Intronic Sequences and 3' Splice Sites Control Rous Sarcoma Virus

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Transcript of Intronic Sequences and 3' Splice Sites Control Rous Sarcoma Virus

  • JOURNAL OF VIROLOGY, Jan. 1992, p. 6-110022-538X/92/010006-06$02.00/0Copyright C 1992, American Society for Microbiology

    Intronic Sequences and 3' Splice Sites Control RousSarcoma Virus RNA SplicingMARK T. McNALLY AND KAREN BEEMON*

    Vol. 66, No. 1

    Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218

    Received 29 July 1991/Accepted 27 September 1991

    cis-acting sequences of Rous sarcoma virus (RSV) RNA involved in control of the incomplete splicing that ispart of the retroviral life cycle have been studied. The 5' and two alternative 3' splice sites, as well as a negativeregulator of splicing element in the intron, have been introduced into chimeric constructs, and their respectiveroles in splicing inhibition have been evaluated by transient transfection experiments. Although the RSV 5'splice site was used efficiently in these assays, substrates containing either the RSV env or the RSV src 3' splicesite were not spliced completely, resulting in 40 to 50% unspliced RNA. Addition of the negative regulator ofsplicing element to substrates containing RSV 3' splice sites resulted in greater inhibition of splicing (70 to 80%unspliced RNA), suggesting that the two elements function independently and additively. Deletion of sequencesmore than 70 nucleotides upstream of the src 3' splice site resulted in efficient splicing at this site, suggestingthat inefficient usage is not inherent in this splice site but is instead due to sequences upstream of it. Insertionof these upstream sequences into the intron of a heterologous pre-mRNA resulted in partial inhibition of itssplicing. In addition, secondary structure interactions were predicted to occur between the src 3' splice site andthe inhibitory sequences upstream of it. Thus, RSV splicing control involves both intronic sequences and 3'splice sites, with different mechanisms involved in the underutilization of the env and src splice acceptor sites.

    Retroviral replication requires the production of subge-nomic mRNAs from the primary RNA transcript throughRNA splicing. In the case of Rous sarcoma virus (RSV),these spliced mRNAs are formed from a single 5' splice sitethat is alternatively spliced to one of two 3' splice sites,resulting in mRNA for the env and src genes. In addition tothe spliced RNA, substantial amounts of unspliced RNA arealso required to serve as genomic RNA and as mRNA for thegag and pol genes. Thus, there must be mechanisms tocontrol the level of splicing such that as much as 70 to 80%of the RNA remains unspliced and is transported to thecytoplasm. This is in contrast to cellular pre-mRNAs, whichare usually spliced to completion and whose unsplicedRNAs are not detectable in the cytoplasm. This control canbe envisioned to occur at one or more levels (for a review,see references 7 and 22). Proteins encoded by the virus mightfunction to block splicing directly or indirectly by shuntingthe RNA away from the splicing machinery. Indeed, humanimmunodeficiency virus and human T-cell leukemia virusencode proteins (rev and rex) that appear to be necessary forcytoplasmic expression of unspliced and singly spliced viralRNAs (24); however, most retroviruses do not encode suchregulatory proteins (7). Another possibility is that signals atthe splice junctions are inefficiently recognized or utilized,resulting in limited splicing. Alternatively, cis-acting se-quences elsewhere in the RNA might render splicing ineffi-cient, by generating detrimental structures or by acting asdecoys for splicing factors, for example.

    Studies in several laboratories indicate that RSV proteinsare not involved in splicing regulation (1, 14, 23). However,several cis-acting sequences have been identified as impor-tant in this process. Insertion of a short oligonucleotide closeto the env splice site results in a large increase in splicing atthat site (13). Additional studies of this mutant virus andrevertants isolated from it indicate that the wild-type branch

    * Corresponding author.

    point is suboptimal; splicing increases in vivo and in vitrowhen a better branch point is provided (9, 14). Thus, itappears that poor utilization of a splice acceptor site is onestrategy adopted by RSV to control splicing. In a recentstudy, deletions upstream of the src acceptor site in RSVresulted in elevated production of src mRNA (3). Removal ofthese upstream sequences does not appear to be involved ingenerating a better branch site, and the mechanism of actionis unclear. A third sequence which affects splicing has beenidentified in the gag gene between nucleotides (nt) 703 and1006 (1, 19, 23). Deletions in this area result in oversplicing,suggesting that it contains a negative regulator of splicing(NRS) (1). The NRS element can confer a low-splicingphenotype on heterologous introns when present in thesense orientation, and it seems to function best when locatedin the intron near the 5' splice site (19). None of theseelements appears to be sufficient to control RSV splicingalone, since significant amounts of unspliced RNA remainsubsequent to the deletion or modification of the elements (1,3, 23).

    In an effort to elucidate the respective contributions of thegag NRS and of the splice sites in RSV splicing control, weperformed a series of splice site shuffle experiments, usingprimary transcripts containing the RSV splice sites in com-bination with the chicken c-myc second intron and flankingexon sequences. Splicing of these substrates was assayed invivo both in the presence and in the absence of the gag NRSelement. We found that both RSV 3' splice sites are ineffi-ciently utilized in these chimeric substrates and that the NRScan function independently and additively with them togenerate higher levels of unspliced RNA. The RSV donorsite does not appear to be involved in control, as it wasefficiently used and did not significantly affect levels ofspliced RNA when paired with any of the 3' splice sites. Onesrc acceptor region construct, which included sequences 70nt upstream of the splice junction, was spliced efficiently. Incontrast, a construct that contained sequences 184 nt up-stream of the src acceptor functioned poorly. The sequence



    present between nt -70 and -184 with respect to the srcacceptor site was capable of partially inhibiting splicingwhen inserted into a heterologous intron, although thisinhibition was weaker than that observed with the gag NRSelement. We also noted computer-predicted base-pairingbetween the src 3' splice site and this upstream sequence.These results demonstrate that control of RSV splicinginvolves multiple cis-acting sequences and that differentmechanisms are involved in the underutilization of the envand src 3' splice sites.


    Plasmid constructions. We constructed an expression vec-tor, pMyc23, that contains part of chicken cellular myc geneexons 2 and 3 flanking the complete myc intron 2. Initially,we generated pRSV1 by a three-way ligation of a fragmentof pRSVNeo-int (16) containing the RSV promoter (BglI-HindIII), a pBR322 EcoRI-BglI fragment, and the EcoRI-HindIII polylinker from pGEM-3Z (Promega Biotec). AHinfl-BamHI fragment containing the simian virus 40 (SV40)polyadenylation signal from pSVlcat (10) was then insertedinto the SmaI site of pRSV1 to give pRSV2. Thus, transcrip-tion of a test gene inserted in the polylinker is driven by theRSV promoter and terminated by the SV40 poly(A) site. TheSV40 small t intron is not present in this vector. pMyc23 wasgenerated by inserting into the repaired HindIII site ofpRSV2 a SacI-SspI fragment of the chicken c-myc genecontaining most of exon 2 (514 nt), all of intron 2 (970 nt),and most of exon 3 (618 nt). The chicken c-myc gene was agift from Maxine Linial. The portion of the myc gene exon 3included in pMyc23 does not include sequences at the 3' endinvolved in rapid turnover of myc mRNA (12).Other viral DNA fragments were obtained from plasmid

    pATV-8K (6), which contains DNA of the Prague-C strain ofRSV. The RSV sequence coordinates are those determinedby Schwartz et al. (21). RSV NRS sequences within anMroI-SphI (MS) fragment (nt 707 to 1006) were introducedinto the intron of pMyc23 at the SacII site (340 nt fromthe splice donor) by replacing the intron-located Eco47III-HindIll fragment, which encompasses the SacII site, withthe corresponding fragment from pRSVNeo-int that containsthe MS sequence in the SacII site (19).The myc 5' splice site was replaced by the RSV 5' splice

    site by removing the pMyc23 site with BssHII and insertingthe RSV PstI-SmaI fragment (nt 263 to 520) encompassingthe 5' splice site at nt 397. The myc 3' splice site wasremoved by digestion with AflIl and ClaI and replaced withthe RSV env and src 3' splice site regions. The env fragmentwas KpnI-XhoI (nt 500 to 5258), while two src fragmentswere used: srcS has a shorter region upstream of the 3' splicesite (XhoI-MstII, nt 6984 to 7154), and srcL has a longerupstream region (SacI-NcoI, nt 6870 to 7131).The RSV SacI-XhoI fragment (SX fragment; nt 6870 to

    6987) fragment located upstream of the src 3' splice site wastested for its ability to inhibit splicing in a heterologous assayby using pRSVNeo-int. It was cloned in both orientationsinto three sites in the myc intron of the vector: BstXI, SacII,and AflII, which are 167, 340, and 803 nt, respectively, fromthe 5' splice site. A plasmid containing the gag NRS (MSfragment) in the SacII site was used as a control for splicinginhibition (19).

    Cell culture and DNA transfection. Secondary chickenembryo fibroblasts (CEFs) were maintained in medium 199containing 2% tryptose phosphate, 1% calf serum, and 1%chick serum (all from GIBCO). Cells were transfected with

    plasmid DNA at 5 to 8 pug/ml in medium 199 containing 200,ug of DEAE-dextran per ml. After 5 to 12 h, cells weresubjected to a 2- to 5-min shock with 10% dimethyl sul