Protein-free small nuclear RNAs catalyze a two-step splicing reaction

Valadkhan, Saba; Mohammadi, Afshin; Jaladat, Yasaman; Geisler, Sarah

doi:10.1073/pnas.0902020106

Cited by 62 publications

(69 citation statements)

References 39 publications

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“…The same can be said of the spliceosome (Will and Lührmann 2010). Although a detectable level of catalysis of an isolated step of RNA splicing can be achieved with pure snRNAs (Valadkhan et al 2009), the efficient and regulated splicing of an entire genome's collection of primary transcripts requires the collaboration of almost 200 proteins and five snRNAs in the modern spliceosome. Telomerase represents another paradigm, as it includes a canonical protein enzyme (TERT) that operates in intimate collaboration with RNA (Blackburn and Collins 2010)-so it appears to derive from more recent evolution, after protein enzymes and DNA chromosomes were well established.…”

Section: The Contemporary Rna Worldmentioning

confidence: 99%

The RNA Worlds in Context

Cech

2011

Cold Spring Harbor Perspectives in Biology

188

136

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SUMMARYThere are two RNAworlds. The first is the primordial RNAworld, a hypothetical era when RNA served as both information and function, both genotype and phenotype. The second RNA world is that of today's biological systems, where RNA plays active roles in catalyzing biochemical reactions, in translating mRNA into proteins, in regulating gene expression, and in the constant battle between infectious agents trying to subvert host defense systems and host cells protecting themselves from infection. This second RNA world is not at all hypothetical, and although we do not have all the answers about how it works, we have the tools to continue our interrogation of this world and refine our understanding. The fun comes when we try to use our secure knowledge of the modern RNAworld to infer what the primordial RNAworld might have looked like.

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Section: The Contemporary Rna Worldmentioning

confidence: 99%

The RNA Worlds in Context

Cech

2011

Cold Spring Harbor Perspectives in Biology

188

136

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“…1B) that led to the formation of a linear RNA product ( Fig. 1B; Valadkhan et al 2009). In the first step of this reaction, an internal phosphodiester bond in the Exon1 substrate is cleaved in a U6/U2-catalyzed hydrolysis reaction.…”

Section: Introductionmentioning

confidence: 99%

Metal binding and substrate positioning by evolutionarily invariant U6 sequences in catalytically active protein-free snRNAs

Lee¹,

Jaladat²,

Mohammadi³

et al. 2010

RNA

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We have previously shown that a base-paired complex formed by two of the spliceosomal RNA components, U6 and U2 small nuclear RNAs (snRNAs), can catalyze a two-step splicing reaction that depended on an evolutionarily invariant region in U6, the ACAGAGA box. Here we further analyze this RNA-catalyzed reaction and show that while the 59 and 39 splice site substrates are juxtaposed and positioned near the ACAGAGA sequence in U6, the role of the snRNAs in the reaction is beyond mere juxtaposition of the substrates and likely involves the formation of a sophisticated active site. Interestingly, the snRNA-catalyzed reaction is metal dependent, as is the case with other known splicing RNA enzymes, and terbium(III) cleavage reactions indicate metal binding by the U6/U2 complex within the evolutionarily conserved regions of U6. The above results, combined with the structural similarities between U6 and catalytically critical domains in group II self-splicing introns, suggest that the base-paired complex of U6 and U2 snRNAs is a vestigial ribozyme and a likely descendant of a group II-like self-splicing intron.

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“…The relevance of using engineered, protein-free RNA systems to study splicing has been recently argued (5,6). In a recent issue of PNAS, Valadkhan et al (7) demonstrated that an RNA complex derived from the spliceosome can perform a reaction that resembles splicing (Fig. 1B).…”

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confidence: 99%

The spliceosome as ribozyme hypothesis takes a second step

Butcher

2009

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

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T he spliceosome is a massive assembly of 5 RNAs and many proteins that, together, catalyze precursor-mRNA (pre-mRNA) splicing. The splicing mechanism involves 2 steps: (i) cleavage of the 5Ј exon-intron phosphodiester bond and formation of a new 2Ј-5Ј bond resulting in an intron ''lariat,'' and (ii) exon ligation (Fig. 1A). This 2-step phosphoryl transfer mechanism is suspiciously identical to the reaction catalyzed by the group II self-splicing introns, which are ribozymes. The precedent for a ribozymecatalyzed splicing mechanism, combined with the fact that the spliceosome has an RNA core that has been highly conserved for Ͼ1 billion years, led researchers to hypothesize that the spliceosome may use an RNA-based catalytic mechanism (1). However, the spliceosome as ribozyme hypothesis has been exceedingly difficult to prove, for 2 major reasons. First, the spliceosome contains many proteins that are essential for splicing (2). Second, the uncatalyzed rate of RNA ligation in vitro is significant when measured over many hours (3). This latter point makes it difficult to establish whether an inefficient reaction is actually caused by a catalytic rate enhancement or occurs spontaneously because of template-driven proximity. This is a particular concern for 1-step phosphoryl transfer reactions. Furthermore, it is possible to serendipitously obtain unrelated activities from RNAs, perhaps because a significant region of RNA conformational space retains some degree of catalytic activity (4). Therefore, it is important to be very cautious when interpreting an inefficient RNA-based reaction. The relevance of using engineered, protein-free RNA systems to study splicing has been recently argued (5, 6). In a recent issue of PNAS, Valadkhan et al. (7) demonstrated that an RNA complex derived from the spliceosome can perform a reaction that resembles splicing (Fig. 1B). This finding is significant because the reaction appears to be identical to the second step of splicing and is Ϸ10-fold more efficient than previous studies that used similar ).An interesting aspect of this new reaction is that the first step seems to be generated via hydrolysis rather than by a 2Ј-5Ј branching reaction (Fig. 1B). This finding is different from spliceosomecatalyzed pre-mRNA splicing (Fig. 1 A), but has precedence among the group II ribozmes (11). The second step of the reaction, however, results in exon ligation and formation of a new 5Ј-3Ј bond, just as in splicing. This reaction also has sequence requirements that closely parallel those of the spliceosome and requires magnesium. These data support, but do not prove, the idea that the reaction is related to pre-mRNA splicing. In future experiments, it will be important to investigate this reaction further for its mechanistic parallels to splicing. Currently, it is not clear how the substrate RNAs are coordinated in the reaction, and whether or not the system faithfully recapitulates the interactions in the spliceosome remains to be determined. Another important question concerns how the cata...

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Protein-free small nuclear RNAs catalyze a two-step splicing reaction

Cited by 62 publications

References 39 publications

The RNA Worlds in Context

The RNA Worlds in Context

Metal binding and substrate positioning by evolutionarily invariant U6 sequences in catalytically active protein-free snRNAs

The spliceosome as ribozyme hypothesis takes a second step

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