A critical assessment of the utility of protein-free splicing systems.
TL;DR: In this article, the authors discuss the advances in both protein-free and fully spliceosomal systems that would be required to conclude that the reactions observed to be catalyzed by protein free snRNAs are related to splicing and question the reliability of snRNA-only systems as tools for mechanistic splicing research.
Abstract: U2 and U6 snRNAs form part of the catalytic spliceosome and represent strong candidates for components of its active site. Over the past decade it has become clear that these snRNAs are capable of catalyzing several different chemical reactions, leading to the widespread conclusion that the spliceosome is a ribozyme. Here, we discuss the advances in both protein-free and fully spliceosomal systems that would be required to conclude that the reactions observed to be catalyzed by protein-free snRNAs are related to splicing and question the reliability of snRNA-only systems as tools for mechanistic splicing research.
Citations
More filters
TL;DR: The extensive interplay of RNA and proteins in aligning the pre-mRNA's reactive groups, and the presence of both RNA and protein at the core of the splicing machinery, suggest that the spliceosome is an RNP enzyme, but elucidation of the precise nature of its active site awaits the generation of a high-resolution structure of its RNP core.
Abstract: Pre-mRNA splicing is catalyzed by the spliceosome, a multimegadalton ribonucleoprotein (RNP) complex comprised of five snRNPs and numerous proteins. Intricate RNA-RNA and RNP networks, which serve to align the reactive groups of the pre-mRNA for catalysis, are formed and repeatedly rearranged during spliceosome assembly and catalysis. Both the conformation and composition of the spliceosome are highly dynamic, affording the splicing machinery its accuracy and flexibility, and these remarkable dynamics are largely conserved between yeast and metazoans. Because of its dynamic and complex nature, obtaining structural information about the spliceosome represents a major challenge. Electron microscopy has revealed the general morphology of several spliceosomal complexes and their snRNP subunits, and also the spatial arrangement of some of their components. X-ray and NMR studies have provided high resolution structure information about spliceosomal proteins alone or complexed with one or more binding partners. The extensive interplay of RNA and proteins in aligning the pre-mRNA's reactive groups, and the presence of both RNA and protein at the core of the splicing machinery, suggest that the spliceosome is an RNP enzyme. However, elucidation of the precise nature of the spliceosome's active site, awaits the generation of a high-resolution structure of its RNP core.
1,436 citations
TL;DR: The recent advances and applications in SBVS are reviewed with a special focus on docking-based virtual screening and the researchers’ practical efforts in real projects are emphasized by understanding the ligand-target binding interactions as a premise.
Abstract: Structure-based virtual screening (SBVS) has been widely applied in early-stage drug discovery. From a problem-centric perspective, we reviewed the recent advances and applications in SBVS with a special focus on docking-based virtual screening. We emphasized the researchers’ practical efforts in real projects by understanding the ligand-target binding interactions as a premise. We also highlighted the recent progress in developing target-biased scoring functions by optimizing current generic scoring functions toward certain target classes, as well as in developing novel ones by means of machine learning techniques.
471 citations
TL;DR: It is shown that the U6 snRNA catalyses both of the two splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction, indicating that RNA mediates catalysis within the spliceosome.
Abstract: In nuclear pre-messenger RNA splicing, introns are excised by the spliceosome, a dynamic machine composed of both proteins and small nuclear RNAs (snRNAs) Over thirty years ago, after the discovery of self-splicing group II intron RNAs, the snRNAs were proposed to catalyse splicing However, no definitive evidence for a role of either RNA or protein in catalysis by the spliceosome has been reported so far By using metal rescue strategies in spliceosomes from budding yeast, here we show that the U6 snRNA catalyses both of the two splicing reactions by positioning divalent metals that stabilize the leaving groups during each reaction Notably, all of the U6 catalytic metal ligands we identified correspond to the ligands observed to position catalytic, divalent metals in crystal structures of a group II intron RNA These findings indicate that group II introns and the spliceosome share common catalytic mechanisms and probably common evolutionary origins Our results demonstrate that RNA mediates catalysis within the spliceosome
285 citations
TL;DR: In this article, the authors investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo, and found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery.
Abstract: Chronic exposure to drugs of abuse or stress regulates transcription factors, chromatin-modifying enzymes and histone post-translational modifications in discrete brain regions. Given the promiscuity of the enzymes involved, it has not yet been possible to obtain direct causal evidence to implicate the regulation of transcription and consequent behavioral plasticity by chromatin remodeling that occurs at a single gene. We investigated the mechanism linking chromatin dynamics to neurobiological phenomena by applying engineered transcription factors to selectively modify chromatin at a specific mouse gene in vivo. We found that histone methylation or acetylation at the Fosb locus in nucleus accumbens, a brain reward region, was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery. This approach allowed us to relate the epigenetic landscape at a given gene directly to regulation of its expression and to its subsequent effects on reward behavior.
176 citations
TL;DR: It is reported that both the 5' m(7)G cap and 3' poly(A) tail are essential for maximum miRNA repression and the cis- and trans-acting factors that modulate miRNA efficacy are defined.
Abstract: MicroRNAs (miRNAs) regulate gene expression post-transcriptionally through binding specific sites within the 3' untranslated regions (UTRs) of their target mRNAs. Numerous investigations have documented repressive effects of miRNAs and identified factors required for their activity. However, the precise mechanisms by which miRNAs modulate gene expression are still obscure. Here, we have examined the effects of multiple miRNAs on diverse target transcripts containing artificial or naturally occurring 3' UTRs in human cell culture. In agreement with previous studies, we report that both the 5' m(7)G cap and 3' poly(A) tail are essential for maximum miRNA repression. These cis-acting elements also conferred miRNA susceptibility to target mRNAs translating under the control of viral- and eukaryotic mRNA-derived 5' UTR structures that enable cap-independent translation. Additionally, we evaluated a role for the poly(A)-binding protein (PABP) in miRNA function utilizing multiple approaches to modulate levels of active PABP in cells. PABP expression and activity inversely correlated with the strength of miRNA silencing, in part due to antagonism of target mRNA deadenylation. Together, these findings further define the cis- and trans-acting factors that modulate miRNA efficacy.
72 citations
References
More filters
TL;DR: The number of new proteins emerging with no prior connection to splicing was surprising and it would be premature to label these proteins as bona fide splicing factors, yet many were identified multiple times in complexes purified under diverse conditions or from different organisms.
1,020 citations
TL;DR: Structural and functional analogies support the hypothesis that group II introns and the spliceosome share a common ancestor.
Abstract: Group II introns are self-splicing ribozymes that catalyze their own excision from precursor transcripts and insertion into new genetic locations. Here we report the crystal structure of an intact, self-spliced group II intron from Oceanobacillus iheyensis at 3.1 angstrom resolution. An extensive network of tertiary interactions facilitates the ordered packing of intron subdomains around a ribozyme core that includes catalytic domain V. The bulge of domain V adopts an unusual helical structure that is located adjacent to a major groove triple helix (catalytic triplex). The bulge and catalytic triplex jointly coordinate two divalent metal ions in a configuration that is consistent with a two-metal ion mechanism for catalysis. Structural and functional analogies support the hypothesis that group II introns and the spliceosome share a common ancestor.
437 citations
"A critical assessment of the utilit..." refers background in this paper
...Group II self-splicing introns provide a natural precedent for RNA catalysis of the two chemical reactions of pre-mRNA splicing, and their well-established similarities and likely evolutionary relatedness to snRNAs (Pyle 2008), especially in light of the recent group II intron crystal structure (Toor et al. 2008), provide strong support for the hypothesis that the spliceosome active site is at least par-...
[...]
...Although these core motifs, which are shared between U6 snRNA and group II introns, have recently been demonstrated to form the basis of the group II intron active site (Toor et al. 2008), similar sequences are also found in other ribozymes believed to be unrelated to snRNAs, and the full details of the role(s) of ACAGAGA and AGC in both these ribozymes and the spliceosome remain unclear....
[...]
...…which are shared between U6 snRNA and group II introns, have recently been demonstrated to form the basis of the group II intron active site (Toor et al. 2008), similar sequences are also found in other ribozymes believed to be unrelated to snRNAs, and the full details of the role(s) of…...
[...]
...…and their well-established similarities and likely evolutionary relatedness to snRNAs (Pyle 2008), especially in light of the recent group II intron crystal structure (Toor et al. 2008), provide strong support for the hypothesis that the spliceosome active site is at least partially RNA based....
[...]
TL;DR: It is shown that a protein-free complex of two snRNAs, U2 and U6, can bind and position a small RNA containing the sequence of the intron branch site, and activate the branch adenosine to attack a catalytically critical domain of U6 in a reaction that is related to the first step of splicing.
Abstract: Removal of intervening sequences from eukaryotic messenger RNA precursors is carried out by the spliceosome, a complex assembly of five small nuclear RNAs (snRNAs) and a large number of proteins. Although it has been suggested that the spliceosome might be an RNA enzyme, direct evidence for this has been lacking, and the identity of the catalytic domain of the spliceosome is unknown. Here we show that a protein-free complex of two snRNAs, U2 and U6, can bind and position a small RNA containing the sequence of the intron branch site, and activate the branch adenosine to attack a catalytically critical domain of U6 in a reaction that is related to the first step of splicing. Our data provide direct evidence for the catalytic potential of spliceosomal snRNAs.
252 citations
"A critical assessment of the utilit..." refers background in this paper
...Relatedness of a ribozyme reaction to pre-mRNA splicing has conventionally been concluded from its sensitivity to mutation of the ACAGAGA and/or AGC motifs in the U6 snRNA (or its derivative) component of the reaction (Tuschl et al. 2001; Valadkhan and Manley 2001; Valadkhan et al. 2007)....
[...]
...The ability of snRNA-derived RNA sequences to catalyze chemical reactions including 29–59 phosphodiester bond formation has previously been interpreted as strong evidence for ribozyme catalysis by the spliceosome (Tuschl et al. 2001; Valadkhan and Manley 2001; Valadkhan et al. 2007)....
[...]
TL;DR: The spliceosome is both compositionally and conformationally dynamic, allowing splice site choice to be regulated throughout both the assembly and catalytic phases of the reaction.
153 citations
"A critical assessment of the utilit..." refers background in this paper
...…and splicing in vitro: while many play important roles in the conformational rearrangements required for productive splicing (for review, see Smith et al. 2008), a growing body of evidence suggests that some spliceosomal proteins have a function beyond simple scaffolding of a catalytic RNA…...
[...]
TL;DR: It is shown that 3'-sulfur substitution at the 5' splice site of a group II intron causes a metal specificity switch during the first step of splicing, and striking parallels between the catalytic mechanisms employed by these two systems are provided.
Abstract: The identical reaction pathway executed by the spliceosome and self-splicing group II intron ribozymes has prompted the idea that both may be derived from a common molecular ancestor. The minimal sequence and structural similarities between group II introns and the spliceosomal small nuclear RNAs, however, have left this proposal in question. Mechanistic comparisons between group II self-splicing introns and the spliceosome are therefore important in determining whether these two splicing machineries may be related. Here we show that 3'-sulfur substitution at the 5' splice site of a group II intron causes a metal specificity switch during the first step of splicing. In contrast, 3'-sulfur substitution has no significant effect on the metal specificity of the second step of cis-splicing. Isolation of the second step uncovers a metal specificity switch that is masked during the cis-splicing reaction. These results demonstrate that group II intron ribozymes are metalloenzymes that use a catalytic metal ion for leaving group stabilization during both steps of self-splicing. Furthermore, because 3'-sulfur substitution of a spliceosomal intron has precisely the same effects as were observed during cis-splicing of the group II intron, these results provide striking parallels between the catalytic mechanisms employed by these two systems.
130 citations
"A critical assessment of the utilit..." refers background in this paper
...Magnesium has been directly demonstrated to be important for spliceosomal catalysis (Sontheimer et al. 1999; Gordon et al. 2000), and further investigation should clarify the coordination and precise mechanistic roles of divalent ions in the spliceosome....
[...]