Deletion of a conserved dinucleotide inhibits the second step of group II intron splicing

Mikheeva, Svetlana A.; Murray, Heather L.; Zhou, Hui; Turczyk, Brian M.; Jarrell, Kevin A.

doi:10.1017/s1355838200000972

Cited by 23 publications

(27 citation statements)

References 15 publications

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“…Certain revertants of the triad mutants, and some revertants with nuclear suppressor mutations regain significant splicing activity in vivo and these are interesting because they do not display second-step defects (Boulanger et al 1995;Schmidt et al 1998). Given the role of J2/3 in formation of gÀg9 and recognition of the 39-splice site, it has been suggested that J2/3 is important only in the second step of splicing (Mikheeva et al 2000), which would imply a major active-site rearrangement between the first and second steps of splicing. The fact that revertants of triad mutants do not have second step defects (Boulanger et al 1995;Schmidt et al 1998) suggests that the catalytic triplex persists through both steps of splicing, which is consistent with site-directed crosslinking studies (DeLencastre et al 2005).…”

Section: Biochemical and Genetic Studies On Function Of The Catalyticmentioning

confidence: 99%

A structural analysis of the group II intron active site and implications for the spliceosome

Keating¹,

Toor²,

Perlman³

et al. 2009

RNA

133

203

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Group II introns are self-splicing, mobile genetic elements that have fundamentally influenced the organization of terrestrial genomes. These large ribozymes remain important for gene expression in almost all forms of bacteria and eukaryotes and they are believed to share a common ancestry with the eukaryotic spliceosome that is required for processing all nuclear pre-mRNAs. The three-dimensional structure of a group IIC intron was recently determined by X-ray crystallography, making it possible to visualize the active site and the elaborate network of tertiary interactions that stabilize the molecule. Here we describe the molecular features of the active site in detail and evaluate their correspondence with prior biochemical, genetic, and phylogenetic analyses on group II introns. In addition, we evaluate the structural significance of RNA motifs within the intron core, such as the major-groove triple helix and the domain 5 bulge. Having combined what is known about the group II intron core, we then compare it with known structural features of U6 snRNA in the eukaryotic spliceosome. This analysis leads to a set of predictions for the molecular structure of the spliceosomal active site.

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Section: Biochemical and Genetic Studies On Function Of The Catalyticmentioning

confidence: 99%

A structural analysis of the group II intron active site and implications for the spliceosome

Keating¹,

Toor²,

Perlman³

et al. 2009

RNA

133

203

View full text Add to dashboard Cite

show abstract

“…The second major target of cross-linking from photoactivated nucleotides at the 59-splice site is the linker between domains 2 and 3 (J2/3), which contains two highly conserved nucleotides, A587 and G588, which are important for the catalysis of splicing (Jacquier and Michel 1990;Ho Faix 1998;Mikheeva et al 2000). Specifically, crosslinks in set b from s U4 is found to cross-link strongly to A587 and moderately to G588 (Fig.…”

Section: Mapping Of Cross-linksmentioning

confidence: 99%

“…Previous studies have shown that mutations to A587, G588, and A589 significantly impair the second step of splicing (Jacquier and Michel 1990;Mikheeva et al 2000). On the basis of these studies, J2/3 has generally been assumed to play a role only in the second step.…”

Section: Defining J2/3 As An Active-site Componentmentioning

confidence: 99%

“…Specifically, it may be valuable to consider parallels between J2/3 and the conserved ACA GAGA sequence of U6 in the spliceosome. The trinucleotide A 51 G 52 A 53 of U6 is highly conserved, like A 587 G 588 A 589 of J2/3, and plays an important role in the second step of splicing (Jacquier and Michel 1990;Madhani and Guthrie 1994;Mikheeva et al 2000). Structurally, it has been shown that A 51 in U6 cross-links to the second nucleotide of the pre-mRNA (U 2 ) near the 59-splice site (Sontheimer and Steitz 1993), and genetic analysis indicates that this interaction is specifically important during the second step of splicing (Lesser and Guthrie 1993).…”

Section: Defining J2/3 As An Active-site Componentmentioning

confidence: 99%

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Three essential and conserved regions of the group II intron are proximal to the 5′-splice site

Lencastre¹,

Pyle²

2007

RNA

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Despite the central role of group II introns in eukaryotic gene expression and their importance as biophysical and evolutionary model systems, group II intron tertiary structure is not well understood. In order to characterize the architectural organization of intron ai5g, we incorporated the photoreactive nucleotides s 4 U and s 6 dG at specific locations within the intron core and monitored the formation of cross-links in folded complexes. The resulting data reveal the locations for many of the most conserved, catalytically important regions of the intron (i.e., the J2/3 linker region, the IC1(i-ii) bulge in domain 1, the bulge of D5, and the 59-splice site), showing that all of these elements are closely colocalized. In addition, we show by nucleotide analog interference mapping (NAIM) that a specific functional group in J2/3 plays a role in first-step catalysis, which is consistent with its apparent proximity to other first-step components. These results extend our understanding of active-site architecture during the first step of group II intron self-splicing and they provide a structural basis for spliceosomal comparison.

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“…To test whether the reverse-splicing reaction by linear intron is actually related to the second step of splicing, we created an intron mutant where the consecutive nucleotides G588 and A589, located in the region linking Domains D2 and D3 of ai5g, were deleted (ai5g-DG588,DA589). It was shown previously with a full length forward-splicing construct that this double mutant is almost unable to catalyze the second step of splicing (Mikheeva et al 2000). The concept of microscopic reversibility requires that this mutation will also affect the true reversal of the second splicing step (Ricard 1978).…”

Section: Activity Of the Dg588 Da589 Mutantmentioning

confidence: 97%

The linear form of a group II intron catalyzes efficient autocatalytic reverse splicing, establishing a potential for mobility

Roitzsch

Pyle²

2009

RNA

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Self-splicing group II introns catalyze their own excision from pre-RNAs, thereby joining the flanking exons. The introns can be released in a lariat or linear form. Lariat introns have been shown to reverse the splicing reaction; in contrast, linear introns are generally believed to perform no or only poor reverse splicing. Here, we show that a linear group II intron derived from ai5g can reverse the second step of splicing with unexpectedly high efficiency and precision. Moreover, the linear intron generates dramatically more reverse-splicing product than its lariat equivalent. The finding that linear group II introns can readily undergo the critical first step of mobility by catalyzing efficient reverse splicing into complementary target molecules demonstrates their innate potential for mobility and transposition and raises the possibility that reverse splicing by linear group II introns may have played a significant role in certain forms of intron mobility and lateral gene transfer during evolution.

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Deletion of a conserved dinucleotide inhibits the second step of group II intron splicing

Cited by 23 publications

References 15 publications

A structural analysis of the group II intron active site and implications for the spliceosome

A structural analysis of the group II intron active site and implications for the spliceosome

Three essential and conserved regions of the group II intron are proximal to the 5′-splice site

The linear form of a group II intron catalyzes efficient autocatalytic reverse splicing, establishing a potential for mobility

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