Abstract
Box C/D ribonucleoprotein particles (RNPs) are 2′-O-methylation enzymes required for maturation of ribosomal and small nuclear RNA. Previous biochemical and structural studies of the box C/D RNPs were limited by the unavailability of purified intact RNPs. We developed a bacterial co-expression strategy based on the combined use of a multi-gene expression system and a tRNA-scaffold construct that allowed the expression and purification of homogeneous archaeal and human box C/D RNPs. While the co-expressed and co-purified archaeal box C/D RNP was found to be fully active in a 2′-O-methylation assay, the intact human U14 box C/D RNP showed no detectable catalytic activity, consistent with the earlier findings that assembly of eukaryotic box C/D RNPs is nonspontaneous and requires additional protein factors. Our systems provide a means for further biochemical and structural characterization of box C/D RNPs and their assembly factors.
Highlights
Box C/D small nucleolar ribonucleoprotein particles are one of the two major types of RNPs required for ribosome and spliceosome maturation
Coexpression and purification of intact box C/D RNPs Previously characterized Archaeoglobus fulgidus (Af) sR3 and human U14 RNA were used for co-expression with their respective box C/D proteins (Figure 1). sR3 resides within the intron of Af tRNATrp precursor and was shown to be responsible for methylation of tRNATrp [33]
The single affinity tag incorporated into AfNop5 or HsNOP56 allows isolation of intact RNPs by affinity and size exclusion chromatography methods
Summary
Box C/D small nucleolar ribonucleoprotein particles (snoRNPs) are one of the two major types of RNPs required for ribosome and spliceosome maturation. A box C/D RNP comprises at least three (archaea) or four (eukarya) core proteins and a box C/D RNA. Eukaryotic proteins are more complicated than their archaeal counterparts and contain additional domains such as the GAR domain in fibrillarin and KKE/D repeats in Nop56/58 [18,19]. Unlike their archaeal counterparts, eukaryotic snoRNPs are unable to assemble spontaneously. Eukaryotic snoRNPs are unable to assemble spontaneously They require a complicated process mediated by assembly complexes to assemble [20,21,22,23]
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