To understand the role of U2 RNA structure in pre-mRNA splicing we have characterized several cold-sensitive mutations in an essential stem-loop of yeast U2. Although mutant U2 is stable in vivo after a shift to restrictive temperature, splicing is rapidly inhibited, suggesting a direct effect on U2 function rather than U2 synthesis or snRNP assembly. Splicing complexes form at 23 degrees C in both mutant and wild-type extracts; however, stable association of mutant U2 snRNPs with pre-mRNA in vitro is inefficient at 15 degrees C, a temperature permissive for spliceosome assembly in wild-type extracts, indicating that the cold-sensitive defect is in U2 snRNP association with the assembling spliceosome. In vivo RNA structure probing reveals that the bulk of U2 RNA is misfolded in the mutants, even at permissive temperature. We propose that U2 stem-loop IIa is recognized by an assembly factor that assists U2 snRNP binding to pre-mRNA and that the cold sensitivity is due to a critical deficiency of correctly folded U2 for spliceosome assembly at low temperatures. Evolutionary conservation of the potential to form an interfering alternative RNA structure suggests the possibility that splicing could be regulated negatively at an early step by control of U2 snRNA conformation.