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Structural Characterization of the Recognition of U6 snRNA by the Yeast U6 Biogenesis Protein Usb1
Author(s) -
DeLaitsch Andrew T,
Didychuk Allison L,
Montemayor Eric J,
Larson Matthew A,
Lucarelli Stefani,
Butcher Samuel E
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.910.8
Subject(s) - snrnp , small nuclear rna , spliceosome , prp24 , exoribonuclease , biogenesis , intron , biology , rna , rna splicing , endoribonuclease , biochemistry , microbiology and biotechnology , chemistry , gene , non coding rna , rnase p
The spliceosome is an essential molecular machine responsible for the removal of non‐coding introns from precursor messenger RNA in eukaryotic cells. An essential component of the catalytically active spliceosome, the U6 snRNA, must undergo a biogenesis pathway prior to incorporation into the spliceosome. Maturation of U6 snRNA is mediated in part by the highly conserved U six biogenesis protein (Usb1), a 3′‐5′ exoribonuclease responsible for the post‐transcriptional removal of uridines from U6 RNA. In addition, the enzyme is responsible for leaving behind a specific 3′ end modification in the form of a terminal phosphate, which is required for high affinity binding of a component of the U6 snRNP, the Lsm2–8 protein complex. Mutations in the gene encoding Usb1 give rise to the autosomal recessive disease poikiloderma with neutropenia (PN) in humans. I expressed and purified Usb1 from S. cerevisiae, which crystallized and diffracted X‐ray to 1.8 Å. The structure is remarkably similar to the structure of human Usb1 despite less than 20% sequence identity. A sulfate ion occupies the active site in a manner that may mimic coordination to the terminal phosphate of the RNA. Understanding how Usb1 recognizes RNA will give us insight into the mechanism of Usb1 processing and its role in U6 snRNA biogenesis. To biochemically characterize Usb1, I incubated recombinant Usb1 with a fluorescent RNA and monitored exoribonuclease activity via denaturing polyacrylamide gel electrophoresis. The assays show that activity is inactivated upon mutation of active site residues. In addition, fluorescence polarization binding experiments were used to monitor binding of RNA with variants of Usb1. The results of these experiments will help determine conditions most suitable for structural determination by NMR, SAXS, or x‐ray crystallography methods. I will present our progress towards determining a co‐structure of Usb1 with RNA in its active site with the goal of characterizing how the enzyme recognizes and subsequently processes U6 RNA for incorporation into the spliceosome.