The U1 snRNA hairpin II as a RNA affinity tag for selecting snoRNP complexes.
When isolating ribonucleoprotein (RNP) complexes by an affinity selection approach, tagging the RNA component can prove to be strategically important. This is especially true for purifying single types of snoRNPs, because in most cases the snoRNA is thought to be the only unique component. Here, we present a general strategy for selecting specific snoRNPs that features a high-affinity tag in the snoRNA and another in a snoRNP core protein. The RNA tag (called U1hpII) is a small (26 nt) stem-loop domain from human U1 snRNA. This structure binds with high affinity (K(D)=10(-11)M) to the RRM domain of the snRNP protein U1A. In our approach, the U1A protein contains a unique affinity tag and is coexpressed in vivo with the tagged snoRNA to yield snoRNP-U1A complexes with two unique protein tags-one in the bound U1A protein and the other in the snoRNP core protein. This scheme has been used effectively to select C/D and H/ACA snoRNPs, including both processing and modifying snoRNPs, and the snoRNA and core proteins are highly enriched. Depending on selection stringency other proteins are isolated as well, including an RNA helicase involved in snoRNP release from pre-rRNA and additional proteins that function in ribosome biogenesis. Tagging the snoRNA component alone is also effective when U1A is expressed with a myc-Tev-protein A fusion sequence. Combined with reduced stringency, enrichment of the U14 snoRNP with this latter system revealed potential interactions with two other snoRNPs, including one processing snoRNP involved in the same cleavages of pre-rRNA.
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, USA.
Dorota
D
Piekna-Przybylska
Ben
B
Liu
Maurille J
MJ
Fournier
D000345:Affinity Labels; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D008969:Molecular Sequence Data; D012342:RNA, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar / Q000032:analysis* / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae
D000345:Affinity Labels; D012342:RNA, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; C405483:U1 small nuclear RNA