USB1 is a U6 snRNA-specific 3′-5′ exoribonuclease. (A) Overexpression of U6 snRNA rescues the lethality of USB1 deletion in yeast. USB1 usb1Δ diploid (left) or usb1Δ haploid cells (right) transformed with empty vector (pRS426) or plasmid expressing U6 snRNA (pSNR6). (B) Secondary structure model of human U6 snRNA.13 The 3′ end sequence (G95-U106) is marked in red. (C) Fluorescently labeled oligoribonucleotides used in the figure corresponding in sequence to the 3′ end of U6 snRNA (nucleotides G95-U106) carrying 3 to 7 nontemplated uridine residues (indicated in red). (D) USB1 has distributive 3′-5′ exoribonuclease activity. Oligo(U)7 RNA was incubated with exonuclease T (Exo T) for 1 minute (L, RNA ladder) or with USB1 for the indicated times. C1: oligo(U)7 dissolved in water; C2: oligo(U)7 incubated in buffer alone for 30 minutes. The position on the gel of full-length oligo(U)7 RNA (U113), the initial oligo(U)7 degradation product (U112), and the strong USB1 pause site (U107) are indicated. (E) USB1 trims back oligo(U) tracts to nucleotide U107 independent of their length. The USB1 pause site at nucleotide U107 and the processing termination site at U103 after 30 minutes are indicated. C: RNA incubated with buffer alone for 30 minutes. The major product of Exo T degradation (C99) is indicated. (F) The conserved H-x-S motifs are required for USB1 function in vitro. The oligo(U)7 RNA substrate was incubated with Exo T, wild-type USB1, N-terminally truncated USB1 (USB1Δ, residues G70-K265), or USB1Δ containing active site histidine mutations (USB1Δ-H120A and USB1Δ-H208A).
