Abstract
Post-transcriptional modification of snRNA is central to spliceosome function. Usb1 is an exoribonuclease that shortens the oligo-uridine tail of U6 snRNA, resulting in a terminal 2′,3′ cyclic phosphate group in most eukaryotes, including humans. Loss of function mutations in human Usb1 cause the rare disorder poikiloderma with neutropenia (PN), and result in U6 snRNAs with elongated 3′ ends that are aberrantly adenylated. Here, we show that human Usb1 removes 3′ adenosines with 20-fold greater efficiency than uridines, which explains the presence of adenylated U6 snRNAs in cells lacking Usb1. We determined three high-resolution co-crystal structures of Usb1: wild-type Usb1 bound to the substrate analog adenosine 5′-monophosphate, and an inactive mutant bound to RNAs with a 3′ terminal adenosine and uridine. These structures, along with QM/MM MD simulations of the catalytic mechanism, illuminate the molecular basis for preferential deadenylation of U6 snRNA. The extent of Usb1 processing is influenced by the secondary structure of U6 snRNA.
Highlights
Precursor messenger RNA contains intervening sequences, or introns, that must be removed by the spliceosome, a large and highly dynamic macromolecular complex that contains over a hundred proteins and five small nuclear RNAs (U1, U2, U4, U5 and U6 snRNAs) [1]
We report three high-resolution co-crystal structures of Usb1 complexed with RNA, which show the structural basis for this catalytic preference and unambiguously identify His120 and His208 as catalytic base and acid, respectively
Using a fluorescently labeled oligonucleotide substrate corresponding to the 3 fragment of U6 snRNA, we observe very little product accumulation after 1 h at 37◦C with 1 M substrate in the presence of 0.1 M enzyme, and higher concentrations of enzyme result in heterogenous products (Figure 2A)
Summary
Precursor messenger RNA contains intervening sequences, or introns, that must be removed by the spliceosome, a large and highly dynamic macromolecular complex that contains over a hundred proteins and five small nuclear RNAs (U1, U2, U4, U5 and U6 snRNAs) [1]. Individual snRNAs are recruited to spliceosomes in the form of small nuclear ribonucleoproteins (snRNPs). The maturation pathway of U6 and U6atac snRNAs and their incorporation into snRNPs is distinct from the other snRNAs (Figure 1). U6 and U6atac snRNAs are transcribed by RNA polymerase III and subsequent maturation occurs in the nucleus [3,4,5,6,7]. All other snRNAs are synthesized by RNA polymerase II, transported into the cytoplasm for post-transcriptional processing and are bound by Sm proteins before re-entry into the nucleus [8]
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