Synergistic interaction network between the snR30 RNP, Utp23, and ribosomal RNA during ribosome synthesis

Figures & data

Figure 1. Schematic structure of the snR30 RNP. This representation displays the secondary structure of snR30 comprised of the 5ʹ, internal and 3ʹ hairpins as well as the associated H/ACA proteins (Cbf5 – orange, Nop10 - blue, Gar1 – yellow, and Nhp2 - green). The snR30 RNP also interacts with the ribosome assembly factor Utp23 (purple). The m1 and m2 motifs in the 3ʹ-hairpin base-pair with expansion segment 6 (ES6) of 18S rRNA (rm1 and rm2 motifs, red). Nucleotide sequences for the H and ACA boxes as well as the m1, m2 (snR30) and rm1, and rm2 (18S rRNA) sequences are displayed.

Table 1. Summary of plasmids, primers and DNA products for in vitro transcription

Plasmid	DNA Product	Primer names	Primer sequences 5ʹ-3ʹ
pUC19-SNR30	snR30 full-length	snR30 F snR30 R	GCTAATACGACTCACTATAGGGAACCATAGTCTCGTGCTAGTTCGGTACTATACAGGG mAmGATGTCTGCAGTATGGTTTTACCCAAATGATCATGGACC
pUC19-SNR30	snR30 Δ5ʹ	snR30Δ5ʹ snR30 R	GCTAATACGACTCACTATAGGTAGGACGCATGATCTTGAGCTCTTTTCCTATACTTTG mAmGATGTCTGCAGTATGGTTTTACCCAAATGATCATGGACC
pUC19-SNR30	snR30 Δ5ʹΔIH	snR30Δ5ʹΔIH F snR30 R	GCTAATACGACTCACTATAGGGCAGTATATTCCTAAACACTATGAAAT mAmGATGTCTGCAGTATGGTTTTACCCAAATGATCATGGACC
pUC19-SNR30ΔIH	snR30 ΔIH	snR30 F snR30 R	GCTAATACGACTCACTATAGGGAACCATAGTCTCGTGCTAGTTCGGTACTATACAGGG mAmGATGTCTGCAGTATGGTTTTACCCAAATGATCATGGACC
pUC19-RDN37	C2-H22/23*	C2 F H22/23 R	TAATACGACTCACTATAGGGATTTTTTCGTGTACTGGATTTCCAACGGG GAAAACGTCCTTGGCAAATGCTTTCG
pUC19-RDN37	C2-ES6H1*	C2 F ES6H1 R	TAATACGACTCACTATAGGGATTTTTTCGTGTACTGGATTTCCAACGGG TCCTGGTTCGCCAAGAGCC
pUC19-RDN37	ES6H1-ES6H3*	ES6H1 F ES6H3 R	TAATACGACTCACTATAGGGTTCTGGCTAACCTTGAGTCCTTG mTmCATTACGATGGTCCTAGAAACCAAC
pUC19-RDN37	ES6H2-H22/23*	ES6H2 F H22/23 R	TAATACGACTCACTATAGGGTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCG GAAAACGTCCTTGGCAAATGCTTTCG
pUC19-RDN37	ES6H2-ES6H3*	ES6H2 F ES6H3 R	TAATACGACTCACTATAGGGTTACTTTGAAAAAATTAGAGTGTTCAAAGCAGGCG mTmCATTACGATGGTCCTAGAAACCAAC
pUC19-RDN37	rm1-rm2	Rm1 F Rm2 R	GCTAATACGACTCACTATAGGGCATGGAATAATAGAATAGGACGTTTGGTTC CCTAGAAACCAACAAAATAGAACCAAACGTCCTATTCTATTATTCC
pUC19-RDN37	H22/23*	H22/23 F H22/23 R	TAATACGACTCACTATAGGGTTAATAGGGACGGTCGGGGG GAAAACGTCCTTGGCAAATGCTTTCG
pUC19-RDN37	H25/26*	H25/26 F H25/26 R	TAATACGACTCACTATAGGGCCGACTAGGGATCGGGTGG ACCCAAAGACTTTGATTTCTCGTAAGGTGC

Table 2. Affinity of snR30 full-length and truncations binding to the H/ACA proteins Cbf5, Nop10, Gar1 and Nhp2. Dissociation constants (K_D) were determined by nitrocellulose filtration (Fig. 2) and are reported with the standard deviation

snR30 Variant	Dissociation constant (nM)	Amplitude (% Binding)
snR30 (full-length)	0.9 ± 0.2	95 ± 6
snR30 Δ5ʹ	0.4 ± 0.1	76 ± 4
snR30 ΔIH	0.8 ± 0.1	60 ± 2
snR30 Δ5ʹΔIH	1.4 ± 0.8	23 ± 2

Figure 2. Binding of snR30 to the H/ACA proteins Cbf5-Nop10-Gar1-Nhp2 (CNGP). Nitrocellulose filtration assays were conducted using snR30 full-length and truncations while titrating Cbf5-Nop10-Gar1-Nhp2 protein complex. Fitting to a hyperbolic equation yielded the dissociation constants and binding amplitudes (summarized in Table 2).

Table 3. Affinity of Utp23 for snR30 full-length and truncations. The dissociation constants for were measured by nitrocellulose filtration (Fig. 3) and are reported with the standard deviation

snR30 Variant	Dissociation constant (nM)	Amplitude (% Binding)
snR30 (full-length)	17.5 ± 3.2	76 ± 4
snR30 Δ5ʹ	11.0 ± 2.3	33 ± 2
snR30 ΔIH	12.8 ± 2.4	55 ± 3
snR30 Δ5ʹΔIH	8.1 ± 2.4	28 ± 1
snR81	4.7 ± 0.9	59 ± 2

Figure 3. Determining the affinity of Utp23 for snR30. Utp23 was titrated against full-length snR30 and truncations thereof as well as the modification H/ACA snoRNA snR81 as control. Hyperbolic fitting provided the dissociation constants (see Table 3).

Figure 4. 18S rRNA fragments comprising and surrounding expansion segment 6 (ES6). A Diagram of the 18S rRNA and surrounding regions indicating the various rRNA constructs used in this study. B Secondary structure of the ES6 and surrounding regions as observed in the mature 80S ribosome. C Binding of snR30 full-length complexed with Cbf5-Nop10-Gar1-Nhp2 to 18S rRNA fragments containing the rm1-rm2 motif as determined by nitrocellulose filtration. D Interaction of the full-length snR30 RNP with rRNA constructs lacking the rm1-rm2 motif as well as tRNA as control. E and F Similar nitrocellulose filtration assays using snR30 RNP reconstituted with snR30 Δ5ʹ. All dissociation constants were determined by hyperbolic fitting (smooth lines) and are summarized in Table 4.

Table 4. Affinity of the snR30 RNP complex binding to fragments of ES6 of 18S rRNA. The snRN30 RNP complex was titrated with rRNA fragments and binding was quantified by nitrocellulose filtration (Fig. 4). Dissociation constants are stated with the standard deviation

snR30 Variant	18S rRNA Fragment	Dissociation constant (nM)	Amplitude (% Binding)
snR30	C2-H22/23	84 ± 16	187 ± 11
	C2-ES6H1	405 ± 191	600 ± 140
	ES6H1-ES6H3	54 ± 19	70 ± 7
	ES6H2-ES6H3	77 ± 21	193 ± 16
	rm1-rm2	106 ± 51	48 ± 10
	ES6H2-H22/23	19 ± 10	123 ± 18
	H22/23	276 ± 72	1280 ± 170
	H25/26	225 ± 227	410 ± 190
	Mitochondrial tRNA^Met	746 ± 405	3150 ± 950
snR30 Δ5ʹ	C2-H22/23	40 ± 15	102 ± 10
	C2-ES6H1	560 ± 700	480 ± 400
	ES6H1-ES6H3	ND	ND
	ES6H2-ES6H3	32 ± 18	57 ± 8
	rm1-rm2	50 ± 11	24 ± 2
	ES6H2-H22/23	ND	ND
	H22/23	84 ± 27	430 ± 46
	H25/26	92 ± 39	640 ± 93
	Mitochondrial tRNA^Met	NC	NC

ND means the affinity was not measured. NC means the affinity could not be determined.

Table 5. Affinity of Utp23 binding to 18S rRNA fragments. The dissociation constants and binding amplitudes were determined by nitrocellulose filtration (Fig. 5) and are summarized together with the standard deviation

18S rRNA Fragment	Dissociation constant (nM)	Amplitude (% Binding)
C2-H22/23	2.2 ± 0.8	32 ± 1
ES6H2-ES6H3	4.5 ± 0.7	62 ± 2
rm1-rm2	6.2 ± 1.6	38 ± 2
H22/23	8.8 ± 1.8	72 ± 4
mitochondrial tRNA^Met*	<2 ± 1	36 ± 2
snR34 5ʹ wt sub*	<2 ± 1	32 ± 1

*Dissociation constant is lower than the RNA concentration in the reaction.

Figure 5. Nitrocellulose filtration assays to quantify binding of Utp23 to 18S rRNA fragments. A Utp23 binding to 18S rRNA fragments harbouring the rm1-rm2 motif. B Binding curves for Utp23 interaction with 18S rRNA which lacks the rm1-rm2 motif as well as two control RNAs.

Figure 6. rRNA binding by snR30 RNP in presence of Utp23. Nitrocellulose filtration was used to quantify binding of the snR30 RNP (5 nM) to the region of 18S rRNA comprising the ES6 and flanking helices (C2-H22/23, Fig. 4)) in the presence of 5 nM Upt23. For comparison, binding of snR30 RNP to this rRNA fragment in the absence of Utp23 is also shown (same as in Fig. 4C). Hyperbolic fitting determined that snR30 RNP binds rRNA in the presence of Utp23 with a dissociation constant of 20 ± 5 nM and an amplitude of 101 ± 6%.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.