extracts from WNase or alkali-treated gel tracks proved that the effect is due to RNA rather than to degraded DNA comigrating with the RNA bands (2). The possibility of a nonspecific stirnulatory effect of RNA on transcription is mled out by the finding that only some SnRNA subspecies appear to be active.

The finding sf an active subspecies of SnRNA in normal human tissues indicates that the active SnRNA previously found in SV40-transformed W138 cells ( 1 , 2) is not a unique characteristic of viral transformation. However, we did find marked diRerences in the activity of nsmal and transformed cell SnRNA3s.

The SnRNA9s from normal WT38 and placenta cells are capable of stimulating transcription in heteroIogous tissue nuclei to a larger extent than that observed in homologous nuclei. This finding pro- vided more reliable means for quantitating the as- tivity of RWA9s recovered from gel slices after frac- tionation sf RNA by gradient pslyacrylarnide gel electrophoresis. Since the ""active" RNA's appear to occur in amounts too low for detection by normal optical density means, comparison of the activity of aIiqusts in homologous and heterologous transcrip- tion provides a consistent quantitative method.

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LIU ET AL. 351

Moreover, an additional aliquot can be used in the presence of AMD to detect AMD-resistant trans- cription, and to calculate the amount of stimulation which is chromatin DNA-dependent.

Results have shown that the 66active" RNA" of human placenta and monkey vero cells fall within the same size range as those detected in SV40-trans- formed WI38 SnRNA9s ( 2 ) . However, they ap- pear digerent from the ""active" RNA's of trans- formed cells because ( a ) they are much less active in homologous transcription and ( b ) they do not act on nuclei of a different species. We have found that SnRNA's from nontransformed cells have some stirnulatory activity on their own homologous nuclei, a much higher stirnulatory activity on nuclei sf a different tissue of the same species and a slight in- hibitory activity on nuclei sf another species. On the other hand, SnRNA from transformed human cells (SV-WI38) could stimulate transcription in both human and monkey nuclei, although the stimula- tion observed in human nuclei was considerably higher (2) . The finding that WH38 SnRNA shows considerable stimulation sf transcription of placenta nuclei and vice versa could be explained on the basis of induction of extra genes in the heterologous tis- sue. Placenta is a very active tissue. It functions simultaneously as fetal lung, liver, gut, and kidney (271, therefore it must contain active genes which are normally turned off in WT38 fibroblasts. On the other hand, cycling fibroblasts must contain cell cycle genes and other fibroblast-specific genes which are turned off in placenta. Tt is likely, therefore, that their regulatory RNA's will recognize different regions of the chromatin and induce transcription in those regions. The results obtained by interchang- ing SnRWA9s between two different primate species can be explained on the basis that sequence homology between the RNA5s and the recognition sites in DNA is higher in a homologous than in a heterologous albeit-related species.

When the SnRNA9s from both. human tissues QW138 fibroblasts and placenta) and from monkey cells (vero) were fractionated in acrylamide gradient slab gels and the recovered RNA's were tested for transcriptional activity in the same manner, the dis- criminatory RNA9s which elicited stimulation of transcription in homologous nuclei were found to belong to a similar size class as those found in SV40- transformed WP38 cells as previously reported (2) . As indicated in the results, this 160-175 nucleotide long active RNA is likely to be derived from a larger 300-350 nucleotide species by nicking of the molecule around its center during cell lysis and preparation of the RNAs. We have more recently

been able to prevent this nicking by the combined use of (PVS) and spermine as nuclease inhibitors, and by using greater speed during preparation of the SnRNA up to the addition of proteinase K and sarcosyl Qsr sodium dodecyl sulfate), which more effectively inhibit any remaining nuclease activity (24). This two to one relationship in the size of the active RNAs could be explained if the native molecule has a hairpin configuration with a nuelease sensitive single-stranded loop. Thus upon denatura- tion, intact molecules will stay in the larger 300- 358 nucleotide region while the nicked half mole- cules will migrate in the 160-175 nucleotide region.

This consistency in the size categories of the "active" SnRNA's suggests that recognition may occur at the level of the nucleosome, since the size sf the active RNA halves is similar to that of DNA associated with the kistone bodies (28).

It is likely that the inducing activity in vivo in- cludes SmaRNA complexed with other chromosomal proteins since they occur tightly bound to non- histone proteins and can only be released under harsh protein denaturing conditions (1). This also suggests that when 'kctive" SnRNAs are added to isolated nuclei, they probably interact with nuclear proteins to stimulate transcription. Among the pro- teins, which coextract with SnRNA in 0.35 A4 NaCl, the HMG proteins have been well characterized and it is known that HMG 14 m d I7 associate preferentially with nuclessome cores in transcribable regions of the chromatin (29) . However, HMG proteins do not change from tissue to tissue or have the ability to direct themselves to specific regions of DNA. We suggest that the "a~tive '~ SnRNA might serve as the key to the lock by recognizing pro- moter or regulator sequences in the DNA thus pro- viding the extra accuracy which is essential to the determination and maintenance of tissue differentia- tion in eukaryotes. This hypothesis is supported by the finding that small RNA's are involved in em- bryonic heart diff erentiation (20).

Further purification and characterization of the active SnRNA species is in progress in a system which will allow for a study of its origin and mode sf action in the activation of specific genes.

This work was supported by grants from the Natural Sciences and Engineering Research Cow- ci% of Canada and the National Cancer Institute of Canada.

We wish to thank Karen Gordon for expert as- sistance and cultivation of cells.

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