Protein-free spliceosomal snRNAs catalyze a reaction that resembles the first step of splicing

Valadkhan, Saba; Mohammadi, Afshin; Wachtel, Chaim; Manley, James L.

doi:10.1261/rna.626207

Cited by 40 publications

(40 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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 RNAs (8)(9)(10)). …”

mentioning

confidence: 68%

The spliceosome as ribozyme hypothesis takes a second step

Butcher

2009

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

View full text Add to dashboard Cite

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...

show abstract

mentioning

confidence: 68%

The spliceosome as ribozyme hypothesis takes a second step

Butcher

2009

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

View full text Add to dashboard Cite

show abstract

“…These snRNAs exert crucial catalytic functions in the process [86,88,87] in three distinct splicing machineries. The major spliceosome, containing the snRNAs U1, U2, U4, U5 and U6, is the dominant form in metazoans, plants, and fungi, and removes introns with GT-AG (as well as rarely AT-AC and GC-AG) boundaries.…”

Section: Introductionmentioning

confidence: 99%

Evolution of Spliceosomal snRNA Genes in Metazoan Animals

2008

View full text Add to dashboard Cite

While studies of the evolutionary histories of protein families are common place, little is known on noncoding RNAs beyond microRNAs and some snoRNAs. Here we investigate in detail the evolutionary history of the 9 spliceosomal snRNA families (U1, U2, U4, U5, U6, U11, U12, U4atac, and U6atac) across the completely or partially sequenced genomes of metazoan animals. Representatives of the five major spliceosomal snRNAs were found in all genomes. None of the minor splicesomal snRNAs was detected in Nematodes and in the shotgun traces of Oikopleura dioica, while in all other animal genomes at most one of them is missing. Although snRNAs are present in multiple copies in most genomes, distinguishable paralog groups are not stable over long evolutionary times, although they appear independently in several clades. In general, animal snRNA secondary structures are highly conserved, albeit in particular U11 and U12 in insects exhibit dramatic variations. An analysis of genomic context of snRNAs reveals that they behave like mobile elements, exhibiting very little syntenic conservation.

show abstract

“…1B), during which an internal functional group in Oligo1 formed a covalent linkage with U6 or a substrate containing the 5Ј splice site sequence. Although these reactions resembled the first step of splicing in terms of their sequence and ionic requirements, the chemistry of the reactions was either distinct from splicing or the reaction was so inefficient that it could not be chemically characterized (21)(22)(23). Thus, the role of the spliceosomal RNAs in catalysis of the chemistry of the splicing reaction remains uncertain.…”

mentioning

confidence: 99%

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

Valadkhan

Mohammadi

Jaladat

et al. 2009

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

Self Cite

View full text Add to dashboard Cite

Pre-mRNA splicing is a crucial step in eukaryotic gene expression and is carried out by a highly complex ribonucleoprotein assembly, the spliceosome. Many fundamental aspects of spliceosomal function, including the identity of catalytic domains, remain unknown. We show that a base-paired complex of U6 and U2 small nuclear RNAs, in the absence of the Ϸ200 other spliceosomal components, performs a two-step reaction with two short RNA oligonucleotides as substrates that results in the formation of a linear RNA product containing portions of both oligonucleotides. This reaction, which is chemically identical to splicing, is dependent on and occurs in proximity of sequences known to be critical for splicing in vivo. These results prove that the complex formed by U6 and U2 RNAs is a ribozyme and can potentially carry out RNA-based catalysis in the spliceosome.catalysis ͉ ribozymes ͉ snRNAs ͉ spliceosome ͉ U6 E xtensive mechanistic and structural similarities between spliceosomal small nuclear RNAs (snRNAs) and self-splicing group II introns (1, 2) have led to the hypothesis that the snRNAs are evolutionary descendents of group II-like introns and thus could similarly have a catalytic role in the spliceosome (3-5). However, because of the extreme complexity of the spliceosome (6-9), a dynamic cellular machine composed of more than 200 different proteins in addition to the snRNAs (U1, U2, U4, U5, and U6), it has not been possible to determine whether the snRNAs can indeed catalyze the splicing reaction without the help of spliceosomal proteins.In activated spliceosomes, U6 and U2, which are the only snRNAs required for both steps of splicing, form an extensively base-paired complex (7, 10, 11) (Fig. 1A). Considerable data support the functional importance of both the secondary and tertiary structure of the U6/U2 complex (10-13). Furthermore, an evolutionarily invariant region in U6, the ACAGAGA box ( Fig. 1 A), is in close proximity to the splice sites during splicing catalysis (14-18), and mutagenesis studies have shown that this domain plays a crucial role in catalysis of the splicing reaction (7,10,11). Two other conserved regions, the AGC triad and an asymmetric bulge in the intramolecular stemloop of U6 (ISL), are also thought to play important roles in spliceosomal catalysis (11). Recent structural studies indicate that sequences equivalent to these three regions form the active site of group II introns (19). In both systems, the ACAGAGA sequence and its equivalent in group II introns seem to be positioned in close proximity to a conserved asymmetric loop in the middle of the ISL or domain V, the corresponding stemloop in group II introns (19,20).Previously we provided evidence that in vitro-transcribed, protein-free RNAs containing the conserved central domains of human U6 and U2 could form a base-paired complex in vitro that was similar to the complex formed in activated spliceosomes (13) (Fig. 1 A). In addition, this in vitro-assembled, protein-free U6/U2 complex could catalyze a number of branching-like react...

show abstract

Protein-free spliceosomal snRNAs catalyze a reaction that resembles the first step of splicing

Cited by 40 publications

References 44 publications

The spliceosome as ribozyme hypothesis takes a second step

The spliceosome as ribozyme hypothesis takes a second step

Evolution of Spliceosomal snRNA Genes in Metazoan Animals

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

Contact Info

Product

Resources

About