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

We have previously shown that the presence of a poly(A) tail blocks the act ivity of a highly efficient 3'-->5' exonuclease in HeLa extracts, Similar a ctivities have been implicated in RNA turnover in vivo. It is not clear, ho wever, 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 prote ct this transcript from the exonuclease in vitro, Similar 3' stem-loop stru ctures, or extensive base pairing involving 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 placeme nt of RNA structures at internal positions of substrate transcripts did not affect the activity of the exonuclease or lead to the accumulation of degr adation intermediates, Pol III precursor transcripts contain short poly(U) tracts rather than structure at their 3'-ends, Terminal poly(U) tracts prot ected RNA substrates from the 3'-->5' exonuclease in a protein-dependent fa shion. Although La protein is found associated with the terminal U tracts o f pol III precursor transcripts both in vivo and in vitro, La protein was n ot required for poly(U) to protect RNA substrates from the 3'-->5' exonucle ase. In summary, these data reveal a variety of ways RNAs have evolved to p rotect themselves from this exonuclease.