Action of RNase II and polynucleotide phosphorylase against RNAs containing stem-loops of defined structure

Abstract

The 3'-->5' exoribonucleases, RNase II and polynucleotide phosphorylase (PNPase), play an essential role in degrading fragments of mRNA generated by prior cleavages by endonucleases. We have assessed the ability of small RNA substrates containing defined stem-loop structures and variable 3' extensions to impede the exonucleolytic activity of these enzymes. We find that stem-loops containing five G-C base pairs do not block either enzyme; in contrast, more stable stem-loops of 7, 9, or 11 bp block the processive action of both enzymes. Under conditions where enzyme activity is limiting, both enzymes stall and dissociate from their substrates six to nine residues, on average, from the base of a stable stem-loop structure. Our data provide a clear mechanistic explanation for the previous observation that RNase II and PNPase behave as functionally redundant.

FIG. 1

Schematic diagram of the structures of RNA substrates. (a) Three different classes of 3′ extensions, denoted as SLx, where x is the number of base pairs in the stem-loop. The common 5′ end of all substrates is 5′ pppGGGAAUUCGAGCUCGGUAC. An imperfect inverted repeat in the SLxR RNAs (see the text) is shown by arrows. (b) Primary and predicted secondary structures of the four stem-loops examined in this work.

FIG. 2

Digestion of SLx RNAs with 3′ exonucleases. Digestions were performed as described in Materials and Methods. Aliquots were removed at 0, 2.5, 5, and 10 min of digestion and denatured. RNAs were separated by electrophoresis under denaturing conditions, and the products were visualized by autoradiography or by phosphorimaging (see Materials and Methods). Shown are digestions with RNase II (a) and PNPase (b). Each panel is a separate gel. The schematic in the central margin illustrates the structure of the substrates.

FIG. 3

Digestion of SLxA RNAs with 3′ exonucleases. Digestions were performed as described in Materials and Methods. Aliquots were removed after 0, 2.5, 5, 7.5 (a only), and 10 min of digestion, denatured, and resolved as described in the legend to Fig. 2. Shown are digestions with RNase II (a) and PNPase (b). Brackets in the margins point to intermediates which accumulate during digestion (see the text). Each panel is a different gel; therefore, the relative mobilities of products cannot be compared directly between panels. Lanes with sequence ladders are not shown. The schematic diagrams in the margin denote the structures of the substrate and intermediates. Lane M, undigested SL11A RNA.

FIG. 4

Digestion of SLxR RNAs with 3′ exonucleases. Digestions were performed as described in Materials and Methods. Aliquots were removed at 0, 2.5, 5, 7.5, and 10 min of digestion, denatured, and resolved as described in the legend to Fig. 2. Shown are digestions with RNase II (a) and PNPase (b). Each panel is a separate gel. Lanes with sequence ladders are not shown. Brackets in the margins mark the positions of intermediates presumed to be the result of stalling of the enzyme. The arrowheads show the position of a transient intermediate (see the text). The schematic diagrams in the central margin denote the structures of the substrate and intermediates (see the text).