RNA folding and functions of RNA helicases in ribosome biogenesis

Figures & data

Figure 1. Secondary structure of the 18S rRNA. (A) 18S rRNA with the 5’, central (C), 3’ major (3’M) and 3’ minor (3’m) domains indicated in different colours. All known and predicted snoRNA binding sites [13,19,22,23,114,120,121,129,135] are indicated by black/grey (C/D box) or red/pink (H/ACA box) lines, and modification sites are indicated by circles. In the case of the U3 snoRNA, only the hybridization sites observed in cryo-EM structures are indicated in solid lines, while potential additional hybridization sites suggested by biochemical experiments are indicated as dashed lines. The binding regions of RNA helicases, determined by CRAC, and of the Fal1 helicase cofactor Sgd1 [64,135,178,232,238] are indicated. (B) Successive folding of the 18S rRNA [23]. Already folded rRNA elements are displayed in bright colours (unfolded regions in faint colours).

Figure 2. Secondary structure of the 25S and 5.8S rRNAs. (A) 25S rRNA with domains 0 to VI indicated in different colours. All known and predicted snoRNA binding sites [114,120,121] are indicated by black/grey (C/D box) or red/pink (H/ACA box) lines, and modification sites are indicated by circles. The binding regions of RNA helicases, determined by CRAC [64,69,232,238,265], are indicated. Additionally, the binding sites of Npa1, an interaction partner of RNA helicase Dbp6 [52], are indicated. (B) Successive folding of the 25S rRNA [54,61]. Already folded rRNA elements are displayed in bright colours (unfolded regions in faint colours).

Figure 3. rRNA folding steps shaping the evolving 90S/pre-40S and pre-60S particles. 18S (A) or 5.8S, 25S, and 5S (B) (pre-) rRNA domains are colour coded and the consecutive RNA shaping events illustrated schematically (left panels) or using existing cryo-EM structures of ribosomal maturation intermediates (right (A) and middle panels (B)). Association/dissociation of RNA helicases at distinct pre-60S maturation stages is indicated using the colour codes of their potential rRNA target domains ((B), right panel). PDB codes for 90S/pre-40S structures (A) (from top to bottom): 6ZQA (state A), 6ZQB (state B), 6ZQC (state C/pre-A1), 6LQS (state D/post-A1), 6ZQE (state Dis-A), 6ZQG (state Dis-C), 4v88 (mature 40S). PDB codes for pre-60S structures (B) (from top to bottom): 6EM3 (state A), 6EM4 (state B), 6EM1 (state C), 6ELZ (state D/E), 6YLX (state NE – Nop53 early), 3JCT (state Nog2/F), 6YLG (state LN/Rix1-Rea1), 4v88 (mature 60S).

Table 1. RNA helicases in ribosome biogenesis and their presumed functions

Name	Type	Maturation step	Cofactors	Known or hypothesized function in RNA folding/unwinding
Fal1	DEAD	90S	Sgd1	unknown
Dbp8	DEAD	90S	Esf2	unknown
Dbp4/Hca4	DEAD	90S	unknown	18S rRNA 5’ domain folding; U14 release
Rrp3	DEAD	90S	unknown	unknown
Rok1	DEAD	90S	Rrp5	snR30 release from 90S particles; Rrp5 release from 90S particles
Dhr2	DEAH	90S, (early pre-40S)	unknown	unknown
Dhr1/Ecm16	DEAH	90S, early pre-40S	Utp14	promotion of A1 cleavage; release of the U3 snoRNA from the 18S rRNA, which triggers formation of the central pseudoknot
Prp43	DEAH	90S, pre-40S, pre-60S	Pfa1, Pxr1 (G-patch)	snoRNA (snR72, snR60, others) release; 18S rRNA H44 restructuring
Has1	DEAD	90S, early to late nucleolar pre-60S	unknown	U14 release; Rrp36 release; contact formation between 25S rRNA domain I and 5.8S rRNA
Mtr4	Ski2-like	90S, nucleoplasmic pre-60S	unknown	5’ETS and ITS2 unwinding and recruitment of the nuclear exosome for 3’-5’ degradation of these spacer elements
Dbp6	DEAD	(90S), early nucleolar pre-60S	unknown	unknown
Dbp7	DExD	(90S), early nucleolar pre-60S	unknown	snR190 (possibly also snR34) release
Dbp9	DEAD	early nucleolar pre-60S	unknown	unknown
Dbp3	DEAD	early nucleolar pre-60S	unknown	snoRNA release; ITS1 re-arrangement
Drs1	DEAD	early/intermediate nucleolar pre-60S	unknown	promotion of A2 cleavage; stable Nop7–Erb1–Ytm1 incorporation
Mak5	DEAD	early/intermediate nucleolar pre-60S	unknown	25S rRNA H39 restructuring
Dbp2	DEAD	early/intermediate nucleolar pre-60S	unknown	U8 snoRNA release in higher eukaryotes; re-arrangement of ITS2 hairpin in yeast; both are proposed to regulate formation of the proximal 25S(28S)/5.8S stem
Dbp10	DEAD	intermediate nucleolar pre-60S	unknown	25S rRNA H89-H92 (PTC) folding
Spb4	DEAD	late nucleolar pre-60S	unknown	25S rRNA H62/H63 restructuring

Figure 4. Cryo-EM structures of helicases Dhr1 and Has1 bound to their pre-ribosomal substrate particles. (A) Dhr1 (red) bound to 90S (left panel, PDB: 6ZQD) or pre-40S (right panel, PDB: 6ZQG) particles. The assembly factors Utp14 (cyan) and Pno1 (blue) in proximity to the Dhr1 C-terminus, as well as the U3 snoRNA (black) and 25S rRNA H1 (Orange, right panel) base-pairing with U3 box A are depicted. (B) Has1 (red) binds to pre-60S intermediates (PDB: 5Z3G) on top of the ITS2-containing foot structure close to Nsa3. The two RecA-like domains interact with H16 (purple) of 25S rRNA domain I. The Has1 25S rRNA crosslink site at H21/H22 (Orange) and further proximal assembly factors and -r-proteins are indicated.

Figure 5. Cryo-EM structures of Mtr4 on 90S and pre-60S intermediates channelling its substrate RNAs for exosomal degradation.

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