In vitro RNP assembly and methylation guide activity of an unusual box C/D RNA, cis-acting archaeal pre-tRNA(Trp)

Abstract

Among the large family of C/D methylation guide RNAs, the intron of euryarchaeal pre-tRNA(Trp) represents an outstanding specimen able to guide in cis, instead of in trans, two 2'-O-methylations in the pre-tRNA exons. Remarkably, both sites of methylation involve nucleotides within the bulge-helix-bulge (BHB) splicing motif, while the RNA-guided methylation and pre-tRNA splicing events depend on mutually exclusive RNA folding patterns. Using the three recombinant core proteins of archaeal C/D RNPs, we have analyzed in vitro RNP assembly of the pre-tRNA and tested its site-specific methylation activity. Recognition by L7Ae of hallmark K-turns at the C/D and C'/D' motifs appears as a crucial assembly step required for subsequent binding of a Nop5p-aFib heterodimer at each site. Unexpectedly, however, even without L7Ae but at a higher concentration of Nop5p-aFib, a substantially active RNP complex can still form, possibly reflecting the higher propensity of the cis-acting system to form guide RNA duplex(es) relative to classical trans- acting C/D RNA guides. Moreover, footprinting data of RNPs, consistent with Nop5p interacting with the non-canonical stem of the K-turn, suggest that binding of Nop5p-aFib to the pre-tRNA-L7Ae complex might direct transition from a splicing-competent structure to an RNA conformer displaying the guide RNA duplexes required for site-specific methylation.

Figure 1

(A) Predicted K-turns motifs formed at the C/D and C′/D′ boxes of the pre-tRNA^Trp intron in H.volcanii and P.abyssi (left and right, respectively). The consensus K-turn structural motif derived for C/D RNAs in Eukarya and Archaea (boxed, middle part) harbors a canonical stem I, a non-canonical stem II extending from tandem sheared GA pairs, and an internal asymmetric loop of three nucleotides (20,25). (B) Structure of the two large intronic deletion mutants, Hv Del1 and Hv Del2, and of the exonic deletion Hv ExDel. The intact H.volcanii pre-tRNA^Trp structure is schematized (top line) with indication of sequence coordinates and of major sequence features (5′AE and 3′AE: 5′ and 3′ antisense elements, in blue and red, respectively).

Figure 2

Binding of L7Ae to archaeal pre-tRNA^Trp in vitro assayed by gel mobility shift. The ³²P-labeled RNA was incubated in the presence of increasing concentrations of the P.abyssi protein. Dissociation constants (shown below) for the complex, termed RNP1, were determined by measuring the fraction bound with respect to the total amount of protein. The number of binding sites, n, was determined from the slope of the Hill plot analysis in which the log[fraction bound/fraction non-bound] was plotted versus the log[protein]. K_d and n values were derived from a minimum of three independent experiments. (A) Haloferax volcanii pre-tRNA^Trp. Binding curve fitted to a sigmoidal binding curve for the wild-type RNA (left). Binding was studied for deletion mutants lacking box D, box D′, boxes C′ and D′, or the 5′ and 3′ regions of the 5′ and 3′ tRNA^Trp exons, respectively (right). (B) Pyrococcus abyssi pre-tRNA^Trp. Binding curve fitted to a binding isotherm for the wild-type RNA (left). Bindings were also studied for four different box mutants (right).

Figure 3

(A) Formation of higher order RNP complexes with H.volcanii pre-tRNA^Trp in the presence of the P.abyssi L7Ae concentration sufficient to shift >95% of the RNA into the RNP1 complex, together with increasing concentrations of Nop5p and/or aFib. The Nop5p–L7Ae–pre-tRNA^Trp and aFib–Nop5p–L7Ae–pre-tRNA^Trp complexes are termed RNP2 and RNP3, respectively. The numbers of binding sites were determined as in Figure 2. (B) Same as in (A), but with P.abyssi pre-tRNA^Trp. (C) Higher order complexes of H.volcanii pre-tRNA^Trp formed with Nop5p and aFib in the absence of L7Ae. The Nop5p–pre-tRNA^Trp and aFib–Nop5p–pre-tRNA^Trp complexes are termed RNP2* and RNP3*, respectively.

Figure 4

Footprinting analysis of the L7Ae–pre-tRNA^Trp and aFib–Nop5p–L7Ae–pre-tRNA^Trp complexes using lead acetate as structural probe. (A) Mapping of the lead acetate cleavage sites along H.volcanii pre-tRNA^Trp using a 5′- or 3′-end-labeled transcript (left and right, respectively). After pre-incubation of the transcript at 70°C and formation of the complex as in Figure 3 (see Materials and Methods), lead acetate was used at two different concentrations: 4 mM (+) and 12 mM (++). An RNase T1 ladder and an alkaline hydrolysis pattern (T1 and OH-, respectively) as well as a control experiment (C) without lead acetate were analyzed in parallel. M, molecular weight markers (pBR322 digested with HaeIII and TaqI). The location of major sequence features in the pre-tRNA^Trp is indicated along the gel (5′AE and 3′AE: 5′ and 3′ antisense elements, in blue and red, respectively; 5AE and 3AE: sequences matched by the 5′AE and 3′AE, in blue and red respectively). (B and C) Schematic representation of the lead acetate probing data obtained for H.volcanii and P.abyssi pre-tRNA^Trp, respectively. For each species, the pre-tRNA splicing-competent structure is represented on the left, while an alternative secondary structure involving formation of cis-acting RNA duplex(es) guiding a 2′-O-methylation is schematized on the right. For H.volcanii, the two guide RNA duplexes which might form concomitantly are shown on two separate structures (on top of each other) for clarity, while a single guide duplex involving the C′/D′ motif is shown for P.abyssi pre-tRNA. Strong and weak cleavages are denoted by bars and dots, respectively (data were confirmed by a minimum of three independent experiments). Phosphate backbone positions accessible in the naked pre-tRNA^Trp and protected from cleavage in the RNP1 or RNP3 complexes are indicated by one (O) and three circles (OOO), respectively. Three circles accompanied by an arrow (→OOO) indicate new cleavages appearing in the RNP3 complex only.

Figure 5

In vitro 2′-O-methylation activity of the reconstituted RNP complex (RNP3). (A) Kinetics of labeled methyl incorporation into the H.volcanii (circles) or P.abyssi (squares) pre-tRNA^Trp transcripts. (B) Characterization of the 2′-O-methylation sites by 2D-TLC of an RNase T2 digest of in vitro synthesized H.volcanii pre-tRNA^Trp. The transcript was labeled by incorporation of [α-³²P]UTP and [α-³²P]CTP (top and bottom, respectively) in the absence and presence (left and right panels, respectively) of the three recombinant proteins supplemented with SAM. Chromatographic separation was performed with system B. (C) As in (B) using in vitro synthesized P.abyssi pre-tRNA^Trp. The transcript was labeled by incorporation of [α-³²P]CTP and [α-³²P]ATP (top and bottom, respectively) in the absence and presence (left and right panels, respectively) of the three recombinant proteins supplemented with SAM.

Figure 6

In vitro 2′-O-methylation activity of the reconstituted aFib–Nop5p–H.volcanii pre-tRNA^Trp complex (or RNP3* complex). The pre-tRNA^Trp transcript was labeled by incorporation of [α-³²P]CTP in the absence and presence (left and right panels, respectively) of the two recombinant proteins, aFib and aNop5p, supplemented with SAM, and the sites of 2′-O-methylation were identified by 2D-TLC of an RNase T2 digest of the RNA, as in Figure 5B.