MUTATIONS IN AN ESSENTIAL U2 SMALL NUCLEAR-RNA STRUCTURE CAUSE COLD-SENSITIVE U2 SMALL NUCLEAR RIBONUCLEOPROTEIN FUNCTION BY FAVORING COMPETING ALTERNATIVE U2 RNA STRUCTURES

Mutations in stem-loop IIa of yeast U2 RNA cause cold-sensitive growth and cold-sensitive U2 small nuclear ribonucleoprotein function in vit ro. Cold-sensitive U2 small nuclear RNA adopts an alternative conforma tion that occludes the loop and disrupts the stem but does so at both restrictive and permissive temperatures. To determine whether alternat ive U2 RNA structure causes the defects, we tested second-site mutatio ns in U2 predicted to disrupt the alternative conformation. We find th at such mutations efficiently suppress the cold-sensitive phenotypes a nd partially restore correct U2 RNA folding. A genetic search for addi tional suppressors of cold sensitivity revealed two unexpected mutatio ns in the base of an adjacent stem-loop. Direct probing of RNA structu re in vivo indicates that the suppressors of cold sensitivity act to i mprove the stability of the essential stem relative to competing alter native structures by disrupting the alternative structures. We suggest that many of the numerous cold-sensitive mutations in a variety of RN As and RNA-binding proteins could be a result of changes in the stabil ity of a functional RNA conformation relative to a competing structure . The presence of an evolutionarily conserved U2 sequence positioned t o form an alternative structure argues that this region of U2 is dynam ic during the assembly or function of the U2 small nuclear ribonucleop rotein.