Sequences adjacent to the 5' splice site control U1A binding upstream of the IgM heavy chain secretory poly(A) site

We have recently shown that the stability of the alternatively expressed immunoglobulin M heavy chain secretory mRNA is developmentally regulated by U1A. U1A binds novel non-consensus sites upstream of the secretory poly(A) site and inhibits poly(A) tail addition in undifferentiated cells. U1A's dependence for binding and function upon a stem-loop structure has been extensively characterized for the consensus sites. We therefore probed the structure surrounding the novel U1A binding sites. We show that two of the three novel binding sites represent the major single-stranded regions upstream of the secretory poly(A) site, consistent with a major role at this site. The strength of binding and ability of U1A to inhibit poly(A) polymerase correlate with the accessibility of the novel sites. However, long range interactions are responsible for maintaining them in an open configuration. Mutation of an RNase V1-sensitive site 102 nucleotides upstream, directly adjacent to the competing 5' splice site, changes the structure of one the U1A binding sites and thus abolishes the binding of the second U1A molecule and the ability of U1A ability to inhibit poly(A) polymerase activity at this site. These sites bind U1A via its N-terminal domain but with a 10-fold lower affinity than U1 small nuclear RNA. This lower binding affinity is more conducive to U1A's regulation of poly(A) tail addition to heterologous mRNA.