Eur. J. Riochem. -709,445-452 (1992) FEBS 1992

Regulation of insulin-like-growth-factor-I1 gene expression in rat liver cells Raffaele ZARRILLI

’,Vittorio COLANTUONI’ and Carmelo Bruno RRUNI’

’ Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimcnto di Biologia e Patologia

’

Cellulare c Molccolare “L. Califano” and Dipartimento di Biochimica e Biotecnologie Mediche, Universita di Napoli, Napoli, Italy (Received May 22/July 9, 1992)

-

EJB 92 0716

The rat insulin-like-growth-factor-(1GF)-I1 gene is expressed at high levels during embryonic and fetal life and at low levels in adult animals. To study the regulation of IGF-I1 gene expression, we analyzed the synthesis and localization of the IGF-I1 transcripts in cultured rat liver cells either expressing (BRL3A cells) or not expressing (BRL30E and F A 0 cells) the IGF-I1 mRNA. The IGFI1 gene is transcribed at a similar rate in expressing and non-expressing cells, whereas its nuclear and cytoplasmic RNA levels are diversely distributed in the cells. IGF-I1 RNA is more abundant in the cytoplasmic than in the nuclear RNA fraction of BRL3A cells and is present in the nucleus but not in the cytoplasm of the F A 0 cells. However, both precursor and mature IGF-I1 nuclear RNA levels are reduced in F A 0 cells. Our data indicate that the IGF-I1 gene expression is regulated by mechanisms affecting the subcellular distribution and the abundance of the transcripts.

Insulin-like growth factor (IGF) I1 is a mitogenic polypeptide that is believed to play an important role in embryonic growth of rodents (Daughday and Rotwein, 1989; Humbel, 1990). IGF-I1 peptide is present at high levels in the serum of fetal rats and decreases to low adult levels by 3 weeks of age (Humbel, 1990). IGF-I1 mRNA levels are high in many tissues during embryonic and neonatal life and decline overs 222 days after birth (Brown et al., 1986). In the adult, IGF-I1 expression is confined to the choroid plexus and leptomeninges (Stylianopoulou et al., 1988). Evidence supporting a mitogenic role for IGF-TI during development stems from experiments in which disruption of one IGF-I1 allele determined growth-deficient heterozygous progeny. These experiments also demonstrate that the gene is subject to imprinting, the active allele being the one inherited from the father (De Chiara et al.. 1990; De Chiara et al., 1991). In the rat, IGF-TI is a singlc-copy gene that gives origin to a family of RNA transcripts (Frunzio el al., 1986; Evans el al., 1988). These heterogeneous RNA species are synthesized using three promoters and different polyadenylation sites (Chiariotti et al.: 1988; Evans et al., 1988; Matsuguchi et al., 1990). Coordinate developmental regulation has been demonstrated for all IGF-I1 mRNA species (Graham et al., 1986). Little is known about the molecular mechanisms that regulate IGF-11 gene expression during development. Analysis of the sequenccs of thc three rat promoters did not allow the identification of regulatory elements able to confer developmentalspecific expression (Evans et al., 1988; Matsuguchi ct al., 1990). The P3 proximal promoter, that accounts for more than 90% of the transcriptional activity of the rat gene, has been further characterized and found to span 128 bp, containing an AlA box and four GC boxes binding the general transcription Correspondence to C. B. Bruni, Dipartimento di Biologia e Patologia Cellularc e Molecolare “L. Califano”, Via S. Pansini 5, 1-80131 Napoli, ltaly Fax: +39-81-7703285. Abbreviutiuns. IGF, insulin-like growth factor; IGFBP. insulinlike-growth-factor-binding protein.

Factor Spl (Evans et al., 1988). The P2 upstream promoter also has a very simple structure and consists of an ATA box and two GC-core hexanucleotides (Matsuguchi et al., 1990). A reporter gene, driven by the IGF-I1 promoters, was transiently expressed at comparable levels both in IGF-11-mRNA expressing and non-expressing cell lines (Evans et al., 1988; Matsuguchi et al., 1990). In addition, nuclear extracts from IGF-11-mRN A expressing and non-expressing cells, challenged with DNA fragments spanning distinct segments from the rat promoters, produced similar results in footprint, bandshift and methylation-interference experiments (Evans et al., 1988; Matsuguchi et al., 1990). In the present study, we have investigated the molecular mechanisms of IGF-I1 regulation in a system of cultured rat liver cells either expressing or not expressing IGF-11. Analysis of the total RNA levels and of the relative transcription rates and measurements of steady-state RNA from nuclear and cytoplasmic subcellular fractions indicated that the IGF-I1 gene is transcribed in all cell types. IGF-11-specific transcripts are present in different amounts and are diversely distributed in the cellular compartments of IGF-11-expressing and nonexpressing cells.

MATERIALS AND METHODS Materials [LX-~’P]~ATP (400 Ci/mmol), [ce3’P]dGTP (400 Ci/mmol) and [LX-~’P]UTP(400 Ci/mmol) wcre from Amersham. Guanidinium thiocyanate was purchased from Yluka. Restriction endonucleases were from Boehringer Mannheim. The RNase-protection-assay reagents were all purchased from Promega. Cell cultures The isolation, growth and properties of BRL3A, BRL30E and F A 0 cells have been described elsewhere (Deschatrette

446

9

5’

1

n

1

1 kb M

p3

1

2 IE3

TGA

ATG

n

h

3

4

3’

5

6

I E4

* I

II I

Irn

14

B B E6

rn

Fig. 1. Physical map of rat IGF-I1 gene. Exons arc indicatcd by boxes and numbered according to Daughday and Rotwein, 1989. Empty boxes indicate the untranslated regions and filled boxes mark the coding region for the pre-pro-IGF-11. Arrows denote the sites of transcriplion initiation from promoters PI, l’, or P3.The DNA segments used as probes throughout the experiments and their relative position in the gene are shown at the bottom of the figure.

and Weiss, 1974; Dulak and Temin, 1973; Nissley et al., 1977). All cells were maintained as monolayer culture and were grown in Coon’s-modified Ham’s F-12 medium, supplemented with 5% fetal calf scrum (GIBCO Laboratories).

Hybridizationprobes

The ICF-I1 DNA fragments used in the experiments are shown in Fig. 1. Probe A is an 800-bp cDNA spanning exons 3 - 5 and the 5’ end of exon 6 (Frunzio et al., 1986). Probe B is a 301 -bp BamHl- Sac1 fragment spanning the 3’ end of the A probe. This fragment was cloned in pGEM vector. Probe IE 3 is a 300-bp EcoRI- NcoI fragment spanning the 3‘ end of the second intron and the third exon. Probe E 4 correspond to a 120-bp EcoRI-HinclI fragment of the fourth exon. Probe I 4, derived from the genomic clone B12 (Frunzio et al., 1986), corresponds to a 299-bp PstI - XhoI fragment of rat IGF-I1 fourth intron. Probe E 6 corresponds to a 300-bp Hind111 XhoT fragment of the sixth exon. All the IGF-I1 fragments have been subcloned into a pGEM vector. pGEM plasmid, carrying mouse P-actin cDNA (Tokunaga et al., 1986), was kindly provided by R. Dono. The IGF-I1 800-bp fragment (probe A) and mouse p-actin 200-bp inserts were used as probes in Northern-blot analysis. Antisense transcripts of IGF-11 I 4 and B probes and the mouse p-actin probe were used in RNase protection assays. The following targets were used in the run-on experiments: pA, pB, pIE 3, pE 4, pl 4, pE 6 plasmids carrying A, B, IE 3, E 4, 14, E 6 IGF-I1 DNA fragments (Fig. 1); pIGFBP-1, a plasmid carrying a 1.45-kb EcoRI insert encompassing the full coding region of rat insulin-like-growth-factor-binding protein (1GFBP)-1 cDNA, kindly provided by G. Ooi (Orlowski et al., 1990); pIGFBP2, a plasmid carrying a 1.3-kb Hind111 fragment spanning 1.05-kb of rat IGFBP-2 cDNA and the 0.25-kb fragment from the long arm of IgtlO phage (Brown et al., 1989); p-albumin, a plasmid carrying a PstI insert of rat albumin (Sargent et al., 1979), kindly provided by G. Perozzi; p-ferritin, a plasmid carrying a cDNA complementary to human L chain mRNA (Santoro et al., 1986), kindly provided by V. De Franciscis; p-mouse p-actin (see above); pIGF-I, a plasmid containing a rat IGF-I cDNA (Bucci et al., 1989); pB1R-S, a plasmid carrying a rat repetitive sequence belonging to the class of rcpctitivc sequences known as ‘poly(CA)’ or ‘TG elements’ (Chiariotti et al., 1988); pGem vector (Melton et al., 1984).

Nuclear run-on assays

Preparation of nuclei, RNA elongation and isolation were performed as described (Greenberg and Ziff, 1984), except that the nuclei were frozen and stored at - 80 “C before use and that the 32P-labeled RNA was treated with RNase-free DNase (10 pgjml) for 30 min at 37°C. 2 x lo7 nuclei were used for each assay. RNA, corresponding to about 4 x 106cpm, were hybridized to an excess of DNA targets (10 pg) immobilized on nitrocellulose filters. The hybridization conditions were 50% formamide, 5 x NaCljCit. (1 x NaCl/Cit. was 0.15 M NaCl, 15 mM sodium citrate: pH 7.0), 5 x Denhardt’s solution, 50 mM sodium phosphate, pH 7.0,O.l O/o sodium dodecyl sulfate, 100 pg/ml yeast RNA and 10 pgjml pGEM DNA, at 37°C for 3 days. RNA analysis

Total cellular RNA was isolated from cells by the guanidinium-thiocyanatelacid-phenol procedure (Chomczyinski and Sacchi, 1987). Nuclear and cytoplasmic RNA fractions were isolated by lysing cells in Nonidet P-40 hypotonic buffer as described (Arrigo et al., 1989). Northcrnblot analysis of IGF-I1 mRNA and p-actin mRNA were carried out essentially as described (Church and Gilbert, 1984: Sambrook et al., 1989). Probes were generated by random primer extension (Feinberg and Vogelstein, 1984). RNase protection assays were carried out with antisense RNA probes generated from the various pGEM-derived plasmids with Sp, or T7 RNA polymerases and assays were carried out as dcscribed (Melton et al., 1984) with the following modifications: all hybridization reactions were performed overnight at 46 ”C with probe (2.5 x lo5 cpm); when two probes were used simultaneously, each probe (2 x lo5 cpm) was used; RNase digestion was carried out at 30°C for 1 h. The resulting resistant RKA species were denatured and resolved on a 6% polyacrylamide gel containing 7 M urea and visualized by autoradiography. RNA levels were quantified by densitometric scanning of the autoradiographs using an LKB Ultrascan densitometer. RESULTS

IGF-IT steady-state mRNA species

To study the regulation of the IGF-I1 gene, we used, as a model system, three cell lines all derived from rat liver but

447

A

B

Fig. 2. IGF-I1 mRNA levels in rat liver cells. (A) Northem-blot analysis of the RNA from BRL 3A, BRL30E and F A 0 cells. 10 pg total RNA from each cell line was electrophoresed on a 1.2% agarose/formaldehydc gel, blottcd and the filter was hybridized to the A probe (Fig. 1). IGF-I1 indicates the very slrong band corresponding to thc two major transcripts of 4.6-kb and 3.5-kb. The faster migrating 2.0-kb and 1.0-kb RNA species arc also visiblc. The migration of 28s and 18s ribosomal RNA species, as size markers, is indicated on the right of the figure. The signal produced by hybridization of the same filter to a fi-actin probc, used to verify the amount of RNA loaded in cach lanc, is shown on the bottom. (B) RNase protection analysis of RNA species from the same cells. Serial twofold dilutions of total RNA from BRL3A (lanes 1 -9), F A 0 (lancs 11, 12) and BRL30E (lanes 13, 14) cells were hybridized to an antiscnsc transcript of the IGF-I1 B probe (Fig. 1 ) and the protectcd fragments were analyzed on a 6% denaturing polyacrylamide gel. Transcription from the pGEM-IGF-I1 B construct generates a 366-nucleotide RNA with a spccific 301-nucleotide-protected hybrid. The amounts of RNA analyzed arc indicated on the top of each lane. 30 pg hctcrologous yeast KNA were hybridized to the same probe (lanes 10 and 15); undigcsted probe (lane 16). Sizes of the undigested probe and of the protected hybrid are indicated.

cxpressing different levels of IGF-11. BRL3A are Buffalo rat hepatocytcs that have lost differentiated functions of the adult liver and express high levels of IGF-TI (Dulak and Temin, 1973; Nissley et al., 1977). BRL30E, a thymidine-kinase-derivative (Ambesi, F. S. and Coon, H., unpublished results) of BRL3A2, a cell line also isolated from the Buffalo rat liver (Nissley et al.; 1977), and FAO, a rat hcpatoma cell line (Deschatrette and Weiss, 1974), are both negative for IGF-I1 expression (Nissley et al., 1977 and Fig. 2). F A 0 cells, despite their tumor origin, can be considered differentiated hepatocytes, expressing many adult liver functions (Deschatrette and Weiss, 1974). Fig. 2 shows the analysis of total RNA extracted from the cell lincs described above. IGF-I1 mRNA species wcrc prcscnt at high levels in RRL3A (Fig. 2A, lane l), but were not detectable in BRL30E or F A 0 (Fig. 2A, lanes 2 and 3). Analysis by RNase protection (Fig. 2B) revealed that IGFI1 steady-statc mRNA were 2500 - 5000-fold higher in BRL3A than in BRL30E or F A 0 cells. Both D N A fragments used as probes in the above experiments and indicated as A and B (Fig. 1) span a region of the IGF-I1 gene present in all different transcripts. Thus, all IGF-I1 mRNA species were below the level of detection in the non-expressing cclls. IGF-I1 transcriptional rates

To determine whether the differences in IGF-I1 mRNA levels correlate with different transcription rates, the activity of the IGF-11 gene was measured in nuclear transcription (run-on) assays (Fig. 3). The gene was transcribed to a similar extent in the cells analyzed, its rate of transcription in BRL3A bcing equivalent to that found in F A 0 cells and only 2.5-fold higher than that of BRL3OE cells. The relative transcriptional rates of two other genes known to be transcriptionally regulated (Ott et al., 1984; Brown and Rechler, 1990; Herbst et al., 1991; Tseng et al., 1992) were also analyzcd. IGFBP-2 is a carrier protein for insulin-like growth factors, mainly produced in fetal hepatocytes (Brown et al., 1989); albumin is

one of the major plasma proteins synthesized by differentiated hepatocytes (Sargent et al., 1979). BRL3A cells express high levels of IGFBP-2 (Brown et al., 1989), whereas IGFBP-2 transcripts were undetectable in BRL30E cells (data not shown). The IGFBP-2 transcriptional rate was 10-fold higher in nuclei from BRL3A cells expressing IGF-I1 than in nuclei from RRL30E cells not expressing IGF-11. The albumin transcription rate was approximately 10-fold higher in nuclei from F A 0 cells expressing IGF-I1 than in nuclei from BRL3A cells not expressing IGF-IT (Fig. 3). In the same experiments, two housekeeping genes, ferritin and p-actin, were also used as controls for normalization, since their transcription rates were similar in all three cell types (Fig. 3). The IGF-I gene is not expressed in BRL3A and BRL30E cells (data not shown) and, accordingly, its transcriptional rate was below the level of detection in both cell lines (Fig. 3). IGF-TI gene transcription in isolated nuclei was also measured by using probes spanning different regions of the IGFI1 gene. As shown in Fig. 4, irrespective of the targets, no differences in transcriptional elongation were detected in nuclei isolated from BRL3A and F A 0 cells. In both cell lines, the 5’-proximal IE 3 target gave a stronger signal compared to the others (10-fold over that of the E 4 intermediate probe and twofold over that of the E 6 3’ distal probc). In the same experiments. the transcriptional levels of the IGFSP-1 gene, coding for another carrier protein of the I G F and known to be transcriptionally regulated in hepatoma cells (Orlowski et al., 1990), were 10-fold higher in nuclei from F A 0 than in BRL3A cclls. Transcription of the P-actin gene did not vary between the two cell lincs.

IGF-I1 nuclear and cytoplasmic RNA levels Since the more than 2500-fold difference in IGF-I1 mRNA between BRL3A, BRL30E and F A 0 cells cannot be accounted for by the small differences observed in the transcription rate, other mechanisms of rcgulation were investigated. Nn-

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Fig. 4. IGF-I1 gene transcription elongation analysis in nuclei from BRL3A and F A 0 cells. Autoradiographs of two representative experiments and the relative position of the different targets are shown. The TGF-I1 targets used in the experiments correspond to probe IF 3, E 4 and E 6 of Fig. 1. The E 4 targel gives hybridization signals comparable to background lcvels, possibly due to its short size.

80 & U

2Q

+

60

when the same RNA samples wcrc analyzed in a RNase protection assay, using a ‘sense’ transcript of the B probe (data not shown). This result excluded the possibility that TGF-TI transcripts could derive from the non-coding strand o r that cellular DNA could contaminate the nuclear RNA fractions analyzed.

lI

W

40

2 I-

4w

20

U

IGF - I1

ALBUMIN FERRlTlN

Fig. 3. IGF-I1 transcription rates in BRL3.4, BRL30E and F A 0 cells. Nuclear run-on assays were uscd to examine transcription rates in BRL3A and BRL30E cells (A), and BRL3A and F A 0 cells (B). Autoradiographs of two representative experiments and the relative position of the different targets are shown on the right. The IGF-11 target used in these expcrimcnts corresponds to probe A of Fig. 1. The histograms on the left represent the av-erageof densitometric scan analysis of at least three separate experiments. The relativc transcription rates were calculated by subtracting non-specific vector hybridization values. IGF-11 hybridization values ranged over 2 1Wold over background. To compare relative transcription rates in nuclei purified from different cell lines, the hybridization values of caeh gene were normalized to those of B-actin or of repetitivc sequences (BI R-S) obtaining similar results. The histograms in the figure refer to those obtained with 8-actin normalization.

clear and cytoplasmic RNA fractions of the cells were analyzed by an RNAse protection assay for total IGF-I1 RNA content using an antisense transcript of the B probe (Fig. 1). As shown in Figs 5 and 6, an IGF-11-specific protected hybrid was detectcd in BRL3A cells, its levels being 10-fold higher in the cytoplasmic than in the nuclear RNA fraction. The same hybrid was detected in the nuclear but not in the cytoplasmic fraction of RNA species from both BRL30E and F A 0 cells, its levels being 100-fold and 10-fold lower, respectively, than those found in the nuclear RNA fraction of BRL3A cells (see also Table 1). B-actin produced similar hybridization signals in nuclear and cytoplasmic RNA fractions from all three cell lines (Figs 5 and 6). N o hybridi7ation signals were detected

Analysis of IGF-I1 RNA precursors To establish if the ratio between processed and unprocessed RNA molecules varied between expressing and iionexpressing cells, we measured the abundance of IGF-I1 precursor RNA molecules in the nuclear compartment. Nuclear and cytoplasmic RNA fractions were hybridized to an antisense RNA transcript of the I 4 probe, spanning most of the fourth intron of the gene (see Fig. 1). As illustrated in Fig. 7, a specific protected hybrid was observed in the nuclear fraction of all three cell lines, its levels being higher in BRL3A than in BRL30E and F A 0 cells. Densitometric analysis of the autoradiographs revealed that IGF-11-precursor transcripts were 10-fold and 100-fold more abundant in BRL3A than in F A 0 and BRL30E cells, respectively. Thus, the relative differences found with the intron probe paralleled those obtained with the exon probe (compare Fig. 7 to Figs 5 and 6). As expected, no IGF-I1 RNA precursor band was detected in the cytoplasmic fraction of both the IGF-TI-expressing and non-expressing cells. No hybridization signals were detected when the RNA samples were analyzed in a KNase protection assay, using a ‘sense’ transcript of the I 4 probe (data not shown). DISCUSSION

In this study, we analyzed the expression of the IGF-I1 gene in a model system of rat liver cell lines and found differential post-transcriptional regulation of IGF-II gene expression.

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PROBE IGF-II

1

2

3

4

5

Fig. 6. IGF-I1 lcvcls in nuclear and cytoplasmic RNA fractions from BRL3A and F A 0 cells. Nuclear (lanes N) and cytoplasmic (lanes C) RNA species, isolated from the indicated cells, were analyzed by the RNase protection assay. 10 pg each sample were simultaneously hybridized to antiscnsc transcripts of B rat IGF-I1 probe (top) and of mouse 8-actin cDNA (bottom) and the protected hybrids analyzed o n a 6% polyacrylamidc denaturing gel. 30 pg heterologous yeast RNA (lanc 5) were hybridized to the same probes. Different exposurcs of IGF-I1 (96 h) and B-actin (6 h) protectcd RNA species from the same experiment are shown. The sizes of the protected bands are indicated on the right.

PROBE

ACTIN

1

2

3

5

4

6

7

8 9 1 0 1 1

Fig. 5. IGF-I1 levels in nuclear and cytoplasmic RNA fractions from BRl,3A and BRLSOE cells. (A) Nuclear (lanes N) and cytoplasmic (lanes C) RNA species, isolated from the indicatcd cells, were analyzed by the RNase protection assay. 10 pg (lanes 1, 3 and 5) and 20 ~g (lanes 2, 4 and 6) of the samples wcrc hybridized to an antisense transcript of the IGF-I1 B fragment and the protected hybrids analyzcd on a 6% polyacrylamide denaturing gel. Transcription from the pGEM-IGF-I1 B construct gcncrates a 366-nucleotides RNA with a specific 301-nucleotide-protected hybrid. 30 ~g heterologous yeast RNA (lane 7) and no R N A (lane 8) were hybridizcd to the same probe or an undigested probe (lanc 9). A shorter exposure of the first two lanes of thc autoradiogram is shown on the bottom. (B) 1 pg (lanes 1,3, 5 and 7) and 2 pg (lanes 2,4, 6 and 8) of the samc RNA samples were hybridized lo an antisense transcript of mouse 8-actin cDNA. Transcription from pGEM 8-actin construct generates a 202nucleotides RNA with a specific 145-nucleotide-protcctcd hybrid. 30 pg heterologous yeast RNA (lam 9) and no RNA (lane 10) were hybridized to the same probe or an undigested probe (lane 1 t).

Table 1. IGF-I1 expression in rat liver cells. The mean densitomctric scan values and standard errors of at least three independcnt experiments are reported. Relative transcription ratcs and nuclear RNA levels of BRL3A cells wcrc arbitrarily taken as 100. n.d., not detectable.

Cell type

Mean -t S.D. for relativc transcription rates

BRL3A BRL30E FA0

*

100 5.1 38.2 -t 3.5 106 t 8 . 5

nuclear R N A levels

cytoplasmic RNA levels

100 f 15.2 1.2f 0.8 n.d.

106 t 8 . 5 9.7 f 2.4 n.d.

~

Whether these mechanisms also regulate IGF-I1 expression in the tissues of the developing embryos remains to be established. We found that IGF-I1 transcription rates were not very differcnt in the three cell lines. being equivalent in BRL3A and F A 0 cells and only 2.5-fold lower in BRL30E cells. Although run-on assays are not strictly quantitative, they can easily detect a two-log difference. The observed transcription ratcs could not account for the more than 2500-fold differences in the levels of steady-state mRNA species detected in the same cells. A block to elongation has been reported as responsible for c-myc reduced expression during differentiation (Bentley and Groudine, 1986), as wcll as for modulation of c--0s mRNA

levels in cultured macrophagcs (Collart ct al., 1991). Attenuation and/or premature termination of IGF-I1 gene Iranscription does not appear to be involved, since no differences between IGF-11-cxpressing and non-expressing cells were found when 5’ or 3’ regions of the IGF-11 gene transcription unit wcrc analyzed. Analysis of nuclear and cytoplasmic RNA fractions showed a different subcellular distribution of the IGF-I1 transcripts in TGF-11-expressingcompared to non-expressing cells. High levels were present both in the cytoplasmic and in the nuclear compartment of BRL3A cells, with a ratio of about 10 between thc two fractions. In the non-expressing cells, IGF-I1 RNA species were undetectable in the cytoplasm, while they were present in the nucleus. However, the nuclear TGF-I1 transcripts in F A 0 and BRL30E cells were reduced 10-fold and 100-fold. respectively, compared to BRL3A cells. The high cytoplasmic levels in BRL3A cells suggest that the specific mRNA species accumulate in these cells. This hypothesis is

450 BRLSA

BRLBOE

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0

11

Fig.7. IGF-I1 precursor RNA levels in BRWA, BRL30E and F A 0 cells. Nuclear (lanes N) and cytoplasmic (lanes C) RNA species from the indicated cells were analyzed by RNase protection assay. 10 pg (lanes 1. 4 and 7) and 20 pg (lanes 2. 3, 5 , 6. 8 and 9) of thc samples were simultaneously hybridized to antisense transcripts of I 4 IGF-I1 probe (top) and of mouse /]-actin cDNA (bottom) and the protected hybrids analyzed on a 6% polyacrylamide denaturing gel. Transcription of the pGEM - IGF-I1 14 construct generates a 320-nucleotide R N A and a 299-nucleotide-specific protected hybrid. 30 pg heterologous yeast RNA (lam 10) was hybridized to thc same probes or undigested probes (lane 11). Different exposures of IGF-I1 and P-actin-protected RNA species lirom the same experiment are shown. Thc cxposurc timc was 7 days for IGF-I1 and 6 h for /I-actin, and the undigcsted probes. Arrows indicatc the migration of thc undigested rihoprobes.

further substantiated by the observation that IGF-I1 transcripts are very stable in BRL3A cells, their levels remaining unchanged after 24-h of actinomycin-D treatment (data not shown). Therefore. either positive factors in the IGF-II-expressing cells or ncgative ones in the non-expressing cells, should influence KNA stability. Further evidence supporting this hypothesis stem from the observation that IGF-II transcripts are very abundant not only in BRL3A cells (Fig. 2 and Graham et al., 1986), but also in fetal and neonatal rat tissues (Brown et al., 1986). In addition, the IGF-I1 promoters belong to the so-called class of ‘minimal promoters’, whose transcription is dependent only on general factors (Evans ct al., 1988; Matsuguchi et al., 1990; van Dijk et al., 1991). Many eukaryotic genes are known to be regulated posttranscriptionally. For some, the control is exerted on the mRNA stability in the cytoplasm (Graves et al. 1987; Casey et al., 1988; Brawermann, 1989; Miillner et al., 1989). IGF-I1 expression appears to be only partially controlled at the level of cytoplasmic mRNA stability, since specific mRNA spccies were already reduced in the nuclear compartment of IGF-I1 non-expressing cells. Another possible step for regulation at the post-transcriptional level could be the maturation process, i. e. the transition from the precursor to the mature RNA molecule. A block at this step is involved in the regulation of proliferating-ccllnuclear-antigen gene in senescent human fibroblasts (Chang

et al., 1991). We did not find accumulation of unprocessed KNA species in the IGF-I1 non-expressing cells. Moreover, we showed that the total amount of nuclear RNA was reduced 10 - 100-fold in the non-expressing cell lines compared to those expressing the RNA, but the relative ratio between processed and unprocessed RNA species was the same in all cell lines. These data suggest that RNA processing is not affectcd and that some of the events responsible for the altered RNA accumulation occur early in RNA biogenesis. Only few examples of genes for which the RNA turnover is regulated in the nucleus have been reported. Liver bone kidney alkaline phosphatase mRNA levels are controlled at a very early step after transcription, apparently by intron sequences that destabilize the nascent RNA within the nucleus (Kiledjian and Kadesch, 1991). Also, AU-rich domain(s) in the 3’ untranslated region of several mRNA species have been shown to be targets for both nuclear and cytoplasmic RNA degradation (Vakalopoulou et al., 1991). The possible involvement of a similar mechanism in the regulation of IGF-I1 expression is currently under investigation. Another form of post-transcriptional regulation involves the transport of RNA molecules from the nucleus to the cytoplasm. Cytoplasmic accumulation of the human immunodeficient virus (Arrigo et al., 1989; Pavlakis and Felber, 1990) and late Adenovirus type-5 transcripts (Leppard and Shenk, 1989) is due to such a mechanism. If IGF-I1 gene expression were regulated at the level of RNA transport, an increase in the number of mature RNA inolecules in the nuclci of thc IGF-I1 non-expressing cells would be expected. We did not find a relative accumulation of the IGF-TI transcripts in the nuclei of the latter cells, although we cannot exclude that a selective destabilization of the transcripts might act at the same time. IGF-I1 gene expression appears to be regulated by a hierarchy of regulatory steps. During embryonic development, the gene is subject to paternal imprinting, the active allele being that inhcrited from the fathcr (Dc Chiara ct al., 1991). The mechanism responsible for the inactivation of the maternal allele has been postulated to occur at the transcriptional level, by irreversible modifications or regulatory sequences of the genc that are stably transmitted to the progeny (De Chiara et al., 1991). Several other mechanisms have been implicated in the regulation of the IGF-I1 gene in different systems, both at the transcriptional, post-transcriptional (Straus and Tdkemoto, 1988; Zvibel et al., 1991) and translational (Nielsen et al., 1990) levels. Our data demonstrate that the main regulation of the IGF-I1 gene takes place at the post-transcriptional level, possibly by mechanisms affecting the stability of both the nascent and cytoplasmic RNA species and/or the transport of KNA molecules from the nucleus to the cytoplasm. Such regulatory mechanisms entail the involvement of protein factors that could stabilize the IGF-I1 transcripts in the expressing cells, or, alternatively, destabilize them in the non-expressing cells. Since expression of the rat IGF-I1 gene is not detected in fusions of expressing to non-expressing rat liver cells and the phenomenon occurs at the post-transcriptional level (unpublished results), we belicve that ncgative factor(s) may regulate IGF-I1 RNA stability. We wish to thank Drs V. E. Avvedimento, R. Di L a m , P. P. Di Nocera, R. Frunzio, G. Pcrsico, M. M. Rechlcr and A. Riccio for valuable discussion and critical reading of the manuscript, also F. D’Agnello and M. Rerardone for the art work. This work was supported in part by a grant from Progetto Firinlizzato Zizgegneria Genctira

451 of the Consiglio Nazionale defle Ricerche and by the exchange program between Italian Scientists and the National Institutes of Health, funded by the Italian Department of Education (Ministero dellu Pubblica Istruzione).

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