Retroviral splicing suppressor sequesters a 3' splice site in a 50S aberrant splicing complex.

The generation of proteomic diversity occurs in part by alternative splicing, the process by which a pre-mRNA can be spliced using different combinations of 5′ and 3′ splice sites (42). The regulation of alternative splicing is also important for retroviral replication (39). A simple retrovirus, Rous sarcoma virus (RSV), uses a single 5′ splice site and two 3′ splice sites to generate two singly spliced mRNA species, which are translated into the Env and Src proteins (7). In addition, a large fraction of retroviral primary transcripts are not spliced. Instead, a pool of unspliced pre-mRNA is maintained and used both as mRNA for the Gag and Pol proteins and as packaged genomic RNA. RSV encodes no known accessory proteins; its RNA splicing is regulated by cis-acting RNA sequences within the RSV genome. The env branchpoint is suboptimal; mutations that increase env splicing impair retroviral replication (14, 22). The splicing of the RSV src gene is negatively regulated by two cis-acting elements. The first, known as the suppressor of src splicing, is found upstream of the distal src 3′ splice site (1), and the second, known as the negative regulator of splicing (NRS), is downstream of the viral 5′ splice site within the gag gene (2, 35, 37). The NRS suppresses splicing from an upstream 5′ splice site both in vivo and in vitro (16, 34, 35, 37). It has been shown to bind several splicing factors, including SF2/ASF and U1 and U11 snRNPs (16, 19, 32, 33). The terminal regions of the NRS are both essential for splicing suppression (35). SF2/ASF and hnRNPH bind to the purine-rich 5′ portion of the NRS, which functions as a splicing enhancer (12, 31). U1 snRNP binds to a pseudo-5′ splice site near the 3′ end of the NRS, which contains a 5/8 match to a U2-dependent 5′ splice site consensus sequence (19, 33). Three nonconsensus U's are present at positions −2, +3, and +4 relative to the pseudo-splice site (Fig. ​(Fig.1A).1A). Mutation of any one of these U's to a consensus A converts the NRS into a functional 5′ splice site in vivo (38). Surprisingly, mutation of the two U's at +3 and +4 to nonconsensus C's abolishes splicing suppression (38). U11 snRNP also binds to the NRS RNA in vitro at a perfect 5′ splice site consensus sequence for a U12-dependent intron that begins at +5 relative to the U2-dependent pseudo-5′ splice site (16) (Fig. ​(Fig.1A).1A). U11 snRNP recognizes a minor class of 5′ splice sites (46). However, the role of U11 snRNP in NRS-mediated splicing suppression remains unclear (19, 33). FIG. 1. Mutated NRS sequence (UU/AA) functions as a 5′ splice site in vitro. A) The NRS sequence was fused to the adenovirus major late intron and 3′ splice site (13) to generate a splicing substrate. The 5′ splice site-like sequence of ... When the NRS is inserted into the intron of a heterologous adenovirus splicing substrate, it inhibits its splicing in vitro (16). In the absence of ATP, the NRS interacts with a downstream adenovirus 3′ splice site (Ad3′) to form a commitment complex containing U1 snRNP and ASF/SF2 (8, 15). These data led to the hypothesis that the NRS functions as a decoy 5′ splice site that interacts with a downstream 3′ splice site to prevent its splicing. Here, we investigated whether the NRS can form a larger RNP complex, perhaps even a spliceosome, in the presence of ATP. RNA splicing in eukaryotic cells is carried out by a large, dynamic RNA-protein complex called the spliceosome, which appears to be assembled on the pre-mRNA through a series of intermediates (reviewed in references 4, 36, and 43). The first of these, termed the E or commitment complex, is characterized by ATP-independent binding of U1 snRNP at the 5′ splice site. Secondly, U2 snRNP binds to the branchpoint sequence to form the ATP-dependent pre-spliceosome or A complex. The U4/U6.U5 tri-snRNP complex is then recruited to form the B complex. Recent work has identified a spliceosomal intermediate called BΔU1, in which U1 snRNP has been dissociated but the tri-snRNP has yet to undergo the rearrangements required to form the catalytically active C or B* complex, containing U2, U5, and U6 snRNPs (27). Despite extensive characterization of the intermediates that occur during assembly, the possibility exists that the spliceosome may be recruited to the pre-mRNA as a preformed unit (28, 45). In this model of spliceosome assembly, the tri-snRNP is present but loosely associated during the early stages. In either the stepwise or penta-snRNP model of assembly, multiple, mutually exclusive RNA-RNA interactions occur. To further investigate the mechanism of NRS-mediated splicing suppression, we have studied the interaction between the NRS and a downstream 3′ splice site sequence (Ad3′) in vitro. We have compared a splicing substrate containing the NRS pseudo-5′ splice site with one containing an authentic 5′ splice site, derived from the NRS by UU/AA mutations at positions +3 and +4 (Fig. ​(Fig.1A).1A). Both of these RNAs assembled ATP-dependent, ∼50S RNP complexes, which contained all five major spliceosomal snRNPs. In contrast to the functional spliceosome, the NRS-Ad3′ complex was disrupted by heparin. In addition, the U5 snRNP 220-kDa protein, hPrp8, failed to cross-link to the NRS RNA as it did to the functional 5′ splice site, suggesting that the U5 snRNP was positioned differently than it was in an authentic spliceosome. We propose that the inability of the NRS pseudo-5′ splice site to bind hPrp8 properly prevents it from becoming a functional 5′ splice site. However, the NRS can still generate an aberrant spliceosomal complex that sequesters the 3′ splice site, thus suppressing splicing.