3'-Terminal RNA structures and poly(U) tracts inhibit initiation by a 3'-->5' exonuclease in vitro

Abstract

We have previously shown that the presence of a poly(A) tail blocks the activity of a highly efficient 3'-->5' exonuclease in HeLa extracts. Similar activities have been implicated in RNA turnover in vivo. It is not clear, however, what protects poly(A)-non-mRNAs from the action of this enzyme. A stem-loop structure located at the 3'-end of U11 RNA was required to protect this transcript from the exonuclease in vitro. Similar 3' stem-loop structures, or extensive base pairinginvolving the 3'-end, are present on all mature small stable RNAs. The placement of artificial stem-loop structures at the 3'-end also protected RNA substrates, suggesting that RNA structure alone is sufficient to block the initiation of the exonuclease. The placement of RNA structures at internal positions of substrate trans-cripts did not affect the activity of the exonuclease or lead to the accumulation of degradation intermediates. Pol III precursor transcripts contain short poly(U) tracts rather than structure at their 3'-ends. Terminal poly(U) tracts protected RNA substrates from the 3'-->5' exonuclease in a protein-dependent fashion. Although La protein is found associated with the terminal U tracts of pol III precursor transcripts both in vivo and in vitro, La protein was not required for poly(U) to protect RNA substrates from the 3'-->5' exonuclease. In summary, these data reveal a variety of ways RNAs have evolved to protect themselves from this exonuclease.