Co-Expression and Co-Purification of Archaeal and Eukaryal Box C/D RNPs

<div><p>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.</p></div

Diagrams of co-expression plasmids based on pQlink system.

<p>A). The pQlink-N vector inserted with tRNA-box C/D RNA chimera coding sequence. B). Co-expression plasmids inserted with all proteins and RNA encoding sequences. The region containing inserted genes is highlighted on top for pQafCDtRNA+ and pQhsCDtRNA+, respectively. Other elements of the co-expression plasmid are also shown at the bottom.</p

Co-expression and purification of recombinant <i>Archaeoglobus fulgidus</i> sR3 sRNP.

<p>A) Silver-stained SDS-PAGE gel analysis of purified sR3 sRNP components “Af-3Pro-sR3” denotes the sample expressing Nop5, fibrillarin, L7Ae and sR3 RNA. “Af-3Pro-tRNA-sR3” denotes that expressing Nop5, fibrillarin, L7Ae and sR3-tRNA chimeric RNA. Box C/D proteins and sR3-tRNA chimeric RNA are labeled and indicated by arrows. The sR3 sRNPs were purified by Ni-NTA affinity followed by gel filtration method based on the single histidine tag present in Nop5. B). Polyacrylamide gel analysis of total RNA extracted from cells expressing only box C/D proteins (Af-3Pro) or proteins plus sR3-tRNA chimeric RNA (Af-3Pro-tRNA-sR3). The location of sR3-tRNA chimera is indicated.</p

Quantitative RT-PCR analysis of RNA samples from co-expressing box C/D RNPs.

<p>Delta Rn indicates the magnitude of amplification and is the normalized and base-line corrected fluorescence emission intensity of the reporter dye obtained for each PCR reaction. A). Amplification plots of qRT-PCR of RNA samples extracted from Ni-NTA purified (red) or cell lysate (green) of <i>Archaeoglobus fulgidus</i> sR3 sRNP. B). Amplification plots of qRT-PCR of RNA samples extracted from Ni-NTA purified (red) or cell lysate (green) of human U14 snoRNP.</p

Co-expression and purification of recombinant human U14 box C/D snoRNP.

<p>A). Coomassie blue-stained SDS-PAGE gel analysis of purified U14 snoRNP. The U14 snoRNP was purified by Ni-NTA affinity followed by gel filtration method based on the single histidine tag present in NOP56. B). Total RNA extracted from cells expressing various proteins and RNA under both inducing and noninducing conditions. “H-3Pro” denotes the construct expressing NOP56, NOP58, and fibrillarin, “H-4Pro” denotes the construct expressing NOP56, NOP58, fibrillarin, and 15.5 K, “H-4Pro-U14” denotes the construct expressing NOP56, NOP58, fibrillarin, 15.5 K and U14 RNA, “H-4Pro-tRNA-U14” denotes the construct expressing NOP56, NOP58, fibrillarin, 15.5 K and U14-tRNA chimera RNA. C). Coomassie-stained SDS-PAGE gel analysis of the cell lysates of the samples described in B). Locations of NOP56, NOP58, fibrillarin and 15.5 K are indicated.</p

Schematics of box C/D ribonucleoprotein particles (RNPs) and chimera RNA used in co-expression studies.

<p>A) Eukaryotic box C/D RNPs comprise Nop56, Nop58, fibrillarin (FIB), 15.5 K (Snu13p in yeast), and a box C/D RNA. In archaea, Nop56 and Nop58 have a single homolog, Nop5, and 15.5 K has a homolog, L7Ae. The protein assembly model is based on architecture revealed by archaeal box C/D RNP crystal structures. B) Construction of the tRNA-box C/D RNA chimera. tRNA is divided into two parts (part I and part II) that flank the sequence encoding the box C/D RNA.</p