Thermodynamic Examination of the U6 ISL RNA Bulge

Abstract

Spliceosome is large complex made up of five snRNA and nearly 70 proteins that assembles on pre‐mRNA to excise the introns. The U6 subunit of the spliceosome is required for spliceosome assembly and activation. Spliceosome undergoes number of structural rearrangements that disrupt U4/U6 complex and form U2/U6 complex to become activated. The association of U2/U6 complex results in the formation of a highly conserved internal stem loop (ISL) in U6 RNA. The formation of a 1x2 nucleotide bulge that binds catalytically active Mg2+ at U80 position is essential in the RNA cleavage. The 1x2 bulge contains an A79•C67 non‐canonical base pair, which forms a stronger A+•C base pair in lower pH conditions. Upon the A+•C base pair formation, the ISL adopts a conformation closer to an A‐form helix and the unbound U80 in the bulge is forced out into the major groove. This conformation is mutually antagonistic to the binding of the catalytic Mg2+ ion, and therefore prevents splicing activity. Thermal denaturation experiments were performed on RNA constructs derived to mimic 1x2 bulge of the U6 RNA. We tested constructs containing the conserved bulged nucleotides as well as those with modifications that disrupt spliceosomal function. All constructs were examined in 1 M KCl and in varying Mg2+ concentrations at pH of 5.5 and 7.5. The thermodynamic contributions of the bulged RNA were compared to those of the helical RNA and DNA constructs. Thermodynamics of RNA with modification in and around the 1x2 bulge loop will be presented and influence of pH and ionic conditions will be discussed.