Structural Insights into the Roles of Metazoan-Specific Splicing Factors in the Human Step 1 Spliceosome.

Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core resolution and 4.5-5.7 Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.

Molecular architecture of the human 17S U2 snRNP.

The U2 small nuclear ribonucleoprotein (snRNP) has an essential role in the selection of the precursor mRNA branch-site adenosine, the nucleophile for the first step of splicing1. Stable addition of U2 during early spliceosome formation requires the DEAD-box ATPase PRP52-7. Yeast U2 small nuclear RNA (snRNA) nucleotides that form base pairs with the branch site are initially sequestered in a branchpoint-interacting stem-loop (BSL)8, but whether the human U2 snRNA folds in a similar manner is unknown. The U2 SF3B1 protein, a common mutational target in haematopoietic cancers9, contains a HEAT domain (SF3B1HEAT) with an open conformation in isolated SF3b10, but a closed conformation in spliceosomes11, which is required for stable interaction between U2 and the branch site. Here we report a 3D cryo-electron microscopy structure of the human 17S U2 snRNP at a core resolution of 4.1 Å and combine it with protein crosslinking data to determine the molecular architecture of this snRNP. Our structure reveals that SF3B1HEAT interacts with PRP5 and TAT-SF1, and maintains its open conformation in U2 snRNP, and that U2 snRNA forms a BSL that is sandwiched between PRP5, TAT-SF1 and SF3B1HEAT. Thus, substantial remodelling of the BSL and displacement of BSL-interacting proteins must occur to allow formation of the U2-branch-site helix. Our studies provide a structural explanation of why TAT-SF1 must be displaced before the stable addition of U2 to the spliceosome, and identify RNP rearrangements facilitated by PRP5 that are required for stable interaction between U2 and the branch site.

Multiple RNA-RNA tertiary interactions are dispensable for formation of a functional U2/U6 RNA catalytic core in the spliceosome.

The active 3D conformation of the spliceosome's catalytic U2/U6 RNA core is stabilised by a network of secondary and tertiary RNA interactions, but also depends on spliceosomal proteins for its formation. To determine the contribution towards splicing of specific RNA secondary and tertiary interactions in the U2/U6 RNA core, we introduced mutations in critical U6 nucleotides and tested their effect on splicing using a yeast in vitro U6 depletion/complementation system. Elimination of selected RNA tertiary interactions involving the U6 catalytic triad, or deletions of the bases of U6-U80 or U6-A59, had moderate to no effect on splicing, showing that the affected secondary and tertiary interactions are not required for splicing catalysis. However, removal of the base of U6-G60 of the catalytic triad completely blocked splicing, without affecting assembly of the activated spliceosome or its subsequent conversion into a B*-like complex. Our data suggest that the catalytic configuration of the RNA core that allows catalytic metal M1 binding can be maintained by Protein-RNA contacts. However, RNA stacking interactions in the U2/U6 RNA core are required for productive coordination of metal M2. The functional conformation of the U2/U6 RNA core is thus highly buffered, with overlapping contributions from RNA-RNA and Protein-RNA interactions.

Structure and Conformational Dynamics of the Human Spliceosomal Bact Complex.

The spliceosome is a highly dynamic macromolecular complex that precisely excises introns from pre-mRNA. Here we report the cryo-EM 3D structure of the human Bact spliceosome at 3.4 Å resolution. In the Bact state, the spliceosome is activated but not catalytically primed, so that it is functionally blocked prior to the first catalytic step of splicing. The spliceosomal core is similar to the yeast Bact spliceosome; important differences include the presence of the RNA helicase aquarius and peptidyl prolyl isomerases. To examine the overall dynamic behavior of the purified spliceosome, we developed a principal component analysis-based approach. Calculating the energy landscape revealed eight major conformational states, which we refined to higher resolution. Conformational differences of the highly flexible structural components between these eight states reveal how spliceosomal components contribute to the assembly of the spliceosome, allowing it to generate a dynamic interaction network required for its subsequent catalytic activation.

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.

Cryo-EM structure of a human spliceosome activated for step 2 of splicing.

Spliceosome rearrangements facilitated by RNA helicase PRP16 before catalytic step two of splicing are poorly understood. Here we report a 3D cryo-electron microscopy structure of the human spliceosomal C complex stalled directly after PRP16 action (C*). The architecture of the catalytic U2-U6 ribonucleoprotein (RNP) core of the human C* spliceosome is very similar to that of the yeast pre-Prp16 C complex. However, in C* the branched intron region is separated from the catalytic centre by approximately 20 Å, and its position close to the U6 small nuclear RNA ACAGA box is stabilized by interactions with the PRP8 RNase H-like and PRP17 WD40 domains. RNA helicase PRP22 is located about 100 Å from the catalytic centre, suggesting that it destabilizes the spliced mRNA after step two from a distance. Comparison of the structure of the yeast C and human C* complexes reveals numerous RNP rearrangements that are likely to be facilitated by PRP16, including a large-scale movement of the U2 small nuclear RNP.

Molecular architecture of the Saccharomyces cerevisiae activated spliceosome.

The activated spliceosome (Bact) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D electron cryomicroscopy structure of the Saccharomyces cerevisiae Bact complex at 5.8-angstrom resolution. Our model reveals that in Bact, the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5' splice site (ss) is oriented for step 1 catalysis but occluded by protein. The first-step nucleophile-the branchsite adenosine-is sequestered within the Hsh155 HEAT domain and is held 50 angstroms away from the 5'ss. Our structure suggests that Prp2 adenosine triphosphatase-mediated remodeling leads to conformational changes in Hsh155's HEAT domain that liberate the first-step reactants for catalysis.

Dynamic Contacts of U2, RES, Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes.

Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B(act) spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B(act) crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25's step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome's protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.

RNA structure analysis of human spliceosomes reveals a compact 3D arrangement of snRNAs at the catalytic core.

Although U snRNAs play essential roles in splicing, little is known about the 3D arrangement of U2, U6, and U5 snRNAs and the pre-mRNA in active spliceosomes. To elucidate their relative spatial organization and dynamic rearrangement, we examined the RNA structure of affinity-purified, human spliceosomes before and after catalytic step 1 by chemical RNA structure probing. We found a stable 3-way junction of the U2/U6 snRNA duplex in active spliceosomes that persists minimally through step 1. Moreover, the formation of alternating, mutually exclusive, U2 snRNA conformations, as observed in yeast, was not detected in different assembly stages of human spliceosomal complexes (that is, B, B(act), or C complexes). Psoralen crosslinking revealed an interaction during/after step 1 between internal loop 1 of the U5 snRNA, and intron nucleotides immediately downstream of the branchpoint. Using the experimentally derived structural constraints, we generated a model of the RNA network of the step 1 spliceosome, based on the crystal structure of a group II intron through homology modelling. The model is topologically consistent with current genetic, biochemical, and structural data.

Post-transcriptional spliceosomes are retained in nuclear speckles until splicing completion.

There is little quantitative information regarding how much splicing occurs co-transcriptionally in higher eukaryotes, and it remains unclear where precisely splicing occurs in the nucleus. Here we determine the global extent of co- and post-transcriptional splicing in mammalian cells, and their respective subnuclear locations, using antibodies that specifically recognize phosphorylated SF3b155 (P-SF3b155) found only in catalytically activated/active spliceosomes. Quantification of chromatin- and nucleoplasm-associated P-SF3b155 after fractionation of HeLa cell nuclei, reveals that ~80% of pre-mRNA splicing occurs co-transcriptionally. Active spliceosomes localize in situ to regions of decompacted chromatin, at the periphery of or within nuclear speckles. Immunofluorescence microscopy with anti-P-SF3b155 antibodies, coupled with transcription inhibition and a block in splicing after SF3b155 phosphorylation, indicates that post-transcriptional splicing occurs in nuclear speckles and that release of post-transcriptionally spliced mRNA from speckles is coupled to the nuclear mRNA export pathway. Our data provide new insights into when and where splicing occurs in cells.

Phosphorylation of human PRP28 by SRPK2 is required for integration of the U4/U6-U5 tri-snRNP into the spliceosome.

Several protein kinases, including SRPK1 and SRPK2, have been implicated in spliceosome assembly and catalytic activation. However, little is known about their targets. Here we show that SRPK1 is predominantly associated with U1 small nuclear ribonucleoprotein (snRNP), whereas SRPK2 associates with the U4/U6-U5 tri-snRNP. RNAi-mediated depletion in HeLa cells showed that SRPK2 is essential for cell viability, and it is required for spliceosomal B complex formation. SRPK2 knock down results in hypophosphorylation of the arginine-serine (RS) domain-containing human PRP28 protein (PRP28, also known as DDX23), and destabilizes PRP28 association with the tri-snRNP. Immunodepletion of PRP28 from HeLa cell nuclear extract and complementation studies revealed that PRP28 phosphorylation is required for its stable association with the tri-snRNP and for tri-snRNP integration into the B complex. Our results demonstrate a role for SRPK2 in splicing and reveal a previously unknown function for PRP28 in spliceosome assembly.

GraFix: sample preparation for single-particle electron cryomicroscopy.

We developed a method, named GraFix, that considerably improves sample quality for structure determination by single-particle electron cryomicroscopy (cryo-EM). GraFix uses a glycerol gradient centrifugation step in which the complexes are centrifuged into an increasing concentration of a chemical fixation reagent to prevent aggregation and to stabilize individual macromolecules. The method can be used to prepare samples for negative-stain, cryo-negative-stain and, particularly, unstained cryo-EM.

Binding of the human Prp31 Nop domain to a composite RNA-protein platform in U4 snRNP.

Although highly homologous, the spliceosomal hPrp31 and the nucleolar Nop56 and Nop58 (Nop56/58) proteins recognize different ribonucleoprotein (RNP) particles. hPrp31 interacts with complexes containing the 15.5K protein and U4 or U4atac small nuclear RNA (snRNA), whereas Nop56/58 associate with 15.5K-box C/D small nucleolar RNA complexes. We present structural and biochemical analyses of hPrp31-15.5K-U4 snRNA complexes that show how the conserved Nop domain in hPrp31 maintains high RNP binding selectivity despite relaxed RNA sequence requirements. The Nop domain is a genuine RNP binding module, exhibiting RNA and protein binding surfaces. Yeast two-hybrid analyses suggest a link between retinitis pigmentosa and an aberrant hPrp31-hPrp6 interaction that blocks U4/U6-U5 tri-snRNP formation.

Composition and three-dimensional EM structure of double affinity-purified, human prespliceosomal A complexes.

Little is known about the higher-order structure of prespliceosomal A complexes, in which pairing of the pre-mRNA's splice sites occurs. Here, human A complexes were isolated under physiological conditions by double-affinity selection. Purified complexes contained stoichiometric amounts of U1, U2 and pre-mRNA, and crosslinking studies indicated that these form concomitant base pairing interactions with one another. A complexes contained nearly all U1 and U2 proteins plus approximately 50 non-snRNP proteins. Unexpectedly, proteins of the hPrp19/CDC5 complex were also detected, even when A complexes were formed in the absence of U4/U6 snRNPs, demonstrating that they associate independent of the tri-snRNP. Double-affinity purification yielded structurally homogeneous A complexes as evidenced by electron microscopy, and allowed for the first time the generation of a three-dimensional structure. A complexes possess an asymmetric shape (approximately 260 x 200 x 195 angstroms) and contain a main body with various protruding elements, including a head-like domain and foot-like protrusions. Complexes isolated here are well suited for in vitro assembly studies to determine factor requirements for the A to B complex transition.

The 5' end of U2 snRNA is in close proximity to U1 and functional sites of the pre-mRNA in early spliceosomal complexes.

Recognition and pairing of the correct 5' and 3' splice sites (ss) of a pre-mRNA are critical events that occur early during spliceosome assembly. Little is known about the spatial organization in early spliceosomal complexes of the U1 and U2 snRNPs, which together with several non-snRNP proteins, are involved in juxtapositioning the functional sites of the pre-mRNA. To better understand the molecular mechanisms of splice-site recognition/pairing, we have examined the organization of U2 relative to U1 and pre-mRNA in spliceosomal complexes via hydroxyl-radical probing with Fe-BABE-tethered U2 snRNA. These studies reveal that functional sites of the pre-mRNA are located close to the 5' end of U2 both in E and A complexes. U2 is also positioned close to U1 in a defined orientation already in the E complex, and their relative spatial organization remains largely unchanged during the E to A transition.

Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP.

Two different models currently exist for the assembly pathway of the spliceosome, namely, the traditional model, in which spliceosomal snRNPs associate in a stepwise, ordered manner with the pre-mRNA, and the holospliceosome model, in which all spliceosomal snRNPs preassemble into a penta-snRNP complex. Here we have tested whether the spliceosomal A complex, which contains solely U1 and U2 snRNPs bound to pre-mRNA, is a functional, bona fide assembly intermediate. Significantly, A complexes affinity-purified from nuclear extract depleted of U4/U6 snRNPs (and thus unable to form a penta-snRNP) supported pre-mRNA splicing in nuclear extract depleted of U2 snRNPs, whereas naked pre-mRNA did not. Mixing experiments with purified A complexes and naked pre-mRNA additionally confirmed that under these conditions, A complexes do not form de novo. Thus, our studies demonstrate that holospliceosome formation is not a prerequisite for generating catalytically active spliceosomes and that, at least in vitro, the U1 and U2 snRNPs can functionally associate with the pre-mRNA, prior to and independent of the tri-snRNP. The ability to isolate functional spliceosomal A complexes paves the way to study in detail subsequent spliceosome assembly steps using purified components.

Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions.

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including approximately 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.

RNA structural requirements for the association of the spliceosomal hPrp31 protein with the U4 and U4atac small nuclear ribonucleoproteins.

The kink-turn, a stem I-internal loop-stem II structure of the 5 ' stem-loop of U4 and U4atac small nuclear (sn) RNAs bound by 15.5K protein is required for binding of human Prp31 protein (hPrp31) during U4 and U4atac snRNP assembly. In box C/D snoRNPs a similar kink-turn with bound 15.5K protein is required for selective binding of proteins NOP56 and NOP58. Here we analyzed RNA structural requirements for association of hPrp31 with U4 snRNP in vitro by hydroxyl radical footprinting. hPrp31 induced protection of the terminal penta-loop, as well as of stems I and II flanking the kink-turn. Similar protection was found with U4/U6 snRNA duplex prebound with 15.5K protein. A detailed mutational analysis of the U4 snRNA elements by electrophoretic mobility shift analysis revealed that stem I could not be shortened, although it tolerated sequence alterations. However, introduction of a third Watson-Crick base pair into stem II significantly reduced hPrp31 binding. While stem I of U4atac snRNA showed relaxed binding requirements, its stem II requirements were likewise restricted to two base pairs. In contrast, as shown previously, stem II of the kink-turn motif in box C/D snoRNAs is comprised of three base pairs, and NOP56 and NOP58 require a G-C pair at the central position. This indicates that hPrp31 binding specificity is achieved by the recognition of the two base pair long stem II of the U4 and U4atac snRNAs and suggests how discrimination is achieved by RNA structural elements during assembly of U4/U6 and U4atac/U6atac snRNPs and box C/D snoRNPs.

Proximity of conserved U6 and U2 snRNA elements to the 5' splice site region in activated spliceosomes.

Major structural changes occur in the spliceosome during its catalytic activation, which immediately precedes the splicing of pre-mRNA. Whereas changes in snRNA conformation are well documented at the level of secondary RNA-RNA interactions, little is known about the tertiary structure of this RNA-RNA network, which comprises the spliceosome's catalytic core. Here, we have used the hydroxyl-radical probe Fe-BABE, tethered to the tenth nucleotide (U(+10)) of the 5' end of a pre-mRNA intron, to map RNA-RNA proximities in spliceosomes. These studies revealed that several conserved snRNA regions are close to U(+10) in activated spliceosomes, namely (i) the U6 snRNA ACAGAG-box region, (ii) portions of the U6 intramolecular stem-loop (U6-ISL) including a nucleotide implicated in the first catalytic step (U74), and (iii) the region of U2 that interacts with the branch point. These data constrain the relative orientation of these structural elements with respect to U(+10) in the activated spliceosome. Upon conversion of the activated spliceosome to complex C, the accessibility of U6-ISL to hydroxyl-radical cleavage is altered, suggesting rearrangements after the first catalytic step.

U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes.

The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5' end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5' end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.