More than one way to splice an RNA: Branching without a bulge and splicing without branching in group II introns

Chu, Vi; Liu, Qiaolian; Podar, Mircea; Perlman, Philip S.; Pyle, Anna Marie

doi:10.1017/s1355838298980724

Cited by 63 publications

(74 citation statements)

References 41 publications

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“…The construct used in this study lacks the internal loop above the branch site, but trans-branching assays show an almost wild-type like activity. These results corroborate with phylogenetic data [9,10] that indeed no crucial branch point determinants are present in the omitted nucleotides and that the length of the helix above the branch adenosine is not important for docking to the other domains and branching…”

Section: Discussionsupporting

confidence: 89%

“…In the group II intron ai5γ on the other hand, a base paired branch A does not completely abolish branching. [9] In all eukaryotic spliceosome systems investigated to date, a pseudouridine (Ψ) residue opposite of the branchpoint adenosine has been identified. [36,37] NMR structural analyses have shown that this Ψ is responsible for an extrahelical positioning of the branch adenosine in the isolated branch helix.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, we studied a truncated construct (D6-27) from the wild-type (wt) sequence of D6 of the yeast group II intron ai5γ ( Figure 1A and B). As the internal loop is neither phylogenetically conserved, nor contains important branch point determinants, [9] we removed nucleotides A859-U864 and G871-A876. D6-27 still encompasses the branch adenosine together with a minimum of three neighboring base pairs on either side, and thus metal ion binding at the branch site will not be affected by this mutation.…”

Section: Nmr Spectra Of a Minimal Branch Domainmentioning

confidence: 99%

“…[9,10] Instead, the recent crystal structure of a D56 construct shows a shifted base pairing pattern and a two-nucleotide bulge. [5] To clarify the base pairing geometry in D6 and view the structural features of the branch A we have now solved the solution structure of this region by NMR.…”

Section: Introductionmentioning

confidence: 99%

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Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine

et al. 2007

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Group II intron self-splicing is essential for correct expression of organellar genes in plants, fungi, and yeast, as well as of bacterial genes. Self-excision of these autocatalytic introns from the primary RNA transcript is achieved in a two-step mechanism apparently analogous to the one of the eukaryotic spliceosome. The 2'-OH of a conserved adenosine (the branch point) located within domain 6 (D6) acts as the nucleophile in the first step of splicing. Despite the biological importance of group II introns, little is known about their structural organization and usage of metal ions in catalysis. Here we report the first solution structure of a catalytically active D6 construct encompassing the branch point and the neighboring helical regions from the mitochondrial yeast intron ai5 . The branch adenosine is the single unpaired nucleotide, and, in contrast to the spliceosomal branch site, resides within the helix being partially stacked between the two flanking GU wobble pairs. We identified a novel prominent Mg2+ binding site in the major groove of the branch site. Importantly, Mg2+ addition does not impair stacking of the branch-adenosine, but in contrast rather strengthens the interaction with the flanking uridines, as shown by NMR-and fluorescence studies. This means, that domain 6 presents the branch adenosine in a stacked fashion to the core of group II introns upon folding to the active conformation.

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Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Nmr Spectra Of a Minimal Branch Domainmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine

et al. 2007

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“…The branch-site consensus sequence is poorly conserved among eukaryotes (Wu & Manley, 1989)+ In group II introns, not only are the base pairs flanking the bulged adenosine poorly conserved among these selfsplicing introns, but the first step of splicing apparently occurs if the branch-site adenosine is involved in a Watson-Crick base pair (Chu et al+, 1998)+ This finding may diminish the importance of a pseudouridine residue in the spliceosome that facilitates bulging of this residue+ However, there are many documented differences between branch-site recognition in group II introns and spliceosomes+ Specifically, the N6 of the branch-site adenosine is the major determinant in recognition in group II introns (Liu et al+, 1997) whereas the N1 is the most important substituent in spliceosomes (Query et al+, 1996)+ Also, a cytosine can be tolerated at the spliceosome branch site, but it is not acceptable as a branch point in group II introns+ These differences suggest a different mode of recognition for the branch site in spliceosomes and group II introns+…”

Section: Conservation Of C35mentioning

confidence: 99%

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture

Newby

Greenbaum

2001

RNA

111

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The removal of noncoding sequences (introns) from eukaryotic precursor mRNA is catalyzed by the spliceosome, a dynamic assembly involving specific and sequential RNA-RNA and RNA-protein interactions. An essential RNA-RNA pairing between the U2 small nuclear (sn)RNA and a complementary consensus sequence of the intron, called the branch site, results in positioning of the 29OH of an unpaired intron adenosine residue to initiate nucleophilic attack in the first step of splicing. To understand the structural features that facilitate recognition and chemical activity of the branch site, duplexes representing the paired U2 snRNA and intron sequences from Saccharomyces cerevisiae were examined by solution NMR spectroscopy. Oligomers were synthesized with pseudouridine (c) at a conserved site on the U2 snRNA strand (opposite an A-A dinucleotide on the intron strand, one of which forms the branch site) and with uridine, the unmodified analog. Data from NMR spectra of nonexchangeable protons demonstrated A-form helical backbone geometry and continuous base stacking throughout the unmodified molecule. Incorporation of c at the conserved position, however, was accompanied by marked deviation from helical parameters and an extrahelical orientation for the unpaired adenosine. Incorporation of c also stabilized the branch-site interaction, contributing -0.7 kcal/mol to duplex DG8 37 . These findings suggest that the presence of this conserved U2 snRNA pseudouridine induces a change in the structure and stability of the branch-site sequence, and imply that the extrahelical orientation of the branch-site adenosine may facilitate recognition of this base during splicing.

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Understanding Catalysis of Phosphate‐Transfer Reactions by the Large Ribozymes

Lönnberg

2011

Chemistry A European J

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Large ribozymes are unique among catalytic RNA molecules in that their reactions involve intermolecular nucleophilic attack on an RNA phosphodiester linkage. Crystal structures of near-atomic resolution are now available for the group I and group II self-splicing introns and the RNA subunit of RNase P. The structural data agrees well with the earlier models proposed on the basis of biochemical studies and the evidence at hand suggests that all of the large ribozymes utilize a mechanism in which coordination of Mg(II) ions reduces the negative charge on the scissile phosphodiester linkage, as well as assists both the nucleophilic attack and the departure of the leaving group.

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More than one way to splice an RNA: Branching without a bulge and splicing without branching in group II introns

Cited by 63 publications

References 41 publications

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture

Understanding Catalysis of Phosphate‐Transfer Reactions by the Large Ribozymes

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More than one way to splice an RNA: Branching without a bulge and splicing without branching in group II introns

Cited by 63 publications

References 41 publications

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg2+ Binding Site is Located Close to the Stacked Branch Adenosine

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg2+ Binding Site is Located Close to the Stacked Branch Adenosine

A conserved pseudouridine modification in eukaryotic U2 snRNA induces a change in branch-site architecture

Understanding Catalysis of Phosphate‐Transfer Reactions by the Large Ribozymes

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Product

Resources

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Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine

Solution Structure of Domain 6 from a Self‐Splicing Group II Intron Ribozyme: A Mg²⁺ Binding Site is Located Close to the Stacked Branch Adenosine