No abstract

Cap structures are added cotranscriptionally to all RNA polymerase II transcripts. They affect several processes including RNA stability, pre-messenger RNA splicing, RNA export from the nucleus and translation initiation. The effect of the cap on translation is mediated by the initiation factor eIF-4F, whereas the effect on pre-mRNA splicing involves a nuclear complex (CBC) composed of two cap binding proteins, CBP80 and CBP20. A role for CBC in the nuclear export of capped RNAs has also been proposed. We report here the characterization of human and Xenopus CBP20s. Antibodies against recombinant CBP20 prevent interaction of CBC with capped RNAs in vitro. Following microinjection into Xenopus oocytes, the antibodies inhibit both pre-mRNA splicing and export of U small nuclear RNAs to the cytoplasm. These results demonstrate that CBC mediates the effect of the cap structure in U snRNA export, and provide direct evidence for the involvement of a cellular RNA-binding factor in the transport of RNA to the cytoplasm.

The mechanism by which intron-containing RNAs are recognized by the splicing machinery is only partly understood. A nuclear cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts, has previously been shown to play a role in pre-mRNA splicing. Using a combination of splicing complex and psoralen cross-linking analysis we demonstrate that CBC is required for efficient recognition of the 5' splice site by Ul snRNP during formation of E (early) complex on a pre-mRNA containing a single intron. However, in a pre-mRNA containing two introns, CBC is not required for splicing of the cap distal intron. In this case, the presence of an intact polypyrimidine tract in the cap-proximal intron renders splicing of the cap-distal intron independent of CBC. Removal of introns from pre-messenger RNAs (premRNAs) occurs in a dedicated nucleoprotein complex termed the spliceosome. The spliceosome consists of Ul, U2, and U4/U6.U5 snRNP particles together with a large number of nonsnRNP splicing factors, most of which are poorly characterized (for review, see Lamm and Lamond 1993;Moore et al. 1993;Madhani and Guthrie 1994). The cis-acting elements which define in trons, the 5' splice site, branchpoint sequence, polypyri midine tract, and 3' splice site, have quite degenerate sequences in higher eukaryotes. This poses the problem of how pre-mRNAs are efficiently recognized by the splicing machinery in spite of the limited amount of se quence information.In vitro, several stable intermediate complexes in the spliceosome assembly pathway have been observed. In mammalian nuclear extracts, the first prespliceosomal complex to be observed is the early (E) complex, which forms in the absence of ATP (Michaud and Reed 1991). The E complex contains Ul small nuclear ribonucleoprotein (snRNP) and the splicing factor U2AF (U2 sn RNP auxiliary factor; Zamore and Green 1989) bound at the 5' splice site and polypyrimidine tract, respectively. 'Present address:

Structure‐function relationships in the vertebrate U4‐U6 snRNP have been analysed by assaying the ability of mutant RNAs to form U4‐U6 snRNPs and to function in splicing complementation in Xenopus oocytes. The mutants define three categories of domain within the RNAs. First, domains which are not essential for splicing. These include regions of U6 which have previously been implicated in the capping and transport to the nucleus of U6 RNA as well as, less surprisingly, regions of U4 and U6 which have been poorly conserved in evolution. Second, domains whose mutation reduces U4‐U6 snRNP assembly or stability. This group includes mutations in both the proposed U4‐U6 interaction domain, and also, in the case of U6, in a highly conserve sequence flanking stem I of the interaction domain. These mutants are all defective in splicing. Third, regions not required for U4‐U6 assembly, but required for splicing complementation. This category defines domains which are likely to be required for specific contacts with other components of the splicing machinery. Combinations of mutants in the U4 and U6 interaction domain are used to show that there are not only requirements for base complementarity but also for specific sequences in these regions.

Recombinant baculoviruses were used to express wild‐type serum response factor (SRF) and a mutant, SRF.CKIIA, which lacks all four serine residues in the major casein kinase II (CKII) site at residues 77–90. Purified recombinant SRF binds DNA with an affinity and specificity indistinguishable from that of HeLa cell SRF, and activates transcription in vitro. Comparative phosphopeptide analysis of the wild‐type and mutant proteins demonstrated that the wild‐type protein is phosphorylated at the major CKII site in insect cells. Dephosphorylation of recombinant SRF does not affect its affinity for the c‐fos SRE, and results in only a 3‐fold reduction in binding to the synthetic site ACT.L. However, dephosphorylation does cause a large decrease in the rates of association with and dissociation from either site. These effects are due solely to phosphorylation at the major CKII site: the binding properties of the SRF.CKIIA mutant are identical to those of dephosphorylated wild‐type SRF, and CKII phosphorylation in vitro converts dephosphorylated wild‐type SRF from a slow‐binding to a fast‐binding form without significantly changing binding affinity. CKII phosphorylation thus acts to potentiate SRF‐DNA exchange rates rather than alter equilibrium binding affinity.