RESUMEN
Early spliceosome assembly can occur through an intron-defined pathway, whereby U1 and U2 small nuclear ribonucleoprotein particles (snRNPs) assemble across the intron1. Alternatively, it can occur through an exon-defined pathway2-5, whereby U2 binds the branch site located upstream of the defined exon and U1 snRNP interacts with the 5' splice site located directly downstream of it. The U4/U6.U5 tri-snRNP subsequently binds to produce a cross-intron (CI) or cross-exon (CE) pre-B complex, which is then converted to the spliceosomal B complex6,7. Exon definition promotes the splicing of upstream introns2,8,9 and plays a key part in alternative splicing regulation10-16. However, the three-dimensional structure of exon-defined spliceosomal complexes and the molecular mechanism of the conversion from a CE-organized to a CI-organized spliceosome, a pre-requisite for splicing catalysis, remain poorly understood. Here cryo-electron microscopy analyses of human CE pre-B complex and B-like complexes reveal extensive structural similarities with their CI counterparts. The results indicate that the CE and CI spliceosome assembly pathways converge already at the pre-B stage. Add-back experiments using purified CE pre-B complexes, coupled with cryo-electron microscopy, elucidate the order of the extensive remodelling events that accompany the formation of B complexes and B-like complexes. The molecular triggers and roles of B-specific proteins in these rearrangements are also identified. We show that CE pre-B complexes can productively bind in trans to a U1 snRNP-bound 5' splice site. Together, our studies provide new mechanistic insights into the CE to CI switch during spliceosome assembly and its effect on pre-mRNA splice site pairing at this stage.
Asunto(s)
Exones , Intrones , Empalme del ARN , Empalmosomas , Humanos , Empalme Alternativo , Microscopía por Crioelectrón , Exones/genética , Intrones/genética , Modelos Moleculares , Sitios de Empalme de ARN/genética , Empalme del ARN/genética , Empalmosomas/metabolismo , Empalmosomas/química , Empalmosomas/ultraestructura , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/ultraestructuraRESUMEN
Alternative precursor messenger RNA splicing is instrumental in expanding the proteome of higher eukaryotes, and changes in 3' splice site (3'ss) usage contribute to human disease. We demonstrate by small interfering RNA-mediated knockdowns, followed by RNA sequencing, that many proteins first recruited to human C* spliceosomes, which catalyze step 2 of splicing, regulate alternative splicing, including the selection of alternatively spliced NAGNAG 3'ss. Cryo-electron microscopy and protein cross-linking reveal the molecular architecture of these proteins in C* spliceosomes, providing mechanistic and structural insights into how they influence 3'ss usage. They further elucidate the path of the 3' region of the intron, allowing a structure-based model for how the C* spliceosome potentially scans for the proximal 3'ss. By combining biochemical and structural approaches with genome-wide functional analyses, our studies reveal widespread regulation of alternative 3'ss usage after step 1 of splicing and the likely mechanisms whereby C* proteins influence NAGNAG 3'ss choices.
Asunto(s)
Sitios de Empalme de ARN , Empalmosomas , Humanos , Microscopía por Crioelectrón , Empalme Alternativo , IntronesRESUMEN
Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final three-dimensional folding of the U2/U6 catalytic RNA.
Asunto(s)
Pliegue del ARN , Empalme del ARN , ARN Nuclear Pequeño/química , Proteínas de Unión al ARN/química , Empalmosomas/química , Dominio Catalítico , Microscopía por Crioelectrón , Humanos , Conformación Proteica , ARN Catalítico/química , ARN Nuclear Pequeño/genética , Empalmosomas/genéticaRESUMEN
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.
Asunto(s)
Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , Empalmosomas/metabolismo , Animales , Catálisis , Células HeLa , Humanos , Intrones/genética , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Unión Proteica , Estabilidad Proteica , ARN/química , ARN/metabolismo , Ribonucleoproteínas/metabolismo , Saccharomyces cerevisiae/metabolismo , Especificidad de la Especie , Factores de TiempoRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/ultraestructura , Secuencia de Bases , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , Células HeLa , Humanos , Modelos Moleculares , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Unión Proteica , Conformación Proteica , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Transactivadores/química , Transactivadores/metabolismoRESUMEN
The spliceosome is a highly complex, dynamic ribonucleoprotein molecular machine that undergoes numerous structural and compositional rearrangements that lead to the formation of its active site. Recent advances in cyroelectron microscopy (cryo-EM) have provided a plethora of near-atomic structural information about the inner workings of the spliceosome. Aided by previous biochemical, structural, and functional studies, cryo-EM has confirmed or provided a structural basis for most of the prevailing models of spliceosome function, but at the same time allowed novel insights into splicing catalysis and the intriguing dynamics of the spliceosome. The mechanism of pre-mRNA splicing is highly conserved between humans and yeast, but the compositional dynamics and ribonucleoprotein (RNP) remodeling of the human spliceosome are more complex. Here, we summarize recent advances in our understanding of the molecular architecture of the human spliceosome, highlighting differences between the human and yeast splicing machineries.
Asunto(s)
Conformación de Ácido Nucleico , Precursores del ARN/metabolismo , Empalme del ARN , ARN Mensajero/metabolismo , Catálisis , Microscopía por Crioelectrón , Células Eucariotas , Mutación , Neoplasias/metabolismo , Conformación Proteica , Precursores del ARN/química , ARN Mensajero/química , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismoRESUMEN
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.
Asunto(s)
Simulación de Dinámica Molecular , Empalmosomas/química , Células HeLa , Humanos , Empalmosomas/metabolismo , Empalmosomas/ultraestructuraRESUMEN
Little is known about the spliceosome's structure before its extensive remodeling into a catalytically active complex. Here, we report a 3D cryo-EM structure of a pre-catalytic human spliceosomal B complex. The U2 snRNP-containing head domain is connected to the B complex main body via three main bridges. U4/U6.U5 tri-snRNP proteins, which are located in the main body, undergo significant rearrangements during tri-snRNP integration into the B complex. These include formation of a partially closed Prp8 conformation that creates, together with Dim1, a 5' splice site (ss) binding pocket, displacement of Sad1, and rearrangement of Brr2 such that it contacts its U4/U6 substrate and is poised for the subsequent spliceosome activation step. The molecular organization of several B-specific proteins suggests that they are involved in negatively regulating Brr2, positioning the U6/5'ss helix, and stabilizing the B complex structure. Our results indicate significant differences between the early activation phase of human and yeast spliceosomes.
Asunto(s)
Empalmosomas/química , Núcleo Celular/química , Microscopía por Crioelectrón , Células HeLa , Humanos , Modelos Moleculares , Proteínas de Unión al ARN/química , Ribonucleoproteínas Nucleares Pequeñas/química , Saccharomyces cerevisiae/química , Empalmosomas/ultraestructuraRESUMEN
Small molecule inhibitors of pre-mRNA splicing are important tools for identifying new spliceosome assembly intermediates, allowing a finer dissection of spliceosome dynamics and function. Here, we identified a small molecule that inhibits human pre-mRNA splicing at an intermediate stage during conversion of pre-catalytic spliceosomal B complexes into activated Bact complexes. Characterization of the stalled complexes (designated B028) revealed that U4/U6 snRNP proteins are released during activation before the U6 Lsm and B-specific proteins, and before recruitment and/or stable incorporation of Prp19/CDC5L complex and other Bact complex proteins. The U2/U6 RNA network in B028 complexes differs from that of the Bact complex, consistent with the idea that the catalytic RNA core forms stepwise during the B to Bact transition and is likely stabilized by the Prp19/CDC5L complex and related proteins. Taken together, our data provide new insights into the RNP rearrangements and extensive exchange of proteins that occurs during spliceosome activation.
Asunto(s)
Inhibidores Enzimáticos/aislamiento & purificación , Empalme del ARN/efectos de los fármacos , Empalmosomas/efectos de los fármacos , Empalmosomas/metabolismo , Evaluación Preclínica de Medicamentos , Humanos , Precursores del ARN/metabolismoRESUMEN
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.
Asunto(s)
Microscopía por Crioelectrón , Empalme del ARN , Empalmosomas/metabolismo , Empalmosomas/ultraestructura , Adenosina/metabolismo , Secuencia de Bases , Biocatálisis , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestructura , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/ultraestructura , Exones/genética , Humanos , Intrones/genética , Modelos Moleculares , Movimiento , Dominios Proteicos , Factores de Empalme de ARN/química , Factores de Empalme de ARN/metabolismo , Factores de Empalme de ARN/ultraestructura , Estabilidad del ARN , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/ultraestructura , Ribonucleasa H/química , Ribonucleasa H/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/ultraestructura , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Empalmosomas/químicaRESUMEN
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.
Asunto(s)
ARN Nuclear Pequeño/química , Ribonucleoproteína Nuclear Pequeña U4-U6/química , Ribonucleoproteína Nuclear Pequeña U5/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestructura , Empalmosomas/ultraestructura , Adenosina Trifosfatasas , Biocatálisis , Dominio Catalítico , Microscopía por Crioelectrón , Exones , Conformación Proteica , ARN Helicasas/química , ARN Helicasas/genética , Sitios de Empalme de ARN , Empalme del ARN , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U5/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Empalmosomas/químicaRESUMEN
We have elucidated the spatial arrangement of proteins and snRNP subunits within the purified spliceosomal B(act) complex from Saccharomyces cerevisiae, using negative-stain immunoelectron microscopy. The B(act) spliceosome exhibits a mushroom-like shape with a main body connected to a foot and a steep and a shallow slope. The U5 core components, including proteins Snu114 and Prp8, are located in the main body and foot, while Brr2 is on the shallow slope. U2 snRNP components and the RNA helicase Prp2 were predominantly located in the upper regions of both slopes. While several proteins of the "nineteen complex" are located on the steep slope, Prp19, Cef1, and the U6 snRNA-binding protein Cwc2 are on the main body. Our results also indicate that the catalytic core RNP of the spliceosome resides in its main body. We thus assign distinct domains of the B(act) complex to its snRNP and protein components, and we provide first structural insights into the remodeling events at the spliceosome during its transformation from the B to the B(act) complex.
Asunto(s)
Mapas de Interacción de Proteínas , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , ARN Helicasas DEAD-box/metabolismo , Unión Proteica , ARN Helicasas/metabolismo , Factores de Empalme de ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequeña U5/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/química , Empalmosomas/ultraestructuraRESUMEN
The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5'ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5'ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Biocatálisis , Reactivos de Enlaces Cruzados/metabolismo , Electroforesis en Gel Bidimensional , Humanos , Modelos Biológicos , Mutación/genética , ARN/metabolismo , Empalmosomas/ultraestructuraRESUMEN
The ATP analog ATPγS inhibits pre-mRNA splicing in vitro, but there have been conflicting reports as to which step of splicing is inhibited by this small molecule and its inhibitory mechanism remains unclear. Here we have dissected the effect of ATPγS on pre-mRNA splicing in vitro. Addition of ATPγS to splicing extracts depleted of ATP inhibited both catalytic steps of splicing. At ATPγS concentrations ≥0.5 mM, precatalytic B complexes accumulate, demonstrating a block prior to or during the spliceosome activation stage. Affinity purification of the ATPγS-stalled B complexes (B(ATPγS)) and subsequent characterization of their abundant protein components by 2D gel electrophoresis revealed that B(ATPγS) complexes are compositionally more homogeneous than B complexes previously isolated in the presence of ATP. In particular, they contain little or no Prp19/CDC5L complex proteins, indicating that these proteins are recruited after assembly of the precatalytic spliceosome. Under the electron microscope, B(ATPγS) complexes exhibit a morphology highly similar to B complexes, indicating that the ATPγS-induced block in the transformation of the B to B(act) complex is not due to a major structural defect. Likely mechanisms whereby ATPγS blocks spliceosome assembly at the activation stage, including inhibition of the RNA helicase Brr2, are discussed. Given their more homogeneous composition, B complexes stalled by ATPγS may prove highly useful for both functional and structural analyses of the precatalytic spliceosome and its conversion into an activated B(act) spliceosomal complex.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Empalme del ARN , Empalmosomas/metabolismo , Adenosina Trifosfato/farmacología , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Empalmosomas/efectos de los fármacosRESUMEN
The U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structure of the 1.8-megadalton human tri-snRNP at a resolution of 7 angstroms using single-particle cryo-electron microscopy (cryo-EM). We fit all known high-resolution structures of tri-snRNP components into the EM density map and validated them by protein cross-linking. Our model reveals how the spatial organization of Brr2 RNA helicase prevents premature U4/U6 RNA unwinding in isolated human tri-snRNPs and how the ubiquitin C-terminal hydrolase-like protein Sad1 likely tethers the helicase Brr2 to its preactivation position. Comparison of our model with cryo-EM three-dimensional structures of the Saccharomyces cerevisiae tri-snRNP and Schizosaccharomyces pombe spliceosome indicates that Brr2 undergoes a marked conformational change during spliceosome activation, and that the scaffolding protein Prp8 is also rearranged to accommodate the spliceosome's catalytic RNA network.
Asunto(s)
Ribonucleoproteína Nuclear Pequeña U4-U6/química , Ribonucleoproteína Nuclear Pequeña U5/química , Microscopía por Crioelectrón , Cristalografía por Rayos X , ARN Helicasas DEAD-box/química , Activación Enzimática , Células HeLa , Humanos , Modelos Moleculares , Factores de Elongación de Péptidos/química , Conformación Proteica , ARN Helicasas/química , Proteínas de Unión al ARN/química , Ribonucleoproteínas Nucleares Pequeñas/química , Proteínas de Saccharomyces cerevisiae/química , Schizosaccharomyces/metabolismo , Ubiquitina Tiolesterasa/químicaRESUMEN
The spliceosome assembles on a pre-mRNA intron by binding of five snRNPs and numerous proteins, leading to the formation of the pre-catalytic B complex. While the general morphology of the B complex is known, the spatial arrangement of proteins and snRNP subunits within it remain to be elucidated. To shed light on the architecture of the yeast B complex, we immuno-labelled selected proteins and located them by negative-stain electron microscopy. The B complex exhibited a triangular shape with main body, head and neck domains. We located the U5 snRNP components Brr2 at the top and Prp8 and Snu114 in the centre of the main body. We found several U2 SF3a (Prp9 and Prp11) and SF3b (Hsh155 and Cus1) proteins in the head domain and two U4/U6 snRNP proteins (Prp3 and Lsm4) in the neck domain that connects the main body with the head. Thus, we could assign distinct domains of the B complex to the respective snRNPs and provide the first detailed picture of the subunit architecture and protein arrangements of the B complex.
Asunto(s)
Saccharomyces cerevisiae/ultraestructura , Empalmosomas/metabolismo , Unión Proteica , Transporte de Proteínas , ARN Helicasas/genética , ARN Helicasas/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Empalmosomas/ultraestructuraRESUMEN
Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5'ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5'ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5'ss sequence, most importantly between Prp8 and nucleotides at the exon-intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.
Asunto(s)
Sitios de Empalme de ARN/genética , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Empalmosomas/genética , Línea Celular Tumoral , Exones/genética , Proteínas del Ojo/genética , Células HeLa , Humanos , Intrones/genética , Fosforilación/genética , Empalme del ARN/genéticaRESUMEN
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.
Asunto(s)
Núcleo Celular/metabolismo , Empalme del ARN/fisiología , Empalmosomas/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Microscopía Fluorescente , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/genética , Fosforilación/fisiología , Empalme del ARN/genética , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/metabolismoRESUMEN
A first step in understanding the architecture of the spliceosome is elucidating the positions of individual spliceosomal components and functional centers. Catalysis of the first step of pre-mRNA splicing leads to the formation of the spliceosomal C complex, which contains the pre-mRNA intermediates--the cleaved 5' exon and the intron-3' exon lariat. To topographically locate the catalytic center of the human C complex, we first determined, by DNA oligonucleotide-directed RNAse H digestions, accessible pre-mRNA regions closest to nucleotides of the cleaved 5' splice site (i.e., the 3' end of exon 1 and the 5' end of the intron) and the intron lariat branch point, which are expected to be at/near the catalytic center in complex C. For electron microscopy (EM) localization studies, C complexes were allowed to form, and biotinylated 2'-OMe RNA oligonucleotides were annealed to these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein A-coated colloidal gold were additionally bound. EM analyses allowed us to map the position of exon and intron nucleotides near the cleaved 5' splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C complex and further define a possible location for its catalytic center.
Asunto(s)
Precursores del ARN/ultraestructura , Empalmosomas/ultraestructura , Exones , Oro Coloide/química , Humanos , Intrones , Microscopía Inmunoelectrónica/métodos , Precursores del ARN/genética , Precursores del ARN/metabolismo , Empalme del ARN , Ribonucleasa H/metabolismo , Empalmosomas/metabolismoRESUMEN
The spliceosome excises introns from pre-mRNA in a two-step splicing reaction. So far, the three-dimensional (3D) structure of a spliceosome with preserved catalytic activity has remained elusive. Here, we determined the 3D structure of the human, catalytically active step I spliceosome (C complex) by cryo-electron microscopy (cryo-EM) in vitrified ice. Via immunolabeling we mapped the position of the 5' exon. The C complex contains an unusually salt-stable ribonucleoprotein (RNP) core that harbors its catalytic center. We determined the 3D structure of this RNP core and also that of a post-step II particle, the 35S U5 snRNP, which contains most of the C complex core proteins. As C complex domains could be recognized in these structures, their position in the C complex could be determined, thereby allowing the region harboring the spliceosome's catalytic core to be localized.