Catalytic Role of 2′-Hydroxyl Groups Within a Group II Intron Active Site

Abramovitz, Dana L; Friedman, Richard A.; Pyle, Anna Marie

doi:10.1126/science.271.5254.1410

Cited by 104 publications

(115 citation statements)

References 34 publications

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“…(37), can play important roles in potentiating RNA͞RNA and RNA͞protein interactions (38)(39)(40). For example, methyl modification has been implicated as a possible switch for controlling A͞I editing and splice site selection in brain-specific pre-mRNAs (41,42).…”

Section: Resultsmentioning

confidence: 99%

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

Omer

Ziesche²,

Ebhardt³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

176

223

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The genomes of hyperthermophilic Archaea encode dozens of methylation guide, C͞D box small RNAs that guide 2 -O-methylation of ribose to specific sites in rRNA and various tRNAs. The genes encoding the Sulfolobus homologues of eukaryotic proteins that are known to be present in C͞D box small nucleolar ribonucleoprotein (snoRNP) complexes were cloned, and the proteins (aFIB, aNOP56, and aL7a) were expressed and purified. The purified proteins along with an in vitro transcript of the Sulfolobus sR1 small RNA were reconstituted in vitro, into an RNP complex. The order of assembly of the three proteins onto the RNA was aL7a, aNOP56, and aFIB. The complex was active in targeting S-adenosyl methionine (SAM)-dependent, site-specific 2 -O-methylation of ribose to a short fragment of ribosomal RNA (rRNA) that was complementary to the D box guide region of the sR1 small RNA. The presence of aFIB was essential for methylation; mutant proteins having amino acid replacements in the SAM-binding motif of aFIB were able to assemble into an RNP complex, but the resulting complexes were defective in methylation activity. These experiments define the minimal number of components and the conditions required to achieve in vitro RNA guide-directed 2 -O-methylation of ribose in a target RNA.T he eukaryotic nucleolus is a highly specialized organelle where rRNA is transcribed, processed, folded, and assembled along with ribosomal proteins into small and large ribosomal subunits (1-5). During this process, up to a hundred or more nucleotide modifications are introduced into the ribosomal RNA (rRNA) by two distinct families of small nucleolar ribonucleoprotein (snoRNP) complexes. The snoRNAs in these RNP complexes contain short antisense guide elements that are used to target modifications to specific locations within the rRNAs. One guide family, the C͞D box snoRNPs, targets site-specific 2Ј-O-methylation of ribose (6-9), and the other guide family, the H͞ACA snoRNPs, targets site-specific conversion of uridine to pseudouridine (10).The C͞D box snoRNAs are characterized by a bipartite structure with conserved C box (RUGAUGA) and D box (CUGA) motifs near their respective 5Ј and 3Ј ends and related CЈ (UGAUGA) and DЈ (CUGA) motifs near the center of the molecule. The antisense elements are located upstream of the D or DЈ motifs and are generally 10 or more nucleotides (nt) in length. Methylation is directed to the rRNA nucleotide that participates in a Watson-Crick base pair five nucleotides upstream from the start of the D or DЈ box; this is the N plus five rule (10-12). Although the general mechanism used by these RNP complexes in mediating modification has been deduced from in vivo biochemical and genetic observations, isolation and characterization of the structure and the in vitro activity of these guide complexes have not been described.The human C͞D box snoRNAs associate with several essential proteins, including fibrillarin, NOP56, and NOP58 (paralogous proteins derived from a gene duplication event), and a 15.5-kDa protein (8,(12)...

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Section: Resultsmentioning

confidence: 99%

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

Omer

Ziesche²,

Ebhardt³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

176

223

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“…The current experimental conditions, in fact, identify two nucleotide positions within domain V of particular interest where differential DMS probing indicates novel roles in secondary or tertiary structure+ Interestingly, extensive modification of A2389 is observed under native conditions+ Modification at this position was not expected, because this nucleotide, like the rest of domain V (dV), is well conserved in group II introns and, until now, had been assumed to base pair with U2374 in secondary structure models+ The high reactivity of the N1 position of A2389 is all the more striking because its immediate neighbor on the 39 side, A2390, is fully protected in molecules with a native structure+ The latter nucleotide nevertheless becomes accessible under semi-denaturing conditions or in separate dV or dV-dVI transcripts, so that the two bases are then modified to about the same extent (Figs+ 4A, 5)+ These data suggest a potential tertiary contact involving position A2390 and imply a refinement of the dV secondary structure (see below)+ Substitution of the dV terminal loop results in a concise footprint in domain I, but no change in the rest of dV Whether or not N1 of A2390 is itself engaged in a tertiary contact, its reactivity to DMS thus constitutes a sensitive indicator of the state of the middle part of dV+ These findings are of interest, inasmuch as the small domain V is a central piece of the group II ribozyme structure, with an inordinate number of components that are already known to be involved in interactions with the rest of the molecule and/or to be essential for function (Chanfreau & Jacquier, 1994;Boulanger et al+, 1995;Peebles et al+, 1995;Abramovitz et al+, 1996)+ In the case of the dV terminal loop, the interacting partner has, in fact, been identified and, as mentioned above, consists of the z "receptor" motif in domain I (Costa & Michel, 1995)+ Because a single, rather short helix connects the GAAA dV terminal loop of intron Pl+LSU/2 to its two-nucleotide bulge, it was interesting to determine whether changing the loop sequence would have repercussions on the accessibility of A2389 and A2390+…”

Section: Differential Dms Modification Of Intron Pllsu/2 Identifies mentioning

confidence: 99%

Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V

Costa

Christian

Michel

1998

RNA

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Dimethyl sulfate modification was used to probe for tertiary structural elements in the group II intron Pl.LSU/2 from the mitochondrial pre-ribosomal RNA of the brown alga Pylaiella littoralis. Modification of the lariat form of the intron under conditions that allow both native folding and conformational homogeneity is found to be generally consistent with secondary and tertiary structural features identified previously for group II ribozymes. A comparison of chemical probing at temperatures just below and above the first melting transition illustrates the cooperative unfolding of tertiary structure and identifies novel candidates for tertiary interactions in addition to defining elements of secondary structure. Substitution of the GAAA terminal loop of domain V is shown to be compatible with retention of conformational homogeneity (despite the loss of an important tertiary interaction), but produces a concise methylation footprint in domain I at the site previously shown to harbor the receptor for that loop. The analysis also identified two nucleotide positions in domain V with novel secondary and potential tertiary structural roles. The proposed refinement of domain V secondary structure is supported by an expanded comparative analysis of group II sequences and bears increased resemblance to U2:U6 snRNA pairing in the spliceosome.

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“…1,2 Domain 6 (D6) thereby actively takes part in splicing as this domain contains a highly conserved bulged adenosine whose 2'-OH is the nucleophile in the first step of splicing. [3][4][5][6] We have recently solved the NMR solution structure of a minimal but active branch domain 6 from the yeast mitochondrial group II intron Sc.ai5γ ( Figure 1). 7 This so-called D6-27 construct retains all important branching determinants that lie within the branch-domain itself 8 and has been shown to actively trans-splice in vitro.…”

Section: Introductionmentioning

confidence: 99%

Determination of the Intrinsic Affinities of Multiple Site-Specific Mg²⁺Ions Coordinated to Domain 6 of a Group II Intron Ribozyme

Erat

Sigel

2007

Inorg. Chem.

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Group II introns are large metallo-ribozymes that use divalent metal ions in folding and catalysis. The 3'-terminal domain 6 contains a conserved adenosine whose 2'-OH acts as the nucleophile in the first splicing step. In the hierarchy of folding, D6 binds last into the active site. In order to investigate and understand the folding process to the catalytically active intron structure, it is important to know the individual binding affinities of Mg2+ ions to D6. We recently studied the solution structure of a 27 nucleotide long domain 6 (D6-27) from the mitochondrial yeast group II intron Sc.ai5, identifying also five Mg2+ binding sites including the one at the 5'-terminal phosphate residues. Mg2+ coordination to the 5'-terminal di-and triphosphate groups is strongest (e.g., log KA,TP = 4.55 ± 0.10) and could be evaluated here in detail for the first time. The other four binding sites within D6-27 are filled simultaneously (e.g., log KA,BR = 2.38 ± 0.06) and thus compete for the free Mg2+ ions in solution having a distinct influence on the individual affinities of the various sites. For the first time we take this competition into account to obtain the intrinsic binding constants, describing a method that is generally applicable. Our data illustrates that any RNA undergoing tertiary contacts to a second RNA needs first to be loaded evenly and specifically with metal ions in order to compensate for the repulsion between the negatively charged RNAs. ions in solution having a distinct influence on the individual affinities of the various sites.For the first time we take this competition into account to obtain the intrinsic binding constants, describing a method that is generally applicable. Our data illustrates that any RNA undergoing tertiary contacts to a second RNA needs first to be loaded evenly and specifically with metal ions in order to compensate for the repulsion between the negatively charged RNAs.3

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Catalytic Role of 2′-Hydroxyl Groups Within a Group II Intron Active Site

Cited by 104 publications

References 34 publications

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V

Determination of the Intrinsic Affinities of Multiple Site-Specific Mg²⁺Ions Coordinated to Domain 6 of a Group II Intron Ribozyme

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Catalytic Role of 2′-Hydroxyl Groups Within a Group II Intron Active Site

Cited by 104 publications

References 34 publications

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

In vitro reconstitution and activity of a C/D box methylation guide ribonucleoprotein complex

Differential chemical probing of a group II self-splicing intron identifies bases involved in tertiary interactions and supports an alternative secondary structure model of domain V

Determination of the Intrinsic Affinities of Multiple Site-Specific Mg2+Ions Coordinated to Domain 6 of a Group II Intron Ribozyme

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Determination of the Intrinsic Affinities of Multiple Site-Specific Mg²⁺Ions Coordinated to Domain 6 of a Group II Intron Ribozyme