Figure 1.
Schematics of box C/D ribonucleoprotein particles (RNPs) and chimera RNA used in co-expression studies.
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.
Figure 2.
Diagrams of co-expression plasmids based on pQlink system.
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.
Figure 3.
Co-expression and purification of recombinant Archaeoglobus fulgidus sR3 sRNP.
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.
Figure 4.
Co-expression and purification of recombinant human U14 box C/D snoRNP.
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.
Figure 5.
Quantitative RT-PCR analysis of RNA samples from co-expressing box C/D RNPs.
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 Archaeoglobus fulgidus 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.
Figure 6.
In vitro methylation activity assay results using co-expressed and purified sR3 sRNP and U14 snoRNP.
A). Sequences of the target RNAs complementary to upstream sequences of box D of sR3 and U14 and box D′ of sR3 are shown. Radiolabeled nucleotide is indicated by “*C” and is the methylation target nucleotide. B). Various target RNA containing α-32P-cytidine were incubated with co-expressed and purified sR3 RNP, or reconstituted RNP control, phenol/chloroform extracted, ethanol precipitated, and RNase P1 digested and separated on a thin layer chromatography plate. The loading position and those of cytidine monophosphate (unmethylated or methylated) were indicated by “Origin”, “CMP” and “mCMP”, respectively. The target RNA complementary to box D, D′ guide of sR3 RNA, and to box D guide of U14 RNA are indicated by “D”, “D′”, “U14”, respectively. C). Methylation activity analysis of U14 snoRNP at different temperatures (Lanes 1–3). Co-purified sR3 sRNP with target RNA complementary to its box D is included as a positive control (Lane 4).