Báo cáo khoa học: Deadenylation of interferon-b mRNA is mediated by both the AU-rich element in the 3¢-untranslated region and an instability sequence in the coding region pot

Deadenylation of interferon-b mRNA is mediated by boththe AU-rich element in the 3¢-untranslated region and an instability sequence in the coding region Muriel Paste´, Georges Huez and V

Deadenylation of interferon-b mRNA is mediated by both

the AU-rich element in the 3¢-untranslated region and an instability sequence in the coding region

Muriel Paste´, Georges Huez and Ve´ronique Kruys

Laboratoire de Chimie Biologique, Institut de Biologie et de Me´decine Mole´culaires, Universite´ Libre de Bruxelles, Belgium

Viral infection of fibroblastic and endothelial cells leads to

the transient synthesis of interferon-b (IFN-b) The

down-regulation of IFN-b synthesis after infection results both

from transcriptional repression of the IFN-b gene and rapid

degradation of mRNA As with many cytokine mRNAs,

IFN-b mRNA contains an AU-rich element (ARE) in its

3¢-untranslated region (UTR) AREs are known to mediate

mRNAdeadenylation and destabilization Depending on

the class of ARE, deadenylation was shown to occur

through synchronous or asynchronous mechanisms In this

study, we analysed IFN-b mRNAdeadenylation in natural

conditions of IFN-b synthesis, e.g., after viral infection We

show that human IFN-b mRNAfollows an asynchronous

deadenylation pathway typical of a mRNAcontaining a

class II ARE A deletion analysis of the IFN-b natural

transcript demonstrates that poly(A) shortening can be

mediated by the ARE but also by a 32 nucleotide-sequence

located in the coding region, that was identified previously as

an instability determinant In fact, these elements are able to act independently as both of them have to be removed to abrogate mRNAdeadenylation Our data also indicate that deadenylation occurs independently of mRNAtranslation Moreover, we show that deadenylation of IFN-b mRNAis not under the control of viral infection as IFN-b mRNA derived from a constitutively expressed gene cassette is deadenylated in absence of viral infection Finally, an unidentified nuclear event appears to be a prerequisite for IFN-b mRNAdeadenylation as IFN-b mRNAintroduced directly into the cytoplasm does not undergo deadenylation

In conclusion, our study demonstrates that IFN-b mRNA poly(A) shortening is under the control of two cis-acting elements recruiting a deadenylating machinery whose activity is independent of translation and viral infection but might require a nuclear event

Keywords: mRNAstability; polyadenylation; translation

The transient expression of human interferon-b (IFN-b) in

response to double stranded RNAor viral infection is a

direct consequence of transcriptional activation [1] and leads

to the accumulation of mRNA In contrast, the shutoff of

IFN-b gene expression involves the induction of a

tran-scriptional repressor as well as a rapid decay of IFN-b

mRNA[2,3] The human IFN-b mRNAcontains an

AU-rich element (ARE) in its 3¢-untranslated region (3¢UTR)

AREs were first discovered as highly conserved elements

present in the 3¢UTR of mRNAs encoding cytokines and

oncoproteins [4] These motifs composed of the AUUUA

pentamer, were shown to confer mRNAinstability and to

regulate mRNAtranslation [5] Indeed, Shaw and Kamen

first reported that the ARE located in the 3¢UTR of the

granulocyte macrophage-colony stimulating factor

(GM-CSF) mRNAwas responsible for mRNArapid degradation

[6] Later on, the destabilizing activity of several other AREs was documented (for review, ref [7]) AREs have been classified into three distinct categories based on the number and distribution of AUUUA pentamers Class I AREs are characterized by the presence of one to three pentamers distributed into a large part of the 3¢UTR coupled with a nearby U-rich region Class II AREs have at least two overlapping copies of the nonamer UUAUUU(U/A) (U/A)U in a U-rich environment and class III do not contain any pentamers but present U-rich stretches AREs from all three classes confer mRNAinstability in cultured cells through different mechanisms that all imply mRNA deadenylation (for review, see [7]) Class II AREs (e.g., GM-CSF, TNF-a, and IL-3) induce asynchronous dead-enylation resulting in the accumulation of poly(A)– inter-mediates In contrast, class I and class III AREs (e.g., c-fos and c-jun) direct a synchronous poly(A) shortening Several ARE-binding proteins have been identified, among which AUF1 and the tristetraprolin (TTP) were shown to participate in the destabilization of ARE-containing mRNAs Recently, Chen et al showed that ARE-binding proteins such as AUF1 and TTP were able to interact with a multiprotein complex, called the exosome [8] This complex first discovered in yeast [9], is composed of proteins with ribonuclease activity and is able to direct 3¢)5¢ mRNA degradation Therefore, the recruitment of the exosome by ARE-binding proteins might account for the degradation of ARE-containing mRNAs

Correspondence to V Kruys, Laboratoire de Chimie Biologique,

Institut de Biologie et de Me´decine Mole´culaires, Universite´ Libre

de Bruxelles, 12 rue des Profs Jeener et Brachet, 6041 Gosselies,

Belgium Fax: +32 2 6509800, Tel.: +32 2 6509804,

E-mail: vkruys@ulb.ac.be

Abbreviations: ARE, AU-rich element; IFN-b, interferon-b; UTR,

untranslated region; CRID, coding region instability.

(Received 30 December 2002, revised 18 February 2003,

accepted 20 February 2003)

Based on its sequence, IFN-b ARE belongs to class II.

Moreover, several reports described IFN-b

mRNAinsta-bility and the involvement of the ARE in this process

[10,11] In addition, another destabilizing sequence was

identified within the coding region of IFN-b mRNA[12,13]

Whereas the independent removal of the ARE or the coding

region instability determinant (CRID) result in a moderate

stabilization of the mRNA, replacement of both elements

by control sequences greatly enhances mRNAhalf-life [13]

It should be mentioned however, that these observations

were made in heterologous cell systems using reporter DNA

constructs under the control of heterologous or IFN-b

modified promoters Moreover, the role of the instability

determinants in the control of IFN-b mRNAdeadenylation

was not addressed

In the present study, we analysed the expression of

endogenous IFN-b in human cells upon viral infection We

also investigated the influence of the ARE and the CRID on

the poly(A) status of the human IFN-b mRNAin natural

conditions of IFN-b synthesis

Materials and methods

Reagents

All the reagents and enzymes used in this study were

purchased from Roche Molecular Biochemicals and Life

Technologies Inc., unless specified The Sendaı¨ virus

(Can-tell strain, ATCC VR-907 Parainfluenza 1) was obtained

from Charles River Laboratories [a-32P]UTP, [a-35S]UTP

and [35S]-Met were purchased from Amersham-Pharmacia

Biotech The anti-IFN-b ELISAkit was purchased from

Biosource Rat monoclonal anti-HAIg (clone 3F10) was

purchased from Roche Molecular Biochemicals

Plasmid Construction

The complete sequence of the human IFN-b gene including

the IFN-b promoter (EcoRI/EcoRI fragment described in

reference [14]) was inserted in the pcDNA3 plasmid

(Invitrogen) from which the cytomegalovirus (CMV)

pro-moter was deleted previously In the pIFNHAconstruct,

the IFN-b gene was tagged by PCR using an oligonucleotide

containing three repetitions of the sequence corresponding

to the human influenza Avirus hemaglutinin (HA) epitope

The pIFNHAAU–construct was generated by deleting the

75-nucleotide region corresponding to the ARE (from

nucleotides 740–815 of the mRNA) The pIFNCRIDHA

and pIFNCRIDHAAU– constructs were obtained by

deletion of a 32-nucleotide region (from 513–545) in the

PIFNHAand PIFNHAAU–constructs, respectively

Astable hairpin (hp) structure obtained by

oligomeriza-tion of a SalI linker was inserted in the HincII site of the

pIFNHAconstruct located at the beginning of IFN-b

mRNA5¢UTR To place the IFN-b gene under the

transcriptional control of a constitutive promoter, the

HA-tagged IFN-b cDNAwas inserted between the EcoRI

and BamHI sites of the pSG5 plasmid (Stratagene)

(pSG5IFNHA) downstream of the simian virus 40 (SV40)

promoter

For in vitro transcription, the pBSIFNpAvector was

generated as follows The poly(A) tail was obtained by

hybridization of a 15-Aand a 15-T oligonucleotide The single stranded extremities were filled with the Klenow polymerase before oligomerization The DNAfragments were cloned in the T4 DNApolymerase blunted PstI site of the pSP65 vector The length of the inserted fragments was estimated on agarose gel and the vector containing a 100–

150 nucleotides insert was selected The poly(A)100)150tail was then cloned in the HindIII/SalI sites of the pBluescript

SK (Stratagene) The restriction sites between SacI and PstI were deleted in this pBluescript SK poly(A) and the IFN-b gene without its promoter (EcoRI/BamHI fragment from the pSP65IFNc plasmid described elsewhere [5]) was then cloned between the EcoRI and HindIII sites The deletion of the ARE was performed by inserting a EcoRI/NdeI fragment of the IFN-b gene [5]

Cell culture and treatments The human endometrial adenocarcinoma cells (Hec-1B, ATCC number, HTB-113) were maintained in DMEM containing 10% of fetal bovine serum (FBS; Myoclone Super Plus, Life Technologies) and 1% of penicillin/ streptomycin The cells were infected by addition of

80 UÆmL)1 of Sendaı¨ virus during 2 h Actinomycin D and cycloheximide were used at final concentrations of

5 lgÆmL)1and 10 lgÆmL)1, respectively

Isolation of total RNA and RNase H treatment Total RNAwas prepared by the Trizol method (Life technologies, Inc.) RNase H treatment was performed according to the method described by McGrew et al [15] Northern blot analysis

Northern blot analysis was performed as described by Kruys et al [16] Total RNA(10 lg per lane) was separated

by electrophoresis in a 2.2% agarose gel, electrotransferred

to nylon membrane and cross-linked by UV-irradiation Blots were hybridized with antisense [a-32P]UTP or [a-35S]UTP labelled riboprobes

In vitro Transcription and translation DNAs were linearized at unique restriction sites and capped mRNAwere generated by in vitro transcription with T3 or Sp6 polymerases RNAwas quantified by absorbance at

260 nm and its integrity was verified by agarose gel electrophoresis followed by ethidium bromide staining Translation was carried out in rabbit reticulocyte lysate (Promega) in the presence of35S-labelled Met (Amersham Pharmacia Biotech)

DNA and RNA transfection Hec-1B cells were transfected with DNAusing the Fugene reagent (Life technologies) following the procedure provided

by the supplier RNAtransfections were carried out using the lipofectine reagent (Life technologies) as described by the supplier In brief, cells were grown to 50% confluency in six-well plates before transfection The culture medium was then replaced by serum-free medium and the transfection mix was

added The transfection mix contained 10 lg of RNA

(between 10–100 ng of the in vitro transcribed mRNA

supplemented by a carrier tRNA) and 10 lL of lipofectine in

a total volume of 200 lL of serum free-medium

In both cases, the culture media were harvested to

measure IFN-b concentration by ELISAand the cells were

harvested for total RNAextraction

Metabolic protein labeling and immunoprecipitation

Hec-1B cells were plated in six-well plates at 200 000 cells

per well and were incubated for 6 h before transfection

After transfection, the cells were incubated for another 24 h

and then infected by Sendaı¨ virus for 2 h Cells were washed

and preincubated in a Met and Cys-depleted medium for

1 h Metabolic labeling was performed by adding

500 lCiÆmL)1 of 35S-labelled Met and Cys in the cell

culture for 5 h The cell culture medium was harvested for

immunoprecipitation Immunoprecipitation was performed

in RIPAbuffer (25 mM Tris pH 8.2, 50 mMNaCl, 0.5%

Nonidet P40, 0.5% deoxycholate, 0.1% SDS) using an

anti-HAIg and protein A-Sepharose Proteins were analysed by

SDS/PAGE followed by autoradiography

Results Deadenylation of the human IFN-b mRNA

in virus-infected cells

So far, all the studies aimed at understanding the post-transcriptional regulation of human IFN-b mRNAhave been performed in heterologous cell systems Therefore, we chose to analyse the regulation of human IFN-b mRNAin human cells (Hec-1B) that naturally produce IFN-b upon viral infection [17] We first performed a kinetic analysis of IFN-b production by Hec-1B cells after infection by the Sendaı¨ virus for 2 h IFN-b appeared in the cell culture 2–5 h after the infection, reaching a maximum between 8–11 h and subsequently levels droped at later times (Fig 1A) We then analysed, by Northern blot, the induc-tion and decay of IFN-b transcript in the same condiinduc-tions

A s shown in Fig 1B, IFN-b mRNAwas detectable 4 h after the beginning of the infection, reached a maximum after 6–7 h and then rapidly disappeared thereafter Inter-estingly, two IFN-b mRNAspecies were observed, the shorter form appearing later in the infection process As class II AREs are known to mediate mRNA degradation by

Fig 1 Interferon-b production by Hec-1B cells infected by Sendaı¨ virus Hec-1B cells were infected for 2 h by the Sendaı¨ virus, the cells were then washed with NaCl/P i and fresh medium was added (A) Every 3 h, the supernatant was sampled and replaced by fresh culture medium The IFN-b was quantified in the supernatants by ELISA (B) Cells were harvested for total RNA extraction 0, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 h after infection The amount and the length of IFN-b mRNAwas analysed by Northern blot, using a 32 P-labelled antisense IFN-b riboprobe As a control, the membrane was hybridized with a GAPDH antisense riboprobe (C) Total RNA of cells infected with the Sendaı¨ for various lengths of time was digested (or not) by RNase H in the presence of oligo(dT) Treated and untreated RNAs were analysed by Northern blotting (D) Hec-1B cells were infected by the Sendaı¨ virus and further cultured with cycloheximide (10 lgÆmL)1) At the indicated times after infection, total RNA was extracted and analysed by Northern blot (E) Hec-1B cells were infected by the Sendaı¨ virus and further cultured with cycloheximide (10 lgÆmL)1) and actinomycin D (5 lgÆmL)1) At the indicated times after induction, total RNA was extracted and analysed by Northern blot with a32P-labelled antisense IFN-b riboprobe The results presented in A, B and C are representative of more than five independent experiments Data in D and E are representative of two independent experiments.

promoting deadenylation, we investigated whether the two

transcripts differed by the length of their poly(A) tail

Therefore, total RNAof Hec-1B cells infected by the Sendaı¨

virus was treated (or not) by RNAse H in the presence of

oligo(dT) before the Northern blot analysis with an IFN-b

probe This treatment led to the detection of a single band

comigrating with the short IFN-b transcript in untreated

samples (Fig 1C), indicating that the large and short

transcripts observed in untreated samples corresponded to

polyadenylated and deadenylated mRNAs, respectively

The poly(A) tail of IFN-b mRNAwas estimated to be about

200 nucleotides long based on the difference of

electropho-retic migration between the adenylated and deadenylated

IFN-b mRNA(Fig 1B) We then analysed the effect of

cycloheximide on the accumulation of the two IFN-b

transcripts to determine whether IFN-b

mRNAdeadenyla-tion process required ongoing translamRNAdeadenyla-tion As shown in

Fig 1D, the addition of the translation inhibitor after the

infection of the Hec-1B cells did not prevent the appearance

of the IFN-b short transcript Moreover, as reported

previously, cycloheximide led to a marked increase of

IFN-b mRNAaccumulation at later times of infection

resulting from mRNAstabilization and/or absence of

transcriptional repression [10,12] Treatment by both

actino-mycin D and cycloheximide did not prevent IFN-b mRNA

deadenylation either as the polyadenylated IFN-b transcript

accumulated in response to the viral infection was also

shortened before being degraded Moreover, the

transcrip-tional blockade by actinomycin D abrogated the increase of

mRNAaccumulation due to the cycloheximide (Fig 1E)

This latter observation indicates that increased accumulation

of IFN-b mRNAin cycloheximide-treated cells is due to the

absence of transcriptional repression of the IFN-b promoter

Altogether, these results indicate that viral infection

triggers the synthesis of a polyadenylated IFN-b mRNA

that is deadenylated rapidly before degradation Moreover,

this deadenylation process does not require IFN-b mRNA

translation and/or protein synthesis as it is effective in the

presence of a translational inhibitor

Deadenylation of IFN-b mRNAoccurs when IFN-b

synthesis is induced by other agents such as synthetic

double-stranded polyriboinosinic polyribocitydylic acid

(poly rI.rC) and was observed in other cell types such as Namalwa B cells (data not shown) These observations indicate that deadenylation is a general mechanism con-trolling the length of IFN-b mRNApoly(A) tail

IFN-b mRNA deadenylation is mediated by both the ARE and the CRID

The IFN-b mRNAcontains in its 3¢UTR an AU-rich element (ARE) which is very similar to AREs present in other unstable mRNAs [4] As AREs present in other cytokine mRNAs were demonstrated to induce mRNA degradation by triggering poly(A) shortening, we first analyzed the role of such an element in the deadenylation process of IFN-b mRNA To this end, two DNA constructs were generated in which the IFN-b gene contained or not the ARE (pIFNHA and pIFNHAAU–)

In addition, the sequence encoding the HAepitope was inserted at the end of the IFN-b coding sequence to distinguish the products resulting from the expression of the DNAconstructs and the endogenous gene (Fig 2) These constructs were transfected in Hec-1B cells and the cells were subsequently infected with the Sendaı¨ virus for 2 h Cells were lyzed 3 or 8 h after infection to extract the RNA which was treated (or not) by RNAse H in the presence of oligo(dT) before the Northern blot analysis with a HA antisense riboprobe As shown in Fig 3A (lanes 3 and 7), the HA-tagged IFN-b transcript underwent significant deadenylation 8 h after infection independently of the presence or the absence of the ARE Another RNA instability determinant was identified in the 3¢-end of the IFN-b coding region [13] This element named CRID (coding region instability determinant), has been mapped between nucleotides 513–545, the first nucleotide corres-ponding to the adenosine of the initiation codon Deletion

of this element by itself from the IFN-b gene (pIFNCRI-DHA) did not abolish mRNA deadenylation (Fig 3B, compare lanes 2 and 3) However, deletion of both the ARE and the CRID (pIFNCRIDHAAU-) led to a blockade of the deadenylation process (Fig 3B, compare lanes 6 and 7) These results demonstrate that deadenyla-tion is controlled by both the ARE and the CRID

Fig 2 Schematic representation of the DNA

constructs.

Deadenylation of IFN-b mRNA is uncoupled

from its translation

The role of translation in ARE-mediated mRNA

deadeny-lation and subsequent decay is still a subject of controversy

Indeed, while several reports support a translation-dependent mechanism [18–20], other observations deny any coupling between the recruitment of the mRNAinto polysomes and its deadenylation/degradation [7]

Here, we analysed whether ongoing translation is a prerequisite for IFN-b mRNAdeadenylation Therefore,

we generated a IFN-b gene construct containing a stable hairpin in the 5¢UTR (pIFNHAhp, Fig 2) and the deadeny-lation of the derived mRNAwas compared to that of the mRNA lacking such a secondary structure (pIFNHA) As shown in Fig 4A, the presence of the hairpin in the 5¢UTR does not influence the deadenylation process To verify that the hairpin effectively prevented the translation of the mRNA, the secretion of HA-tagged IFN-b was monitored

in the culture medium of cells transfected with these con-structs Whereas cells transfected with the construct lacking the hairpin produced detectable amounts of HA-tagged IFN-b, no translation product was detectable with the construct containing the hairpin in the 5¢UTR (Fig 4B)

Deadenylation of IFN-b mRNA occurs independently

of viral infection

We then analysed whether IFN-b mRNAdeadenylation resulted from the infection of the cells by the Sendaı¨ virus

In order to ensure the production of IFN-b transcripts in absence of infection, the HA-tagged IFN-b gene was placed downstream of the SV40 early promoter (pSG5IFNHA, Fig 2) Hec-1B cells were transfected with the pSG5IF-NHAconstruct and were subsequently infected (or not) with the Sendaı¨ virus Deadenylation of the HA-tagged IFN-b mRNAwas monitored in the presence of actino-mycin D to block further accumulation of HA-tagged IFN-b mRNA As shown in Fig 5, deadenylation of the HA-IFN-b transcript occurs even in absence of viral infection

Deadenylation of IFN-b mRNA requires a nuclear event

We next determined whether IFN-b mRNAdeadenylation requires a nuclear event To approach this question, a synthetic IFN-b transcript containing a poly(A) tail of

 100–150 residues was generated by in vitro transcription

in the presence of 32P-labelled UTP (see Materials and

Fig 4 Deadenylation of IFN-b mRNA is independent of translation (A) Deadenylation analysis of the PIFNHAand PIFNHAhp transcripts Hec-1B cells were transfected with the PIFNHAand PIFNHAhp DNAconstructs Cells were harvested for RNAextraction 3 h (lanes 1, 2, 5, 6) and 8 h (lanes 3, 4, 7, 8) after infection with the Sendaı¨ virus Half of each RNAsample was treated with RNAse H (lanes 1, 4, 5, 8) before Northern blot analysis with a35S-labelled HAantisense riboprobe (B) Cells transfected with the PIFNHAconstruct (lane 1), and PIFNHAhp construct (lane 2) were cultured in methionine and cysteine-depleted medium in the presence of 500 lCiÆmL)1of 35 S-labelled Met and Cys The supernatants were immunoprecipitated witn the anti-HAIg and the radiolabelled proteins were analysed by SDS/PAGE The results presented in this figure are representative of three independent experiments.

Fig 3 Deadenylation of IFN-b mRNA is abolished upon deletion of

both the ARE and the CRID (A) Hec-1B cells were transfected with

the PIFNHAand PIFNHAAU–DNAconstructs for 24 h and were

subsequently infected during 2 h by the Sendaı¨ virus Cells were

har-vested for RNAextraction 3 h (lanes 1, 2, 5, 6) and 8 h (lanes 3, 4, 7, 8)

after infection with the Sendaı¨ virus Half of each RNAsample was

treated with RNAse H (lanes 1, 4, 5, 8) before Northern blot analysis

with a 35 S-labelled HAantisense riboprobe (B) The PIFNCRIDHA

and PIFNCRIDHAAU–constructs described in Fig 2, were

trans-fected in Hec-1B cells The cells were intrans-fected with the Sendaı¨ virus for

2 h and were harvested 3 (lanes 1, 2, 5 and 6) and 8 h (lanes 3, 4, 7 and

8) after infection for RNAanalysis by Northern blot using a35

S-labelled HAriboprobe Lanes 1, 4, 5 and 8 correspond to deadenylated

RNAs obtained after RNase H treatment The results presented in this

figure are representative of three independent experiments.

methods) (Fig 6A) Hec-1B cells were transfected with this

synthetic transcript for 2 h, and total RNAwas extracted at

various times after transfection to be analysed by agarose

electrophoresis and autoradiography As shown in Fig 6B,

the IFN-b transcript is rapidly degraded without prior

deadenylation, suggesting that IFN-b mRNAmust

origin-ate from the nucleus to be a substrorigin-ate of the deadenylation

process To verify the poly(A) status of the IFN-b

transcript, we compared its migration in agarose gel to

poly(A)– IFN-b transcripts, containing (or not) the ARE

after transfection into Hec-1B cells The migration of the

different transcripts confirmed that the poly(A)+ IFN-b

mRNAbore a 100–150 nucleotides long poly(A) tail

(Fig 6C) Moreover, in order to verify the effective

introduction of the synthetic mRNAinto cells, IFN-b

production was assayed in the culture medium after

transfection As shown in Fig 6D (lane 1), transfection of

polyadenylated mRNAled to IFN-b synthesis (Fig 6D,

lane 1) in contrast to the poly(A)–transcripts which were

poorly translated (Fig 6D lanes 2 and 3) Similar results

were obtained when cells were infected by the Sendaı¨ virus

before RNAtransfection (data not shown)

Discussion

In the present study, we analysed the expression and the

poly(A) status of human IFN-b mRNAin human

endo-thelial Hec-1B cells in response to infection by the Sendaı¨

virus As observed in other cell types, IFN-b synthesis is

transiently induced and results from a strong accumulation

of IFN-b mRNAthat rapidly disappears at later times of

infection [12,21] We showed that the disappearance of

IFN-b mRNAis accompanied by the shortening of its

poly(A) tail As described for certain class II

ARE-containing mRNAs (e.g., GM-CSF, IL-3) [22,23], IFN-b

mRNAis deadenylated asynchronously with the formation

of poly(A)–intermediates However, IFN-b

mRNAdead-enylation is not solely under the control of the ARE Indeed,

poly(A) shortening is abolished only upon deletion of both

the ARE and the CRID (Fig 3) The CRID was identified

previously as an instability determinant that, in

combina-tion with the 3¢UTR, mediates the rapid decay of human

IFN-b mRNAin NDV-infected NIH/3T3 cells [13] Both sequences were shown by UV-crosslinking experiments to recruit a cytosolic 65-kDa protein of unknown identity

Fig 6 Deadenylation of IFN-b mRNA is independent of viral infection (A) Schematic representation of the DNA constructs used to generate

in vitro transcribed IFN-b mRNA (B)32P-labelled IFN-b mRNA containing a 100 nucleotide poly(A) tail was generated by in vitro transcription The RNAwas transfected for 2 h into Hec-1B cells and total RNAwas extracted at the indicated times after the end of transfection 32 P-labelled IFN-b mRNAwas analysed by agarose gel electrophoresis and autoradiography (C) Polyadenylated IFN-b mRNAA U+was transcribed from the pBSIFNpA The poly(A)– IFN-b mRNAs, AU + pA – and AU – pA – , were transcribed from the pSP65IFN construct linearized by BamHI and NdeI, respectively The [32P]–labelled transcripts were transfected into Hec-1B cells for 8 h before total RNAextraction, agarose gel electrophoresis and auto-radiography (D) The IFN-b was assayed in cell culture medium by ELISA The results presented in this figure are representative of four independent experiments.

Fig 5 In vitro transcribed IFN-b mRNA is not deadenylated upon

transfection into Hec-1B cells The pSG5IFNHAconstruct was

transfected into Hec-1B cells The cells were infected (or not) with the

Sendaı¨ virus for 2 h and actinomycin D (5 lgÆmL)1) was added in the

culture medium Total RNAwas extracted at the indicated times and

analysed by Northern blot using a 35 S-labelled HAriboprobe The

RNase H-treated samples position the fully deadenylated mRNA The

same blot was rehybridized with a35S-labelled GAPDH riboprobe.

The results presented in this figure are representative of two

inde-pendent experiments.

These and our observations suggest that the binding of this

65-kDa protein to one of these elements might be required

to induce IFN-b mRNAdeadenylation and subsequent

degradation of the RNAbody

c-fos, c-myc and plasminogen activator inhibitor (PAI-2)

messenger RNAs are other ARE-containing mRNAs

bearing instability determinants in their coding region

[24–27] Moreover, in the case of c-fos, it was shown that

ARE mediates mRNA deadenylation by a

translation-independent mechanism, while the coding region

instabi-lity determinant facilitates mRNAdeadenylation by a

mechanism coupled to translation [25,28] Here, we show

that IFN-b mRNAdeadenylation occurs independently of

the translational status of the mRNA This observation

correlates with the fact that IFN-b mRNAdestabilization

at later times of infection occurs even when mRNA

translation is abrogated by the insertion of a stop codon

immediately after the initiation codon [13] It remains to

be established, however, whether any of the two elements

taken separately is translation-dependent in promoting

mRNAdeadenylation

IFN-b mRNAdeadenylation is a constitutive

mecha-nism Indeed, the IFN-b transcript derived from a

consti-tutively transcribed gene cassette undergoes deadenylation

in absence of viral infection However, poly(A) shortening is

detectable only after addition of actinomycin D that blocks

the accumulation of newly synthesized polyadenylated

IFN-b mRNA(Fig 5) This observation suggests that the

deadenylation machinery is pre-existing in the cells and

deadenylates IFN-b mRNAas soon as its synthesis is

induced by stimulating agents The fact that the 65-kDa

protein binds ARE and CRID in UV-crosslinking

experi-ments, performed with cytosolic extracts from both

non-infected and non-infected cells [13], further supports the

involvement of this protein in the deadenylation process

of IFN-b mRNA Interestingly, IFN-b ARE does not

recruit other ARE-binding factors (data not shown),

thereby emphasizing the role of this 65-kDa RNA-binding

protein [13] whose identity and function remain to be

investigated

IFN-b mRNAdeadenylation seems to be conditioned by

a nuclear event Indeed, a synthetic IFN-b mRNAbearing a

100 nucleotide poly(A) tail escapes the deadenylation

process when transfected in Hec-1B cells (Fig 6) The

nondeadenylation of this synthetic transcript does not

however, protect it from rapid decay, thereby suggesting

that it becomes a target of an alternative

poly(A)-independ-ent degradative pathway Although the nuclear evpoly(A)-independ-ent

conditioning IFN-b mRNAdeadenylation remains to be

established, we provide the first evidence indicating such

requirement for this mRNAdegradative process It seems

however, possible that such a nuclear event might also be

required for other mRNAs undergoing specific

deadenyla-tion Indeed, most RNA-binding proteins mediating

mRNAdeadenylation/degradation shuttle between the

nucleus and the cytoplasm [29] The association of specific

transcripts with such factors in the nuclear compartment

might thus condition their cytoplasmic fate

Altogether, our results and previous observations

[12,13,30] demonstrate clearly that IFN-b mRNAbehaves

similarly to class II ARE-containing mRNA prototypes

(e.g., GM-CSF, IL-3) However, the deadenylation and

degradation of IFN-b mRNAis under the control of two independent elements, one of which is located in the mRNA coding region

The coexistence of two independent but apparently redundant instability determinants might reflect the need for stringent control of IFN-b gene, whose prolonged expression might be detrimental to the organism

Acknowledgements

This work was funded by the EC contract (QLK3-2000-00721), the Fund for Medical Scientific Research (Belgium, grant 3.4618.01), and the Actions de Recherches Concerte´es (grant 00-05/250).

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