Splicing of the adenovirus IIIa mRNA is subjected to a strict temporal regulation during virus infection such that efficient IIIa 3 splice site usage is confined to the late phase of the infectious cycle. Here we show that the adenovirus L4-33K protein functions as a virus-encoded RNA splicing factor that preferentially activates splicing of transcripts with a weak 3 splice site sequence context, a sequence configuration that is shared by many of the late adenovirus 3 splice sites. Furthermore, we show that L4-33K activates IIIa splicing through the IIIa virus infection-dependent splicing enhancer element (3VDE). This element was previously shown to be the minimal element, both necessary and sufficient, for activation of IIIa splicing in the context of an adenovirus-infected cell. L4-33K stimulates an early step in spliceosome assembly and appears to be the only viral protein necessary to convert a nuclear extract prepared from uninfected HeLa cells to an extract with splicing properties very similar to a nuclear extract prepared from adenovirus lateinfected cells. Collectively, our results suggest that L4-33K is the key viral protein required to activate the early to late switch in adenovirus major late L1 alternative splicing.Most late adenovirus proteins are translated from mRNAs originating from the major late transcription unit (MLTU), 3 which extends from the major late promoter (MLP) at coordinate 16.8 to a termination signal close to the right-hand end of the genome (reviewed in Ref. 1). The ϳ28,000-nucleotide pre-mRNA expressed from the MLTU becomes polyadenylated at one of five possible sites, generating five families of mRNAs with co-terminal 3Ј-ends (L1-L5; Fig. 1A). Following selection of a poly(A) site, the primary transcript is spliced in such a way that each mature mRNA receives a common set of three short 5Ј-leader segments, the tripartite leader (Fig. 1A). This leader is then spliced to one of several alternative 3Ј splice sites, generating a total of more than 20 cytoplasmic mRNAs.The accumulation of mRNA from the MLTU is subjected to a temporal regulation at the levels of transcription elongation, poly(A) site choice and alternative 3Ј splice site selection (reviewed in Ref. 1). Thus, during the early phase of infection the MLP is active at a level comparable with the other early transcription units, whereas the same promoter accounts for most of the transcriptional activity at late times of infection (2, 3). However, at early times transcription initiated at the MLP decreases gradually over a large region beginning after the L1 unit, with few RNA polymerases extending beyond the L3 polyadenylation sequence (4). At late times, this block in elongation is alleviated and transcripts initiated at the MLP continue to the right hand end of the genome. The control of MLTU transcription is further regulated by events taking place at the level of poly(A) and alternative 3Ј splice site selection (2, 5, 6). Thus, although nuclear transcription proceeds across at least the L1, L2 and L3 poly(A) sites a...

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U2AF has been characterized as an essential splicing factor required for efficient recruitment of U2 small nuclear ribonucleoprotein to the 3-splice site in a pre-mRNA. The U2AF 65 subunit binds to the pyrimidine tract of the pre-mRNA, whereas the U2AF 35 subunit contacts the 3-splice site AG. Here we show that U2AF 35 appears to be completely dispensable for splicing in nuclear extracts prepared from adenovirus late-infected cells (Ad-NE). As a consequence, the viral IIIa and cellular IgM introns, which both have suboptimal 3-splice sites and require U2AF 35 for splicing in nuclear extracts from uninfected cells, are transformed to U2AF35 -independent introns in Ad-NE. Furthermore, we present evidence that two parallel pathways of 3-splice site recognition exist in Ad-NE. We show that the viral 52,55K intron, which has an extended pyrimidine tract, requires U2AF for activity in Ad-NE. In contrast, the IgM intron, which has a weak 3-splice site sequence context, undergoes the first catalytic step of splicing in U2AF-depleted Ad-NE, suggesting that spliceosome assembly occurs through a novel U2AF-independent pathway in Ad-NE.The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. In the cell, splicing takes place within a large macromolecular complex, the spliceosome, which consists of five small nuclear ribonucleoproteins (snRNPs) 1 and many non-snRNP splicing factors (reviewed in Refs. 1-3). Here we will focus our analysis on the function of splicing factors and snRNPs that are involved in the early recognition of the 3Ј-splice site in a pre-mRNA.Several sequence elements help to define the 3Ј-splice site: the branchpoint sequence, which is followed by a pyrimidinerich sequence (pyrimidine tract) and a conserved AG dinucleotide at the 3Ј-end of the intron. The critical step in 3Ј-splice site definition is the stable recruitment of U2 snRNP to the 3Ј-splice site. U2 snRNP binds to the branchpoint sequence through a base pairing interaction with this sequence and U2 snRNA (reviewed in Ref. 4). Binding of U2 snRNP requires auxiliary splicing factors such as SF1/mBBP and U2AF (5). U2AF is a heterodimer consisting of a 65-and a 35-kDa subunit. The U2AF 65 subunit binds specifically to the pyrimidine tract through its RNA recognition domains (6), whereas U2AF 35 has been shown to make contact with the AG dinucleotide at the 3Ј-splice site (7-9).In fact, introns appear to fall into one of two classes: AG-dependent introns and AG-independent introns (10). The AG-dependent introns typically have weak pyrimidine tracts, which make unstable interactions with U2AF65 . In such introns, the U2AF 35 interaction with the 3Ј-splice site AG dinucleotide becomes important, by stabilizing the U2AF 65 interaction with the pyrimidine tract (11). In contrast, AG-independent introns do not require U2AF 35 for activity. In such introns, binding of U2AF 65 to the strong pyrimidine tract is usually sufficient to aid in the recruitment of U2 snRNP to the branchpoint sequence (7).We are u...