FACILITATION OF HAMMERHEAD RIBOZYME CATALYSIS BY THE NUCLEOCAPSID PROTEIN OF HIV-1 AND THE HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN A1

In order to improve the activity of hammerhead ribozymes in vivo, we h ave analyzed the effect of several prototypical RNA binding proteins o n the ribozyme cleavage reaction: bacteriophage T4 gene 32 protein (gp 32), hnRNP Al (Al) and the nucleocapsid protein of HIV-1 (NCp7). We sh ow that, while gp32 has no effect on the cleavage reaction, Al and NCp 7 affect different steps of the reaction. Moreover, some of these effe cts depend upon the ribozyme - substrate hybrid length. A1 and NCp7 in hibit the reaction of the least stable ribozyme-substrate complexes, w hich have 12 bp of duplex. NCp7, but not Al, inhibits the cleavage of substrates that have long ribozyme-substrate duplexes (17 or 20 bp), w hile cleavage of complexes having shorter duplexes (13 or 14 bp) is no t affected. NCp7 and Al enhance the turnover of ribozymes by increasin g the rate of product dissociation, but only when both cleavage produc ts are bound with less than or equal to 7 bp. Al and NCp7 enhance ribo zyme binding to long substrates, such as mRNAs, the structure of which otherwise limits ribozyme binding. Therefore, the effects of Al or NC p7 on the different steps of the cleavage reaction define a length of the ribozyme-substrate duplex which allows enhancement of the rate of binding and product release without inhibiting the cleavage step. Inte restingly, this duplex length (14 bases, or 7 on each side of the clea vage site) is identical for Al and NCp7. Since Al is thought to intera ct with most, if not all mRNAs in vivo, it may enhance the intracellul ar activity of ribozymes targeted against any mRNA. On the other hand, since retroviral nucleocapsid (NC) proteins interact only with the vi ral genomic RNA in vivo, these results raise the exciting possibility that NCp7 and other NC proteins may specifically enhance the activity of ribozymes targeted against their cognate retroviruses. Overexpressi on of Al or NCp7, and targeting the ribozyme to a cellular compartment rich in Al or NCp7 may also enhance ribozyme activity in vivo.