The bipartite architecture of the sRNA in an archaeal box C/D complex is a primary determinant of specificity

Abstract

The archaeal box C/D sRNP, the enzyme responsible for 2'-O-methylation of rRNA and tRNA, possesses a nearly perfect axis of symmetry and bipartite structure. This RNP contains two platforms for the assembly of protein factors, the C/D and C'/D' motifs, acting in conjunction with two guide sequences to direct methylation of a specific 2'-hydroxyl group in a target RNA. While this suggests that a functional asymmetric single-site complex complete with guide sequence and a single box C/D motif should be possible, previous work has demonstrated such constructs are not viable. To understand the basis for a bipartite RNP, we have designed and assayed the activity and specificity of a series of synthetic RNPs that represent a systematic reduction of the wild-type RNP to a fully single-site enzyme. This reduced RNP is active and exhibits all of the characteristics of wild-type box C/D RNPs except it is nonspecific with respect to the site of 2'-O-methylation. Our results demonstrate that protein-protein crosstalk through Nop5p dimerization is not required, but that architecture plays a crucial role in directing methylation activity with both C/D and C'/D' motifs being required for specificity.

Figure 1

(a) Model of the full box C/D sRNP complex. (b) Model of the box C/D sRNP hemi-complex. L7Ae (red), Nop5p or mNop5p (blue) and fibrillarin (green).

Figure 2

Schematic representations of RNA constructs used. The guide sequence is labeled, the target sequence is labeled and shown in red, and the box C/D and C′/D′ motifs are labeled and shown in green. The site of predicted methylation (five bases upstream from the D or D′ motif) is marked with * for each RNA. (a) Hemi-RNA. (b) Two piece hemi-RNA. (c) Full RNA. (d) RNA with two box C/D motifs and one guide sequence. (e) RNA with one box C/D motif and two guide sequences.

Figure 3

(a) Size exclusion chromatography data showing the monomeric state of Nop5p upon removal of the coiled-coil domain. Circles represent size exclusion standards of 1.35, 17, 44 and 158 kDa. (b) Size exclusion chromatograph showing that mNop5p is capable of binding to fibrillarin. (c) SDS polyacrylamide gel electrophoresis of peaks 1 and 2 from size exclusion data shown in (b). Peak 1 contains mNop5p and fibrillarin while peak 2 contains only fibrillarin. (d) Assembly of the box C/D hemi-complex occurs through discrete, well-defined intermediates. Lane 1 shows hemi-RNA (3′ end labeled with Fluorescein). Lane 2 shows hemi-RNA–L7Ae complex. Lane 3 shows hemi-RNA–L7Ae–mNop5p complex. Lane 4 shows hemi-RNA–L7Ae–mNop5p–fibrillarin complex.

Figure 4

(a) Hemi-RNA/L7Ae gel mobility shift. Lane 1 was without L7Ae. L7Ae concentrations for lanes 2–15 ranged from 684 pM to 5.6 μM. Flourescein labeled hemi-RNA was at 2 nM. (b) Hemi-RNA/L7Ae binding curve. (K_d for three experiments = 12 ± 5 nM). (c) Hemi-RNA-L7Ae/mNop5p-fibrillarin gel mobility shift. Lane 1 was without mNop5p/fibrillarin. Nop/Fib concentrations for lanes 2–15 ranged from 488 pM to 4 μM. Secondary shifts were observed at very high mNop5p concentrations (lane 15) due to higher order complex formation/aggregation. Fluorescein labeled hemi-RNA was at 2 nM. (d) Hemi-RNA-L7Ae/mNop5p-fibrillarin binding curve. (K_d for three experiments = 33 ± 6 nM).

Figure 5

(a) Maximum tritium incorporated versus SAM concentration for wild-type box C/D complex. (Apparent K_d = 22 μM). (b) The activity of the hemi-complex exhibited a strong salt dependence. The salt dependence was explored by increasing the KCl concentration incrementally from 0.2 to 2.5 M and observing the incorporation of tritiated S-adenosylmethionine over time. The magnesium concentration was maintained at 100 mM for all reactions. (c) The hemi-complex showed peak activity at ∼75°C. (d) Non Watson–Crick pairs at positions 3–5 within the target-antisense duplex had pronounced effects on the maximal activity observed for the hemi-complex. Maximum methyl incorporation is shown as fraction of the wild-type value.

Figure 6

Tritium incorporation with various deoxy containing RNA targets under varying conditions. (a) A comparison between complexes set up with the hemi-RNA, L7Ae, mNop5p and fibrillarin (Black) and complexes set up with the hemi-RNA, L7Ae, Full Nop5p and fibrillarin (White). Both reactions were conducted at 2 M KCl, 100 mM MgCl₂. Restoration of coiled coil domain in Nop5p does not impart specificity in half RNA complexes. (b) A comparison between complexes set up with the full RNA, L7Ae, Full Nop5p and fibrillarin (Black) at 2.0 M KCl, 100 mM MgCl₂ and complexes set up with the full RNA, L7Ae, Full Nop5p and fibrillarin (White) at 0.2 M KCl, 10 mM MgCl₂. Increased KCl and MgCl₂ concentration does not significantly alter specificity. (c) A comparison between complexes set up with the full RNA, L7Ae, mNop5p and fibrillarin (Black) and complexes set up with the full RNA, L7Ae, Full Nop5p and fibrillarin (White). Both reactions were conducted at 2 M KCl, 100 mM MgCl₂. Restoration of Full RNA imparts significant specificity in both full and mNop5p complexes.

Figure 7

A comparison between complexes set up with the full RNA, L7Ae, full Nop5p and fibrillarin (Black) and complexes set up with the 2B1T RNA, L7Ae, Full Nop5p and fibrillarin (White). Both reactions were conducted at 0.2 M KCl, 10 mM MgCl₂. Target RNAs used were either unaltered ‘wild-type’ (WT), or contained single 2′-hydroxyl deoxy substitutions at positions 3–7 upstream from the D box motif (designated d3–d7). Wild-type specificity is achieved with an asymmetric RNA containing a C′/D′ motif but devoid of a D′ directed targeting sequence.