First Step Of Splicing Research Articles

Visualizing group II intron dynamics between the first and second steps of splicing

Group II introns are ubiquitous self-splicing ribozymes and retrotransposable elements evolutionarily and chemically related to the eukaryotic spliceosome, with potential applications as gene-editing tools. Recent biochemical and structural data have captured the intron in multiple conformations at different stages of catalysis. Here, we employ enzymatic assays, X-ray crystallography, and molecular simulations to resolve the spatiotemporal location and function of conformational changes occurring between the first and the second step of splicing. We show that the first residue of the highly-conserved catalytic triad is protonated upon 5’-splice-site scission, promoting a reversible structural rearrangement of the active site (toggling). Protonation and active site dynamics induced by the first step of splicing facilitate the progression to the second step. Our insights into the mechanism of group II intron splicing parallels functional data on the spliceosome, thus reinforcing the notion that these evolutionarily-related molecular machines share the same enzymatic strategy.

Rapid block of pre-mRNA splicing by chemical inhibition of analog-sensitive CRK9 in Trypanosoma brucei.

Trypanosoma brucei CRK9 is an essential cyclin-dependent kinase for the parasite-specific mode of pre-mRNA processing. In trypanosomes, protein coding genes are arranged in directional arrays that are transcribed polycistronically, and individual mRNAs are generated by spliced leader trans-splicing and polyadenylation, processes that are functionally linked. Since CRK9 silencing caused a decline of mRNAs, a concomitant increase of unspliced pre-mRNAs and the disappearance of the trans-splicing Y structure intermediate, CRK9 is essential for the first step of splicing. CRK9 depletion also caused a loss of phosphorylation in RPB1, the largest subunit of RNA polymerase (pol) II. Here, we established cell lines that exclusively express analog-sensitive CRK9 (CRK9AS ). Inhibition of CRK9AS in these cells by the ATP-competitive inhibitor 1-NM-PP1 reproduced the splicing defects and proved that it is the CKR9 kinase activity that is required for pre-mRNA processing. Since defective trans-splicing was detected as early as 5min after inhibitor addition, CRK9 presumably carries out reversible phosphorylation on the pre-mRNA processing machinery. Loss of RPB1 phosphorylation, however, took 12-24hr. Surprisingly, RNA pol II-mediated RNA synthesis in 24hr-treated cells was upregulated, indicating that, in contrast to other eukaryotes, RPB1 phosphorylation is not a prerequisite for transcription in trypanosomes.

Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo

ABSTRACTPre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this ‘feed-back’ effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.

Architecture and Distribution of Introns in Core Genes of Four Fusarium Species.

Removal of introns from transcribed RNA represents a crucial step during the production of mRNA in eukaryotes. Available whole-genome sequences and expressed sequence tags (ESTs) have increased our knowledge of this process and revealed various commonalities among eukaryotes. However, certain aspects of intron structure and diversity are taxon-specific, which can complicate the accuracy of in silico gene prediction methods. Using core genes, we evaluated the distribution and architecture of Fusarium circinatum spliceosomal introns, and linked these characteristics to the accuracy of the predicted gene models of the genome of this fungus. We also evaluated intron distribution and architecture in F. verticillioides, F. oxysporum, and F. graminearum, and made comparisons with F. circinatum. Results indicated that F. circinatum and the three other Fusarium species have canonical 5′ and 3′ splice sites, but with subtle differences that are apparently not shared with those of other fungal genera. The polypyrimidine tract of Fusarium introns was also found to be highly divergent among species and genes. Furthermore, the conserved adenosine nucleoside required during the first step of splicing is contained within unique branch site motifs in certain Fusarium introns. Data generated here show that introns of F. circinatum, as well as F. verticillioides, F. oxysporum, and F. graminearum, are characterized by a number of unique features such as the CTHAH and ACCAT motifs of the branch site. Incorporation of such information into genome annotation software will undoubtedly improve the accuracy of gene prediction methods used for Fusarium species and related fungi.

Yeast Prp2 liberates the 5' splice site and the branch site adenosine for catalysis of pre-mRNA splicing.

The RNA helicase Prp2 facilitates the remodeling of the spliceosomal Bact complex to the catalytically activated B* complex just before step one of splicing. As a high-resolution cryo-EM structure of the B* complex is currently lacking, the precise spliceosome remodeling events mediated by Prp2 remain poorly understood. To investigate the latter, we used chemical structure probing to compare the RNA structure of purified yeast Bact and B* complexes. Our studies reveal deviations from conventional RNA helices in the functionally important U6 snRNA internal stem–loop and U2/U6 helix Ib in the activated Bact complex, and to a lesser extent in B*. Interestingly, the N7 of U6–G60 of the catalytic triad becomes accessible to DMS modification in the B* complex, suggesting that the Hoogsteen interaction with U6–A52 is destabilized in B*. Our data show that Prp2 action does not unwind double-stranded RNA, but enhances the flexibility of the first step reactants, the pre-mRNA's 5′ splice site and branch site adenosine. Prp2 therefore appears to act primarily as an RNPase to achieve catalytic activation by liberating the first step reactants in preparation for catalysis of the first step of splicing.

A close-up look at the spliceosome, at last

Major developments in cryo-electron microscopy in the past three or four years have led to the solution of a number of spliceosome structures at high resolution, e.g., the fully assembled but not yet active spliceosome (Bact), the spliceosome just after the first step of splicing (C), and the spliceosome activated for the second step (C*). Therefore 30 years of genetics and biochemistry of the spliceosome can now be interpreted at the structural level. I have closely examined the RNase H domain of Prp8 in each of the structures. Interestingly, the RNase H domain has different and unexpected roles in each of the catalytic steps of splicing.

Diverse mechanisms for spliceosome-mediated 3' end processing of telomerase RNA.

The 3′ end of Schizosaccharomyces pombe telomerase RNA (SpTER1) is generated by spliceosomal cleavage, a reaction that corresponds to the first step of splicing. The observation that the spliceosome functions in 3′ end processing raised questions about the evolutionary origin and conservation of this mechanism. We now present data in support of spliceosomes generating 3′ ends of telomerase RNAs in other fungi. Strikingly, the mechanistic basis for restricting spliceosomal splicing to the first transesterification reaction differs substantially among species. Unlike S. pombe, two other fission yeasts rely on hyperstabilization of the U6 snRNA—5′ splice site interaction to impede the 2nd step of splicing. In contrast, a non-canonical 5′ splice site blocks the second transesterification reaction in Aspergillus species. These results demonstrate a conserved role for spliceosomes functioning in 3′ end processing. Divergent mechanisms of uncoupling the two steps of splicing argue for multiple origins of this pathway.

In vitro splicing reactions in Drosophila Kc nuclear extracts.

This protocol describes how to generate and analyze products and intermediates in a pre-mRNA splicing reaction. The reaction relies on the use of labeled, capped, synthetic pre-mRNAs, prepared by in vitro transcription, and Drosophila Kc cell culture nuclear extracts. The pre-mRNA substrate is incubated in the nuclear extract under splicing conditions for 1-2 h. The products of the reaction are purified by phenol:chloroform extraction and precipitation with ethanol, and then loaded directly onto a denaturing urea-acrylamide gel. Visualization of the splicing reactions will reveal the pre-mRNA, the spliced mRNA, and the intermediates generated by the first step of splicing. For inefficient reactions, a more sensitive detection method, such as RNase protection, primer extension, or RT-PCR (reverse transcription-polymerase chain reaction), may be required.

Visualizing Group II Intron Catalysis through the Stages of Splicing

Group II introns are self-splicing ribozymes that share a reaction mechanism and a common ancestor with the eukaryotic spliceosome, thereby providing a model system for understanding the chemistry of pre-mRNA splicing. Here we report 14 crystal structures of a group II intron at different stages of catalysis. We provide a detailed mechanism for the first step of splicing, we describe a reversible conformational change between the first and the second steps of splicing, and we present the ligand-free intron structure after splicing in an active state that corresponds to the retrotransposable form of the intron. During each reaction, the reactants are aligned and activated by a heteronuclear four-metal-ion center that contains a metal cluster and obligate monovalent cations, and they adopt a structural arrangement similar to that of protein endonucleases. Based on our data, we propose a model for the splicing cycle and show that it is applicable to the eukaryotic spliceosome.

Crystal structure of a group II intron in the pre-catalytic state

Group II introns are self-splicing catalytic RNAs that are thought to be ancestral to the spliceosome. Here we report the 3.65-Å crystal structure of the group II intron from Oceanobacillus iheyensis in the pre-catalytic state. The structure reveals the conformation of the 5' splice site in the catalytic core and represents the first structure of an intron prior to the first step of splicing.

Protein splicing facilitated by highly similar inteins from two extreme thermophiles

Protein splicing is a post‐translational event by which an intervening polypeptide, the intein, facilitates its own excision from the flanking polypeptides, the exteins, and the ligation of the exteins. Splicing of the Pyrococcus abyssi (Pab) PolII intein is initiated by an increase in temperature, which allows us to measure the rate of individual steps of the splicing reaction in vitro. We explored the rate of the first step of splicing for versions of the Pab PolII intein modified by mutation at conserved residues Asp74 and Ser166. We found no influence on the rate of the first step of splicing, but mutations at Ser166 influence the efficiency of the overall splicing reaction. The Pyrococcus horikoshii PolII intein has 71% sequence identity with the Pab PolII intein, but lacks an unstructured loop found in the Pab PolII intein. We hypothesize that this loop plays a role either in protein stability or in allowing for a mechanism‐linked conformational change to coordinate the steps of splicing. We will compare the temperature dependence of the rate and extent of splicing reactions and of the overall fold for both inteins.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.

Non‐canonical inteins: Protein splicing by alternate mechanisms

Protein splicing is the post‐translational excision of an intervening polypeptide (intein) from its flanking domains (the exteins), concomitant with extein ligation. The first step of splicing is an amide‐to‐thioester rearrangement of the peptide bond linking the N‐extein and intein. Class three inteins bypass this step. The Clostridium thermocellum TerA intein is class three, whereas the Thermobifida fusca Tfu2914 intein is a traditional intein despite having class three sequence motifs. The third step of splicing is Asn cyclization coupled to cleavage of the peptide bond linking the intein and C‐extein. Both the Methanoculleus marisnigri (Mma) and the Pyrococcus abyssi (Pab) PolII inteins promote efficient splicing with C‐terminal Gln in place of Asn. The activity of the Mma PolII intein may be regulated by disulfide bond formation. The Pab PolII intein splices only at elevated temperatures, allowing for isolation of a stable precursor and study each step of splicing in vitro. An NMR solution structure of the Pab PolII intein reveals a particularly rigid structure, a disordered loop absent in a highly similar P. horikoshii intein, and a β‐hairpin specific to thermophilic inteins.This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and the Camille and Henry Dreyfus Foundation.

Telomerase RNA biogenesis involves sequential binding by Sm and Lsm complexes.

In most eukaryotes, the progressive loss of chromosome-terminal DNA sequences is counteracted by the enzyme telomerase, a reverse transcriptase that uses part of an RNA subunit as template to synthesize telomeric repeats. Many cancer cells express high telomerase activity and mutations in telomerase subunits are associated with degenerative syndromes including dyskeratosis congenita and aplastic anaemia. The therapeutic value of altering telomerase activity thus provides ample impetus to study the biogenesis and regulation of this enzyme in human cells and model systems. We have previously identified a precursor of the fission yeast telomerase RNA subunit (TER1)1 and have demonstrated that the mature 3′ end is generated by the spliceosome in a single cleavage reaction akin to the first step of splicing2. Directly upstream and partially overlapping with the spliceosomal cleavage site is a putative Sm protein binding site. Sm and Like-Sm (LSm) proteins belong to an ancient family of RNA binding proteins represented in all three domains of life3. Members of this family form ring complexes on specific sets of target RNAs and play critical roles in their biogenesis, function and turnover. We now demonstrate that the canonical Sm ring and the Lsm2-8 complex sequentially associate with fission yeast TER1. The Sm ring binds to the TER1 precursor, stimulates spliceosomal cleavage and promotes the hypermethylation of the 5′ cap by Tgs1. Sm proteins are then replaced by the Lsm2-8 complex, which promotes the association with the catalytic subunit and protects the mature 3′ end of TER1 from exonucleolytic degradation. Our findings define the sequence of events that occur during telomerase biogenesis and characterize roles for Sm and Lsm complexes as well as for the methylase Tgs1.

Non‐canonical inteins: Alternate mechanisms for protein splicing

Protein splicing involves the post-translational excision of an intervening polypeptide (the intein) from flanking domains (the exteins), as well as ligation of the exteins. The first step of splicing is an amide-ester rearrangement of the peptide bond that links the N-terminal extein and intein. However, the intein that interrupts a putative phage terminase in C. thermocellum lacks an N-terminal nucleophile and should not be able to promote this step. We show that this intein can splice as a class three intein, using an internal Cys to attack the peptide bond at the N-terminal splice junction. This Cys is part of a covariant non-contiguous Trp-Cys-Thr triplet found in class three inteins. We also show that an intein from Thermobifida fusca that has the conserved triplet, but also has an N-terminal nucleophile, cannot bypass the first step of splicing, and that the internal Cys is not essential for splicing. This material is based upon work supported by the National Science Foundation under grant MCB-0950245 and by the Camille and Henry Dreyfus Foundation.

Structural model of the p14/SF3b155 · branch duplex complex.

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.

Exon Definition Complexes Contain the Tri-snRNP and Can Be Directly Converted into B-like Precatalytic Splicing Complexes

The first step in splicing of pre-mRNAs with long introns is exon definition, where U1 and U2 snRNPs bind at opposite ends of an exon. After exon definition, these snRNPs must form a complex across the upstream intron to allow splicing catalysis. Exon definition and conversion of cross-exon to cross-intron spliceosomal complexes are poorly understood. Here we demonstrate that, in addition to U1 and U2 snRNPs, cross-exon complexes contain U4, U5, and U6 (which form the tri-snRNP). Tri-snRNP docking involves the formation of U2/U6 helix II. This interaction is stabilized by a 5' splice site (SS)-containing oligonucleotide, which can bind the tri-snRNP and convert the cross-exon complex into a cross-intron, B-like complex. Our data suggest that the switch from cross-exon to cross-intron complexes can occur directly when an exon-bound tri-snRNP interacts with an upstream 5'SS, without prior formation of a cross-intron A complex, revealing an alternative spliceosome assembly pathway.

A kinetic analysis of each step of protein splicing of the Pyrococcus abyssi PolII intein

Protein splicing is a post‐translational event by which an intervening polypeptide, called an intein, facilitates its own excision from the flanking polypeptides, called the exteins, and the ligation of the exteins. The intein that interrupts the Pyrococcus abyssi PolII protein can be purified as part of a precursor fusion protein. Splicing can be initiated by an increase in temperature, which allows for detailed kinetic analysis of the overall splicing process as well as the individual steps. We have studied the influence of the residue immediately upstream of the intein on the rate of the first step of splicing, an amide to thioester rearrangement. Modifications leading to changes of that rate can result in splicing side reactions. We also have examined the influence of residues constituting a putative oxyanion hole on the rate of the first step of the reaction and the extent of splicing. Finally, we present data on the influence of two conserved downstream His residues on the rate of step three of splicing, Asn cyclization coupled to peptide bond cleavage.This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.

Non‐canonical inteins: Alternate mechanisms for protein splicing

Protein splicing involves the post‐translational excision of an intervening polypeptide (the intein) from flanking domains (the exteins), as well as ligation of the exteins. The first step of splicing is an amide‐ester rearrangement of the peptide bond that links the N‐terminal extein and intein. However, the intein that interrupts a putative phage terminase in C. thermocellum lacks an N‐terminal nucleophile and should not be able to promote this step. We show that this intein can splice, and have examined the influence of conserved residues on efficient splicing. The third step of splicing is cyclization of Asn coupled to peptide bond cleavage. However, the PolII intein from P. abyssi has a C‐terminal Gln and can still promote splicing. We compare the effects of C‐terminal Asn and Gln on splicing efficiency, as well as the influence of conserved residues on the catalysis of splicing.This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.

Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing.

Eukaryotic pre-mRNA splicing is a complex process requiring the precise timing and action of >100 trans-acting factors. It has been known for some time that the two steps of splicing chemistry require three DEAH-box RNA helicase-like proteins; however, their mechanism of action at these steps has remained elusive. Spliceosomes arrested in vivo at the three helicase checkpoints were purified, and first step-arrested spliceosomes were functionally characterized. We show that the first step of splicing requires a novel ATP-independent conformational change. Prp2p then catalyzes an ATP-dependent rearrangement displacing the SF3a and SF3b complexes from the branchpoint within the spliceosome. We propose a model in which SF3 prevents premature nucleophilic attack of the chemically reactive hydroxyl of the branchpoint adenosine prior to the first transesterification. When the spliceosome attains the proper conformation and upon the function of Prp2p, SF3 is displaced from the branchpoint allowing first step chemistry to occur.

The RNA binding protein Cwc2 interacts directly with the U6 snRNA to link the nineteen complex to the spliceosome during pre-mRNA splicing

Intron removal during pre-messenger RNA (pre-mRNA) splicing involves arrangement of snRNAs into conformations that promote the two catalytic steps. The Prp19 complex [nineteen complex (NTC)] can specify U5 and U6 snRNA interactions with pre-mRNA during spliceosome activation. A candidate for linking the NTC to the snRNAs is the NTC protein Cwc2, which contains motifs known to bind RNA, a zinc finger and RNA recognition motif (RRM). In yeast cells mutation of either the zinc finger or RRM destabilize Cwc2 and are lethal. Yeast cells depleted of Cwc2 accumulate pre-mRNA and display reduced levels of U1, U4, U5 and U6 snRNAs. Cwc2 depletion also reduces U4/U6 snRNA complex levels, as found with depletion of other NTC proteins, but without increase in free U4. Purified Cwc2 displays general RNA binding properties and can bind both snRNAs and pre-mRNA in vitro. A Cwc2 RRM fragment alone can bind RNA but with reduced efficiency. Under splicing conditions Cwc2 can associate with U2, U5 and U6 snRNAs, but can only be crosslinked directly to the U6 snRNA. Cwc2 associates with U6 both before and after the first step of splicing. We propose that Cwc2 links the NTC to the spliceosome during pre-mRNA splicing through the U6 snRNA.