Dual function for U2AF(35) in AG-dependent pre-mRNA splicing

The splicing factor U2AF is required for the recruitment of U2 small nuclea r RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(6 5)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, wh ile the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U 2AF35 and the 3' splice site AG can stabilize U2AF(65) binding to weak Py t racts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interacti ons with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) bindin g. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs , only the presence of U2AF65. In contrast, splicing of a mouse immunoglobu lin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we h ave investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results i ndicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(3 5) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear t o be dispensable, because the purine-rich ESE present in exon M2 is not ess ential for U2AF(35) activity and because a truncation mutant of U2AF(35) co nsisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other a ctivities of U2AF(35) do not correlate with increased cross-linking of U2AF (65) to the Py tract. Collectively, the results argue that interaction of U 2AF(35) with a consensus 3' splice site triggers events in spliceosome asse mbly in addition to stabilizing U2AF(65) binding, thus revealing a dual fun ction for U2AF(35) in pre-mRNA splicing.