In vitro reconstitution of yeast splicing with U4 snRNA reveals multiple roles for the 3′ stem–loop

U4 deletion mutants are defective in triple-snRNP formation. Non-denaturing Northern blot analysis of unextracted (i.e., protein-containing) samples. Untreated extract (lane 1), mock-depleted extract (lane 2), and depleted extract reconstituted with nothing (lane 3) or with IVT U4 variants (283 or 566 nM, lanes 4–17). Location of snRNP species indicated by labels at left. U4*/U6 indicates aberrantly migrating U4/U6 di-snRNP seen in all reconstituted extracts.