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We constructed a viable insertion mutant (ins 5) of polyomavirus which contains, upstream of the L‐strand polyadenylation signal, a 94‐nt fragment of rabbit beta‐globin DNA. Included in this fragment are all of the sequence elements required for efficient cleavage and polyadenylation of rabbit beta‐globin RNA. The beta‐globin signal was efficiently recognized by the cleavage/polyadenylation machinery in mouse 3T6 cells infected with ins 5, signalling greater than 90% of the polyadenylation events on L‐strand RNAs. Furthermore, the presence of this efficient polyadenylation signal resulted in a 1.4‐ to 2.5‐fold increase in the fraction of virus‐specific RNAs that were polyadenylated. Most importantly, termination of transcription by RNA polymerase II on ins 5 DNA was also increased compared with wild‐type virus; nearly 100% of polymerases terminated per traverse of the ins 5 genome. These findings demonstrate that the rabbit beta‐globin insert, which contains a strong polyadenylation signal, also contains at least part of a signal for termination of transcription by RNA polymerase II. These results also show that the multiple, spliced leaders on polyomavirus L‐strand mRNAs, which arise as a result of inefficient termination and polyadenylation, are not necessary for efficient virus replication.

Three exons in the fibronectin primary transcript are alternatively spliced in a tissue-and developmental stage-specific manner. One of these exons, EDA, has been shown previously by others to contain two splicing regulatory elements between 155 and 180 nucleotides downstream of the 3-splice site: an exon splicing enhancer and a negative element. By transient expression of a chimeric ␤-globin/fibronectin EDA intron in COS-7 cells, we have identified two additional exonic splicing regulatory elements. RNA generated by a construct containing the first 120 nucleotides of the fibronectin EDA exon was spliced with an efficiency of approximately 50%. Deletion of most of the fibronectin EDA exon sequences resulted in a 20-fold increase in the amount of spliced RNA, indicative of an exon splicing silencer. Deletion and mutagenesis studies suggest that the fibronectin exon splicing silencer is associated with a conserved RNA secondary structure. In addition, sequences between nucleotides 93 and 118 of the EDA exon contain a non-purine-rich splicing enhancer as demonstrated by its ability to function in a heterologous context.Higher eukaryotes have evolved alternative splicing as a common means of producing several different mRNAs and polypeptides from a single primary transcript (for review, see Ref. 1). Transcripts that undergo alternative splicing can produce mRNAs and proteins with different structures, properties, and functions. The utilization of alternative splice sites can be regulated to prevent the inappropriate accumulation of one or more alternative products. To date, several examples of cisacting regulatory elements, distinct from bona fide splice sites, have been identified which modulate splicing in a diverse assortment of genes from viruses (2-10), Drosophila (11-14), and higher eukaryotes (15-33).The fibronectin gene is a classical example of a gene that undergoes alternative splicing (34, 35). The fibronectin gene is evolutionarily conserved and is predicted to yield up to 20 different mRNAs in human cells. The generation of this remarkable number of different mRNAs, and consequently polypeptides, is made possible by alternative splicing in three different coding regions of the fibronectin primary transcript (for review, see Refs. 36 and 37). One of the alternatively spliced regions encompasses the EDA (also referred to as EIIIA or EDI) exon. This exon is excluded selectively in fibronectin mRNAs produced by hepatocytes, but it is included to various extents by other cell types (Fig. 1A). EDA exon splicing is also subject to developmental regulation as EDA exon inclusion generally declines with age and differentiation (36, 37). In addition to tissue-specific and developmental regulation, inclusion of the EDA exon is modulated during the process of tissue repair and in certain diseases.For these reasons, regulation of fibronectin EDA exon splicing has been the subject of several studies. In fact, the EDA exon was the first exon with which it was demonstrated that proper alternative splicing could occur in...

The size of virus-specific RNA synthesized in cultured mouse kidney cells infected with polyoma virus was estimated by electrophoresis and sedimentation analysis of RNA extracts from whole cells. Newly synthesized "late" polyoma-specific RNA appears as "giant" molecules of heterogeneous size, up to several times larger than a strand of polyoma DNA (1.5 X 106 daltons). Treatment with dinethylsulfoxide or urea showed that the large size of these molecules is not due to aggregation. Giant polyoma-specific RNA is strikingly similar in size distribution to "nuclear messenger-like" RNA ("heterogeneous nuclear" RNA) of the host cell. Subsequent to its synthesis, some of the giant polyoma-specific RNA appears to be cleaved to at least three smaller species.During lytic infection of mouse kidney cell cultures with polyoma virus, virus-specific RNA can be detected by hybridization with polyoma DNA (1, 2). Viral RNA transcribed after the onset of polyoma DNA replication has been designated as "late" RNA (2). The rate of synthesis of late RNA increases rapidly beyond 12 hr after infection and approaches a maximum at about 30 hr.Earlier experimental results suggested that late polyomaspecific RNA is the transcript of most or all of the genetic information contained in a strand of polyoma DNA (2). We undertook the present experiments to define more precisely the size of late virus-specific RNA found in polyoma-infected cells. Total RNA was extracted about 30 hr after infection from ['HJuridine-labeled cultures under conditions that minimize breakdown, and was analyzed by gel electrophoresis and sucrose or Me2SO-sucrose gradient centrifugation. RNA from each gel or gradient fraction was hybridized with an excess of polyoma DNA to determine the amount of virus-specific RNA present. The results show that the bulk of late polyomaspecific RNA is synthesized as "giant" molecules larger than the viral genome, and that some of this giant RNA is subsequently cleaved to smaller RNAs of specific sizes. Within 10-30 sec of lysis, total cellular RNA was extracted with redistilled phenol; this treatment was repeated twice, for a total of 8 min at 60-650C (5). Nucleic acids were precipitated with ethanol, redissolved, and incubated at 00C for 30 min with 10 jhg/ml of DNase I (ribonuclease-free, electrophoretically purified; Worthington Biochemical Corp.) inl 0.01 M sodium acetate (pH 5.1)-2 mM MnCl2. Macaloid, 100 Mg/ml, was added to adsorb DNase and was removed by centrifugation; the purified RNA was reprecipitated. One Petri dish yielded 100-150 ug RNA (taking 1 Am0 Unit to equal 42 ug RNA.) DNA-RNA hybridization Highly purified polyoma DNA I, prepared as the 53 S form by sedimentation at pH 12.5 (6), was converted into single strands by boiling for 30 min in 0.1 X SSC [SSC: 0.15 M NaCl-0.015 M Na citrate (pH 7.4)], cooled rapidly by dilution in ice-cold 6 X SSC, and fixed onto 25-mm membrane filters (Schleicher and Schuell, B6) by filtration (7). Filters 3.5 mm in diameter were cut from dried and baked (800C, 2 hr) filters. A ...

The rate and efficiency of polyadenylation of late polyomavirus RNA in the nucleus of productively infected mouse kidney cells were determined by measuring incorporation of [3H]uridine into total and polyadenylated viral RNAs fractionated by oligodeoxythymidylic acid-cellulose chromatography. Polyadenylation is rapid: the average delay between synthesis and polyadenylation of viral RNA in the nucleus is 1 to 2 min. However, only 10 to 25% of viral RNA molecules become polyadenylated. Polyadenylated RNAs in the nucleus are a family of molecules which differ in size by an integral number of viral genome lengths (5.3 kilobases). These RNAs are generated by repeated passage of RNA polymerase around the circular viral DNA, accompanied by addition of polyadenylic acid to a unique 3' end situated 2.2 + n(5.3) kilobases from the 5' end of the RNAs (n can be an integer from 0 to at least 3). Between 30 and 50% of the sequences in nuclear polyadenylated RNA are conserved during processing and transport to the cytoplasm as mRNA. This is consistent with the molar ratios of nuclear polyadenylated RNAs in the different size classes, and it suggests that most polyadenylated nuclear RNA is efficiently processed to mRNA. Thus, the low overall conservation of viral RNA sequences between nucleus and cytoplasm is explained by (i) low efficiency of polyadenylation of nuclear RNA and (ii) removal of substantial parts of polyadenylated RNAs during splicing. The correlation between inefficient termination of transcription and inefficient polyadenylation of transcripts suggests that these two events may be causally linked.During the late phase of infection of mouse cells by polyomavirus, only about 5% of the viral RNA synthesized in the nucleus is successfully processed and transported to the cytoplasm as mRNA (2). This low efficiency of conservation of viral RNA during processing can be explained partly by the fact that RNA polymerase II may traverse the circular viral DNA genome (5,292 nucleotide pairs [11,35]) several times before terminating transcription (1,3). This leads to production of giant RNAs containing several tandemly repeated copies of the entire nucleotide sequence of one of the strands of the viral DNA. Only a single mRNA body sequence per precursor RNA molecule is conserved during RNA splicing (8,15,21) I have confirmed that only a small fraction (10 to 25%) of nuclear viral RNA is polyadenylated, that it is polyadenylated within less than 2 min of its synthesis, and that the remaining 75 to 90% of nuclear viral RNA is apparently never polyadenylated and cannot therefore serve as a precursor to viral mRNA in the cytoplasm. Nuclear viral RNA which is polyadenylated exists as molecules of discrete lengths of about 2.2 + n(5.3) kilobases (kb). These RNAs represent a series which have the same 5' and 3' ends but differ in the number of genome traverses made by RNA polymerase II before terminating. These results are consis-722 tent with the idea that most polyadenylated nuclear RNAs are successfully processed into mature mR...

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ABSTRACr The bulk of late virusspecific RNA synthesized in polyoma virus-infected mouse cells is larger than a single strand of polyoma DNA. The arrangement of viral nucleotide sequences in these giant polyoma RNAs was studied by electron microscopy of hybrids between purified high molecular weight viral RNA and the Hindu-i fragment of polyoma DNA, which contains 91% of the viral genome. Hybridimolecules containing a short single-stranded gap (corresponding to the 9% of viral sequences not present in Hind]-i), flanked by double-stranded regions, were photographed and measured. The majority of hybrid molecules contained no single-stranded loops or branches, showing that all viral sequences are transcribed contiguously and that no nonviral sequences are present in the RNA. Hybrid molecules, containing RNA up to 3.5 times the genome length, had a repeating structure of single-stranded gaps 8% of genome length interspersed with double-stranded regions 89% of genome length, showing that giant polyoma RNAs contain tandem repeats of the nucleotide sequence of the entire viral DNA. A small proportion of hybrid molecules contained single-stranded branches or deletion loops in characteristic positions, indicating that RNA "splicing" may occur on high molecular weight nuclear polyoma RNA.Transcription of polyoma DNA during the late phase of productive infection (for review, see ref. 1) gives rise to "giant" RNA molecules up to 3-4 times the size of the viral genome (2-5). These giant polyoma-specific RNAs are limited to the nucleus (6) where transcription takes place; viral mRNAs in the cytoplasm are one-quarter to one-half the size of the viral DNA (6-8). Most late nuclear polyoma-specific RNA is transcribed from the L strand of viral DNA (9); all sequences of the L strand are represented in late nuclear transcripts (10,11).To try to understand the mechanism by which these giant RNA molecules are synthesized, I have hybridized this RNA with a fragment (HindII-l) of polyoma DNA consisting of 91% of the genome (12,13) (Amersham,25 Ci/mmol) per culture dish in 1 ml of reinforced Eagle's medium. Cells were scraped into cold 10 mM triethanolamine, pH 8.5/10 mM NaCl/1.5 mM MgCl2 and lysed by addition of Nonidet P40 (Shell) to 0.5% final concentration. Nuclei were pelleted and resuspended in the same buffer. RNA was extracted by addition of 1 vol of 10 mM triethanolamine (pH 8.5)/100 mM NaCl/10 mM EDTA/4% triisopropylnaphthylenesulfonate (Serva) (14, 15) and 2 vol of phenol/chloroform/isoamyl alcohol, 50:50:1 (vol/vol). After three successive extractions at room temperature, nucleic acids in the aqueous phase were precipitated three times in succession with ethanol and then incubated at 00 for 20 min with DNase I (RNase-free; Worthington; 10,Mg/ml) in 10 mM Na acetate, pH 5.1/100 mM NaCI/2 mM MnCI2. RNA was extracted with phenol/chloroform in the presence of 1% sodium dodecyl sulfate (NaDodSO4) and ethanol-precipitated two times.Preparative Hybridization of RNA to Polyoma DNA. Polyoma form I DNA was denatured and bound to nitr...