Abstract
U6 snRNA undergoes post-transcriptional 3′ end modification prior to incorporation into the active site of spliceosomes. The responsible exoribonuclease is Usb1, which removes nucleotides from the 3′ end of U6 and, in humans, leaves a 2′,3′ cyclic phosphate that is recognized by the Lsm2–8 complex. Saccharomycescerevisiae Usb1 has additional 2′,3′ cyclic phosphodiesterase (CPDase) activity, which converts the cyclic phosphate into a 3′ phosphate group. Here we investigate the molecular basis for the evolution of Usb1 CPDase activity. We examine the structure and function of Usb1 from Kluyveromyces marxianus, which shares 25 and 19% sequence identity to the S. cerevisiae and Homo sapiens orthologs of Usb1, respectively. We show that K. marxianus Usb1 enzyme has CPDase activity and determined its structure, free and bound to the substrate analog uridine 5′-monophosphate. We find that the origin of CPDase activity is related to a loop structure that is conserved in yeast and forms a distinct penultimate (n – 1) nucleotide binding site. These data provide structural and mechanistic insight into the evolutionary divergence of Usb1 catalysis.
Highlights
Usb1 is a member of the 2H phosphodiesterase superfamily of enzymes, which contain two vicinal active site HxS/T motifs that are essential for catalysis
The 2H superfamily can be sub-divided into HxT and HxS enzymes, the latter of which contains Usb1. 2H superfamily enzymes act on myriad RNAs and nucleotide substrates, and are capable of many catalytic activities, including 2,5 RNA ligase or nuclease, 2,5 or 3,5 -phosphodiesterase, and 1,2 cyclic or 2,3 -cyclic phosphodiesterase (CPDase) activities
When 1.0 M substrate is incubated in the presence of equimolar or ten-fold excess of KmUsb1 enzyme, multiple products of heterogeneous length are observed (Fig. 1B). This behavior is highly similar to the human enzyme, but differs from S. cerevisiae Usb1 which predominantly removes a single nucleotide [5,14]
Summary
Usb is a member of the 2H phosphodiesterase superfamily of enzymes, which contain two vicinal active site HxS/T motifs that are essential for catalysis. 2H superfamily enzymes act on myriad RNAs and nucleotide substrates, and are capable of many catalytic activities, including 2 ,5 RNA ligase or nuclease, 2 ,5 or 3 ,5 -phosphodiesterase, and 1 ,2 cyclic or 2 ,3 -cyclic phosphodiesterase (CPDase) activities [1]. These divergent activities are all thought to utilize two catalytic histidines within the central HxS/T motifs that act as a general acid and base, while the serine or threonine residues help coordinate substrates and assist in transition state stabilization [2,3,4,5]. ScUsb has 2 CPDase activity that hydrolyzes the 2 ,3 -cyclic phosphate product into a 3 monophosphate, an activity that is not present in the human enzyme
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