A ROLE FOR SRP54 DURING INTRON BRIDGING OF SMALL INTRONS WITH PYRIMIDINE TRACTS UPSTREAM OF THE BRANCH POINT

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Sacchar omyces cerevisiae) to the 3' splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (S R) family of splicing factors as well as splicing factor 1 (SF1 or bra nch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site fo r U2AF is the pyrimidine tract located between the branch point and 3' splice site. Many small introns, especially those in nonvertebrates, lack a classical 3' pyrimidine tract. Here we show that a 59-nucleotid e Drosophila melanogaster intron contains C-rich pyrimidine tracts bet ween the 5' splice site and branch point that are needed for maximal b inding of both U1 snRNPs and U2 snRNPs to the 5' and 3' splice site, r espectively, suggesting that the tracts are the binding site for an in tron bridging factor. The tracts are shown to bind both U2AF and the S R protein SRp54 but not SF1. Addition of a strong 3' pyrimidine tract downstream of the branch point increases binding of SF1, but in this c ontext, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative ear ly recognition mode to SF1-directed bridging for small introns, sugges ting gene-specific early spliceosome assembly.