20S RNA is a noninfectious viral single-stranded RNA found
in most laboratory strains of the yeast Saccharomyces
cerevisiae. 20S RNA encodes a protein of 91 kDa (p91)
that contains the common motifs found among RNA-dependent
RNA polymerases from RNA viruses. p91 and 20S RNA are noncovalently
associated in vivo, forming a ribonucleoprotein complex.
We detected an RNA polymerase activity in p91/20S RNA
complexes isolated by high-speed centrifugation. The activity
was not inhibited by actinomycin D nor α-amanitin.
The majority of the in vitro products was 20S RNA and the
rest was the complementary strands of 20S RNA. Because
the extracts were prepared from cells accumulating 20S
RNA over its complementary strands, these in vitro products
reflect the corresponding activities in vivo. When the
p91/20S RNA complexes were subjected to sucrose gradient
centrifugation, the polymerase activity cosedimented with
the complexes. Furthermore, an RNA polymerase activity
was detected in the complex by an antibody-linked polymerase
assay using anti-p91 antiserum, suggesting that p91 is
present in the active RNA polymerase machinery. These results
together indicate that p91 is the RNA-dependent RNA polymerase
or a subunit thereof responsible for 20S RNA replication.