Spliceosome Catalysis Research Articles

Crystal structure of Prp16 in complex with ADP.

DEAH-box helicases play a crucial role in pre-mRNA splicing as they are responsible for major rearrangements of the spliceosome and are involved in various quality-ensuring steps. Prp16 is the driving force during spliceosomal catalysis, remodeling the C state into the C* state. Here, the first crystal structure of Prp16 from Chaetomium thermophilum in complex with ADP is reported at 1.9 Å resolution. Comparison with the other spliceosomal DEAH-box helicases Prp2, Prp22 and Prp43 reveals an overall identical domain architecture. The β-hairpin, which is a structural element of the RecA2 domain, exhibits a unique position, punctuating its flexibility. Analysis of cryo-EM models of spliceosomal complexes containing Prp16 reveals that these models show Prp16 in its nucleotide-free state, rendering the model presented here the first structure of Prp16 in complex with a nucleotide.

Saccharomyces cerevisiae Ecm2 Modulates the Catalytic Steps of pre-mRNA Splicing.

Genetic, biochemical, and structural studies have elucidated the molecular basis for spliceosome catalysis. Splicing is RNA catalyzed and the essential snRNA and protein factors are well-conserved. However, little is known about how nonessential components of the spliceosome contribute to the reaction and modulate the activities of the fundamental core machinery. Ecm2 is a nonessential yeast splicing factor that is a member of the Prp19-related complex of proteins. Cryo-electron microscopy (cryo-EM) structures have revealed that Ecm2 binds the U6 snRNA and is entangled with Cwc2, a factor previously found to promote a catalytically active conformation of the spliceosome. These structures also indicate that Ecm2 and the U2 snRNA likely form a transient interaction during 5′ splice site (SS) cleavage. We have characterized genetic interactions between ECM2 and alleles of splicing factors that alter the catalytic steps in splicing. In addition, we have studied how loss of ECM2 impacts splicing of pre-mRNAs containing nonconsensus or competing SS. Our results show that ECM2 functions during the catalytic stages of splicing. Our data are consistent with Ecm2 facilitating the formation and stabilization of the first-step catalytic site, promoting second-step catalysis, and permitting alternate 5′ SS usage. We propose that Cwc2 and Ecm2 can each fine-tune the spliceosome active site in unique ways. Their interaction network may act as a conduit through which splicing of certain pre-mRNAs, such as those containing weak or alternate splice sites, can be regulated.

Ecm2 Functions to Support U6 snRNA Structure and Dynamics during Spliceosome Catalysis

Spliceosomes catalyze the removal of non‐coding regions, or introns, from pre‐mRNA. Assembly of the small nuclear RNA (snRNA) core of spliceosomes occurs anew on each intron via a series of carefully coordinated conformational rearrangements. However, the functional importance of many of the snRNA‐protein interactions guiding these rearrangements remain unknown. The splicing factor Ecm2 integrates into the spliceosome adjacent to the active site, where it interacts near a region of the U6 snRNA forming the catalytic RNA core of the spliceosome. By combining mutations in the U6 snRNA that perturb splicing catalysis with Ecm2 deletion in yeast, we have determined that Ecm2 functions during the chemical steps of splicing. While Ecm2 deletion or U6 mutations can be tolerated by yeast individually, we demonstrate strong synthetic interactions between these factors. We have further characterized the impact of Ecm2 deletion on splicing in vivo using a reporter pre‐mRNA. Neither Ecm2 deletion nor the U6 mutations impact splicing of pre‐mRNAs containing strong splice sites. However, both Ecm2 deletion and U6 mutation change splicing when the pre‐mRNAs contain weak, non‐consensus splice sites. Moreover, many changes observed with U6 and Ecm2 are dependent on both factors. For example, Ecm2 deletion promotes splicing of pre‐mRNAs containing a substitution at the +5 position of the 5'SS but mutation of U6 eliminates this effect. We propose that Ecm2 stabilizes U6 conformation so that it may re‐arrange to support the catalytic steps of splicing.

The use of simple model systems to study spliceosomal catalysis

Since direct analysis of many aspects of spliceosomal function is greatly hindered by the daunting complexity of the spliceosome, the development of functionally validated simple model systems can be of great value. The critical role played by a base-paired complex of U6 and U2 snRNAs in splicing in vivo suggests that this complex could be a suitable starting point for the development of such a simple model system. However, several criteria must be satisfied before such a snRNA-based in vitro system can be considered a valid model for the spliceosomal catalytic core, including similarities at the level of reaction chemistry and cationic and sequence requirements. Previous functional analyses of in vitro assembled base-paired complexes of human U2 and U6 snRNAs have been promising, providing insight into catalysis. Furthermore, they strongly suggest that with further optimization, these RNAs might indeed be able to recapitulate the function of the spliceosomal catalytic core, thus opening the door to several lines of study not previously possible.

The Receptor for Branch-Site Docking within a Group II Intron Active Site

The distinguishing feature of group II introns, and the property that links them with spliceosomal catalysis, is their ability to undergo splicing through branching. In this reaction, the 2'-hydroxyl group of a specific adenosine within intron domain 6 serves as the nucleophile for attack on the 5' splice site. We know less about branching than any other feature of group II intron catalysis, largely because the receptor structure for activating the branch site is unknown. Here, we identify the intronic region that binds the branch site of a group IIB intron. Located in domain 1, close to receptors for intron domain 5 and both splice sites, we demonstrate that the branch-site receptor is a functional element required for transesterification. Furthermore, we show that crosslinked branch sites can carry out both steps of splicing, suggesting that the conformational state of the intron core is set early and that it persists throughout the entire splicing process.

Hierarchical, clustered protein interactions with U4/U6 snRNA: a biochemical role for U4/U6 proteins.

During activation of the spliceosome, the U4/U6 snRNA duplex is dissociated, releasing U6 for subsequent base pairing with U2 snRNA. Proteins that directly bind the U4/U6 interaction domain potentially could mediate these structural changes. We thus investigated binding of the human U4/U6-specific proteins, 15.5K, 61K and the 20/60/90K protein complex, to U4/U6 snRNA in vitro. We demonstrate that protein 15.5K is a nucleation factor for U4/U6 snRNP assembly, mediating the interaction of 61K and 20/60/90K with U4/U6 snRNA. A similar hierarchical assembly pathway is observed for the U4atac/U6atac snRNP. In addition, we show that protein 61K directly contacts the 5' portion of U4 snRNA via a novel RNA-binding domain. Furthermore, the 20/60/90K heteromer requires stem II but not stem I of the U4/U6 duplex for binding, and this interaction involves a direct contact between protein 90K and U6. This uneven clustering of the U4/U6 snRNP-specific proteins on U4/U6 snRNA is consistent with a sequential dissociation of the U4/U6 duplex prior to spliceosome catalysis.