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Conformational change within the spliceosome is required between the first catalytic step of pre-mRNA splicing, when the branch site (BS) attacks the 5' splice site, and the second step, when the 5' exon attacks the 3' splice site, yielding mRNA and lariat-intron products. A genetic screen for suppressors of BS A-to-G mutants, which stall between the two steps, identified Prp8, the highly conserved spliceosomal factor. prp8 suppressors facilitate the second step for multiple intron mutants and interact functionally with first step suppressors, alleles of PRP16 and U6 snRNA. Genetic interactions among prp8, prp16, and U6 alleles suggest that these factors control a common stage in first-to-second step transition. We propose that mutant substrates are utilized by alteration of the equilibrium between first/second step conformations, resembling tRNA miscoding caused by altered equilibrium between open/closed ribosomal conformations. This mechanistic commonality suggests that alteration of rearrangements represents an evolutionarily convenient way of modulating substrate selectivity.

The splicing of messenger RNA precursors in vitro proceeds through an intermediate that has the 5' end of the intervening sequence joined to a site near the 3' splice site. This lariat structure, which has been characterized for an adenovirus 2 major late transcript, has a branch point, with 2'-5' and 3'-5' phosphodiester bonds emanating from a single adenosine residue. The excised intervening sequence retains the branch site and terminates in a guanosine residue with a 3' hydroxyl group. The phosphate group at the splice junction between the two exons originates from the 3' splice site at the precursor.

The spliceosome is both compositionally and conformationally dynamic. Each transition along the splicing pathway presents an opportunity for progression, pausing, or discard, allowing splice site choice to be regulated throughout both the assembly and catalytic phases of the reaction.

The functional coupling of transcription and splicing has been reported both in vivo and in vitro, but the molecular mechanisms governing these interactions remain largely unknown. Here we show that p54 nrb , a transcription/ splicing factor, associates with the 5 0 splice site (SS) within large complexes present in HeLa cell nuclear extracts, in which the hyperphosphorylated form of RNA polymerase II (RNAPIIO) is associated with U1 or U1 and U2 snRNPs. These RNAPIIO-snRNP complexes also contain other transcription/splicing factors, such as PSF and TLS, as well as transcription factors that interact with RNAPIIO during elongation, including P-TEFb, TAT-SF1 and TFIIF. The presence of these factors in functional elongation complexes, demonstrated using an immobilized DNA template assay, strongly suggests that the RNAPIIO-snRNP complexes reflect physiologically relevant interactions between the transcription and splicing machineries. Our finding that both p54 nrb and PSF, which bind the C-terminal domain of the largest subunit of RNAPII, can interact directly with the 5 0 SS indicates that these factors may mediate contacts between RNAPII and snRNPs during the coupled transcription/splicing process.

We have analyzed the pathway of mammalian spliceosome assembly in vitro using a mobility retardation assay. The binding of splicing complexes to both wild-type and mutant [3-globin pre-RNAs was studied. Three kinetically related, ATP-dependent complexes, a, ~, and % were resolved with a wild-type [3-globin substrate. These complexes formed, both temporally and in order of decreasing mobility, ~ -, [3 --, ~/. All three complexes contained U2 snRNA. The RNA intermediates of splicing, i.e., free 5' exon and intron lariat + 3' exon, were found predominantly in the ~/complex. The RNA products of splicing, i.e., ligated exons and fully excised intron lariat, were found in separate, postsplicing complexes which appeared to form via breakdown of ~/. Mutations of the 5' splice site, which caused an accumulation of splicing intermediates, also resulted in accumulation of the ~/complex. Mutations of the 3' splice site, which severely inhibited splicing, reduced the efficiency and altered the pattern of complex formation. Surprisingly, the analysis of double mutants, with sequence alterations at both the 5' and 3' splice sites, revealed that the 5' splice site genotype was important for the efficient formation of a U2 snRNA-containing a complex at the 3' splice site. Thus, it appears that a collaborative interaction between the separate 5' and 3' splice sites promotes spliceosome assembly. Splicing of nuclear mRNA precursors (pre-RNA) involves the joining of two specific splice sites that can be widely separated, often by thousands of bases. The mechanism of splice site selection is poorly understood but must involve sequence recognition at both the 5' and 3' splice sites. Mutational studies have demonstrated the importance of the conserved sequences located at the intron-exon junctions of mammalian preRNAs, e.g., a comprehensive analysis has been made for a large collection of mutants in the large intron of rabbit [3-globin (Aebi et al. 1986). This has shown that mutants with alterations in the highly conserved GU or AG dinucleotides, located, respectively, at the 5' and 3' splice sites, promote the utilization in vivo of previously inactive cryptic splice sites. In most cases the level of spliced RNA appearing in the cytoplasm is also reduced. When the same mutants were analyzed in vitro, they were found to be processed in two separate ways. This either generated spliced RNAs utilizing the same cryptic sites observed in vivo or caused an accumulation of the splicing intermediates, with the reaction arrested after the initial 5' cleavage event. Presumably these latter RNAs are degraded in vivo.Small nuclear ribonucleoprotein particles (snRNPs) are an integral part of the nuclear splicing mechanism.