RNA-BINDING ACTIVITY OF HETERODIMERIC SPLICING FACTOR U2AF - AT LEASTONE RS DOMAIN IS REQUIRED FOR HIGH-AFFINITY BINDING

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3' splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3' splice site and targets U2 sn RNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF(65)) and small (hU2AF( 35)) subunits, hU2AF(65) contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs), hU2AF(35) has a degenerate RR M and a carboxyl-terminal RS domain. Genetic studies have recently sho wn that the RS domains on the Drosophila U2AF subunit homologs are eac h inessential and might have redundant functions in vivo. The site-spe cific pyrimidine tract binding activity of the U2AF heterodimer has pr eviously been assigned to hU2AF(65). While the requirement for the thr ee RRMs on hU2AF(65) is firmly established, a role for the large-subun it RS domain in RNA binding remains unresolved. We have analyzed the R NA binding activity of the U2AF, heterodimer in vitro, When the Drosop hila small-subunit homolog (dU2AF(38)) was complexed with the large-su bunit (dU2AF(50)) pyrimidine tract, RNA binding activity increased 20- fold over that of free dU2AF(50), We detected a similar increase in RN A binding activity when we compared the human U2AF heterodimer and hU2 AF(65), Surprisingly, the RS domain on dU2AF(38) was necessary for the increased binding activity of the dU2AF heterodimer. In addition, rem oval of the RS domain from the Drosophila large-subunit monomer (dU2AF (50)Delta RS) severely impaired its binding activity. However, if the dU2AF(38) RS domain was supplied in a complex with dU2AF(50)Delta RS, high-affinity binding was restored. These results suggest that the pre sence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consiste nt with redundant roles for the U2AF RS domains in vivo.