U1 SMALL NUCLEAR-RNA CHIMERIC RIBOZYMES WITH SUBSTRATE-SPECIFICITY FOR THE REV PRE-MESSENGER-RNA OF HUMAN-IMMUNODEFICIENCY-VIRUS

The in vivo effectiveness of ribozymes strongly depends on the correct choice of the vector molecule, High levels of expression, stability, active conformation, and correct cellular localization are the most im portant features for a ribozyme vector, We have exploited the utilizat ion of the U1 small nuclear RNA (snRNA) as a vector for specifically t argeting a ribozyme into the nucleus. The Rev pre-mRNA of human immuno deficiency virus type 1 was chosen as target for testing the activity of the U-1-ribozyme. The catalytic core of the hammerhead motif, plus the recognition sequences, substituted the stem-loop III of the U1 snR NA, The resulting construct displays efficient cleavage activity in vi tro. In addition, in the in vivo system of Xenopus laevis oocytes, the Ill-chimeric ribozyme accumulates in large amounts in the nucleus and produces a considerable reduction of Rev pre-mRNA levels, The Rev-spe cific ribozyme was also inserted in a derivative of the U1 snRNA mutat ed in the region of pairing with the 5' splice site, such as to match it with the suboptimal splice junction of the Rev precursor, This cons truct shows more efficient reduction of Rev pre-mRNA in vivo than the wild-type U1 vector.