Introns as Mobile Genetic Elements

Lambowitz, Alan M.; Belfort, Marlene

doi:10.1146/annurev.bi.62.070193.003103

Cited by 599 publications

(431 citation statements)

References 2 publications

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“…This suggests that an efficient first-step proofreading mechanism may be unnecessary in nature; indeed, our results directly show that first-step reversibility does not contribute to the fidelity of 5Ј-splice site selection (6). We agree with the previous suggestion (6) that first-step reversibility likely evolved to enable retrotransposition (40,41), and not for any proofreading function. In contrast to the irreversibility of the first step for the mis-spliced RNAs, our data directly demonstrate the second proofreading mechanism for improper 5=-splice site selection (Figure 4).…”

Section: Assaying Mis-spliced Rnas For the Reverse Of The First Step supporting

confidence: 92%

Experimental Tests of Two Proofreading Mechanisms for 5′-Splice Site Selection

Wang

Silverman

2006

ACS Chem. Biol.

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Self-splicing group II intron RNAs catalyze a two-step process in which the intron is excised as a lariat by two successive phosphodiester exchange reactions. The reversibility of the first step has been hypothesized to act as a proofreading mechanism for improper 5=-splice site selection. However, without synthetic access to mis-spliced RNAs, this hypothesis could not be tested. Here, we used a deoxyribozyme to synthesize several branched RNAs that are derived from the ai5␥ group II intron and mis-spliced at the 5=-splice site. Unlike the correctly spliced ai5␥ RNAs, the mis-spliced RNAs are observed not to undergo the reverse of the first step. This is well-controlled negative evidence against the hypothesis that first-step reversibility is a proofreading mechanism for 5=-splice site selection. In a reaction equivalent either to the hydrolytic first step of splicing or to the hydrolytic reverse of the second step of splicing, a mis-spliced 5=-exon can be "trimmed" to its proper length by the corresponding mis-spliced intron, and in one case, the trimmed 5=-exon was observed to proceed correctly through the second step of splicing. These findings are the first direct evidence that this second proofreading mechanism can occur with a group II intron RNA that is misspliced at the 5=-splice site. On the basis of the likely structural and evolutionary relationship between group II introns and the spliceosome, we suggest that this second proofreading mechanism may be operative in the spliceosome.T he RNA self-splicing pathway catalyzed by group II intron RNAs (1-5) has two reaction steps ( Figure 1, panel a). In the first step, an internal adenosine 2=-hydroxyl group from domain 6 attacks the 5=-splice site phosphodiester linkage, forming a lariat-3=-exon intermediate with departure of the 5=-exon. In the second step, the 5=-exon attacks the 3=-splice site, forming the ligated exons and excising the intron as a lariat. Although the first step of splicing is reversible (6), the underlying explanation for this reversibility is unclear. The lack of natural 5=-splice site mis-splicing (7-9) suggests that a proofreading mechanism may exist. One specific hypothesis is that first-step reversibility is itself a proofreading mechanism: any intron that mis-splices at the first step by choosing an improper 5=-splice site can return to the initial unspliced state by the reverse of the first step (6). This would provide the RNA another opportunity to splice correctly, rather than waste the RNA molecule in a dead-end route or lead to improperly spliced exons after subsequently proceeding through the second step of splicing. This first proofreading mechanism requires that mis-spliced introns which have been formed by use of the improper 5=-splice site during the first step will be competent to proceed through the reverse of the first step. However, this mechanism has never been tested experimentally, because the required mis-spliced RNAs could not be synthesized using previously available methods.Because a mis-spliced 5=-e...

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Section: Assaying Mis-spliced Rnas For the Reverse Of The First Step supporting

confidence: 92%

Experimental Tests of Two Proofreading Mechanisms for 5′-Splice Site Selection

Wang

Silverman

2006

ACS Chem. Biol.

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“…Proteins containing LAGLIDADG motifs are derived from a family of DNA endonucleases, whose ORFs appear to have invaded group I introns on multiple occasions (22)(23)(24). A subset of LA-GLIDADG proteins additionally function as RNA maturases; this RNA binding and splicing facilitation activity is thought to have evolved from the existing DNA-binding function (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…A subset of LA-GLIDADG proteins additionally function as RNA maturases; this RNA binding and splicing facilitation activity is thought to have evolved from the existing DNA-binding function (Fig. 6A) (16,22,39). The maturase domain (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The maturase is a member of a large family of DNA endonucleases identifiable, in part, by conserved LAGLIDADG motifs (20). The presence of the bI3 maturase ORF within the intron probably represents an early colonization event by a LAGLI-DADG DNA endonuclease precursor (21)(22)(23)(24). The RNAdirected maturase, or splicing facilitation, activity is likely a subsequent innovation.…”

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confidence: 99%

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Recruitment of intron-encoded and co-opted proteins in splicing of the bI3 group I intron RNA

Bassi

Oliveira

White

et al. 2002

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

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Detectable splicing by the Saccharomyces cerevisiae mitochondrial bI3 group I intron RNA in vitro is shown to require both an intron-encoded protein, the bI3 maturase, and the nuclearencoded protein, Mrs1. Both proteins bind independently to the bI3 RNA. The bI3 maturase binds as a monomer, whereas Mrs1 is a dimer in solution that assembles as two dimers, cooperatively, on the RNA. The active six-subunit complex has a molecular mass of 420 kDa, splices with a k cat of 0.3 min ؊1 , and binds the guanosine nucleophile with an affinity comparable to other group I introns. The functional bI3 maturase domain is translated from within the RNA that encodes the intron, has evolved a high-affinity RNAbinding activity, and is a member of the LAGLIDADG family of DNA endonucleases, but appears to have lost DNA cleavage activity. Mrs1 is a divergent member of the RNase H fold superfamily of dimeric DNA junction-resolving enzymes that also appears to have lost its nuclease activity and now functions as a tetramer in RNA binding. Thus, the bI3 ribonucleoprotein is the product of a process in which a once-catalytically active RNA now obligatorily requires two facilitating protein cofactors, both of which are compromised in their original functions. M any small ribozymes, both those that occur naturally (1) and those that are products of in vitro selection experiments (2), display an impressive array of catalytic activities. Although these simple structures are capable of performing catalysis, most cellular ribozymes have undergone a process of elaboration in which potentially simple RNA active sites are bolstered by additional RNA structural domains (3, 4) or protein cofactors (5-9).One example of this process is the group I introns. The group I intron RNA core consists of two extended and roughly coaxially stacked helices that form a catalytic cleft, which binds, in turn, the 5Ј and 3Ј splice site helices and the guanosine cofactor (guanosine 5Ј-monophosphate, pG) (4, 5, 10). Although the catalytic core is relatively compact and can independently form a functional active site (11), most group I introns have acquired additional peripheral structures that function to stabilize the RNA core (4, 12, 13). Moreover, whereas a large number of group I introns have been identified by sequence and structural analysis (4, 14), both anecdotal and experimental evidence suggests that many have lost their self-splicing activity (15). It appears that group I intron RNAs commonly recruit protein cofactors and now function as obligatory ribonucleoproteins (8, 16).The Saccharomyces cerevisiae mitochondrial cytochrome b bI3 group I intron represents an especially intriguing case of elaboration by accretion of peripheral RNA domains and protein cofactors. First, the bI3 RNA potentially spans two group I intron subgroups, IA2 and IB4, characterized by RNA sequence insertions between helices P7 and P3 and adjacent to P9 and by an extension of the P5 helix, respectively (4) (Fig. 1A, boxed structures). The bI3 intron also contains two tertiary inter...

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“…Many of the group I introns contain an open reading frame (ORF) encoding a protein proposed to be involved either in intron splicing as an RNA maturase, or in intron mobility as a highly specific DNA endonuclease (1). The translation of the intronic ORFs is generally achieved in continuity with the upstream exon or initiated at an intronic initiation codon (2).…”

Section: Introductionmentioning

confidence: 99%

Thein vivouse of alternate 3′-splice sites in group I introns

Sellem

Belcour

1994

Nucl Acids Res

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(5,6) and in vitro experiments (7), to depend upon either one or both of two long distance pairings involving nucleotides immediately preceeding -P9-0-(8, 9, 10) and following -PlO-(6, 11) the invariable G terminal nucleotide residue of the group I introns. The alternative selection of an internal 3'-splice site, concerning only a minority of the primary transcripts, would bring the discontinous ORF in frame with the upstream exon and thus ensure a low level translation of the intron encoded protein. Even though the presence of alternate P9-0 and Pl0 signals at the begining of several group I introns free standing ORFs has allowed precise prediction of alternative splicing events (12), it remained to be shown that they do operate in vivo.For this purpose, we have employed Polymerase Chain Reaction (PCR) analysis to search for molecules resulting from

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Introns as Mobile Genetic Elements

Cited by 599 publications

References 2 publications

Experimental Tests of Two Proofreading Mechanisms for 5′-Splice Site Selection

Experimental Tests of Two Proofreading Mechanisms for 5′-Splice Site Selection

Recruitment of intron-encoded and co-opted proteins in splicing of the bI3 group I intron RNA

Thein vivouse of alternate 3′-splice sites in group I introns

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