Henning Urlaub

Isolation of an active step I spliceosome and composition of its RNP core

Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome’s active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of added factors. Comparisons of the composition of the precatalytic versus the catalytic spliceosome revealed a marked exchange of proteins during the transition from the B to the C complex, with apparent stabilization of Prp19–CDC5 complex proteins and destabilization of SF3a/b proteins. Disruption of purified C complexes led to the isolation of a salt-stable ribonucleoprotein (RNP) core that contained both splicing intermediates and U2, U5 and U6 small nuclear RNA plus predominantly U5 and human Prp19–CDC5 proteins and Prp19-related factors. Our data provide insights into the spliceosome’s catalytic RNP domain and indicate a central role for the aforementioned proteins in sustaining its catalytically active structure.

Protein composition of human prespliceosomes isolated by a tobramycin affinity-selection method.

Detailed knowledge of the composition and structure of the spliceosome and its assembly intermediates is a prerequisite for understanding the complex process of pre-mRNA splicing. To this end, we have developed a tobramycin affinity-selection method that is generally applicable for the purification of native RNP complexes. By using this method, we have isolated human prespliceosomes that are ideally suited for both biochemical and structural studies. MS identified >70 prespliceosome-associated proteins, including nearly all known U1 and U2 snRNP proteins, and expected non-snRNP splicing factors. In addition, the DEAD-box protein p68, RNA helicase A, and a number of proteins that appear to perform multiple functions in the cell, such as YB-1 and TLS, were detected. Several previously uncharacterized proteins of unknown function were also identified, suggesting that they play a role in splicing and potentially act during prespliceosome assembly. These data provide insight into the complexity of the splicing machinery at an early stage of its assembly.

Importin 8 is a gene silencing factor that targets argonaute proteins to distinct mRNAs.

Small regulatory RNAs including small interfering RNAs (siRNAs) and microRNAs (miRNAs) guide Argonaute (Ago) proteins to specific target RNAs leading to mRNA destabilization or translational repression. Here, we report the identification of Importin 8 (Imp8) as a component of miRNA-guided regulatory pathways. We show that Imp8 interacts with Ago proteins and localizes to cytoplasmic processing bodies (P bodies), structures involved in RNA metabolism. Furthermore, we detect Ago2 in the nucleus of HeLa cells, and knockdown of Imp8 reduces the nuclear Ago2 pool. Using immunoprecipitations of Ago2-associated mRNAs followed by microarray analysis, we further demonstrate that Imp8 is required for the recruitment of Ago protein complexes to a large set of Ago2-associated target mRNAs, allowing for efficient and specific gene silencing. Therefore, we provide evidence that Imp8 is required for cytoplasmic miRNA-guided gene silencing and affects nuclear localization of Ago proteins.

Protein Composition and Electron Microscopy Structure of Affinity-Purified Human Spliceosomal B Complexes Isolated under Physiological Conditions

ABSTRACT 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 ∼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 Å 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.

CASK Functions as a Mg2+-independent neurexin kinase

CASK is a unique MAGUK protein that contains an N-terminal CaM-kinase domain besides the typical MAGUK domains. The CASK CaM-kinase domain is presumed to be a catalytically inactive pseudokinase because it lacks the canonical DFG motif required for Mg2+ binding that is thought to be indispensable for kinase activity. Here we show, however, that CASK functions as an active protein kinase even without Mg2+ binding. High-resolution crystal structures reveal that the CASK CaM-kinase domain adopts a constitutively active conformation that binds ATP and catalyzes phosphotransfer without Mg2+. The CASK CaM-kinase domain phosphorylates itself and at least one physiological interactor, the synaptic protein neurexin-1, to which CASK is recruited via its PDZ domain. Thus, our data indicate that CASK combines the scaffolding activity of MAGUKs with an unusual kinase activity that phosphorylates substrates recuited by the scaffolding activity. Moreover, our study suggests that other pseudokinases (10% of the kinome) could also be catalytically active.

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‐mRNAs 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 ∼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 (∼260 × 200 × 195 Å) 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.

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.

Sm protein–Sm site RNA interactions within the inner ring of the spliceosomal snRNP core structure

Seven Sm proteins, E, F, G, D1, D2, D3 and B/B′, assemble in a stepwise manner onto the single‐stranded Sm site element (PuAU4–6GPu) of the U1, U2, U4 and U5 spliceosomal snRNAs, resulting in a doughnut‐shaped core RNP structure. Here we show by UV cross‐linking experiments using an Sm site RNA oligonucleotide (AAUUUUUGA) that several Sm proteins contact the Sm site RNA, with the most efficient cross‐links observed for the G and B/B′ proteins. Site‐specific photo‐cross‐linking revealed that the G and B/B′ proteins contact distinct uridines (in the first and third positions, respectively) in a highly position‐specific manner. Amino acids involved in contacting the RNA are located at equivalent regions in both proteins, namely in loop L3 of the Sm1 motif, which has been predicted to jut into the hole of the Sm ring. Our results thus provide the first evidence that, within the core snRNP, multiple Sm protein–Sm site RNA contacts occur on the inner surface of the heptameric Sm protein ring.

Conservation of the Protein Composition and Electron Microscopy Structure of Drosophila melanogaster and Human Spliceosomal Complexes

ABSTRACT Comprehensive proteomics analyses of spliceosomal complexes are currently limited to those in humans, and thus, it is unclear to what extent the spliceosomes highly complex composition and compositional dynamics are conserved among metazoans. Here we affinity purified Drosophila melanogaster spliceosomal B and C complexes formed in Kc cell nuclear extract. Mass spectrometry revealed that their composition is highly similar to that of human B and C complexes. Nonetheless, a number of Drosophila-specific proteins were identified, suggesting that there may be novel factors contributing specifically to splicing in flies. Protein recruitment and release events during the B-to-C transition were also very similar in both organisms. Electron microscopy of Drosophila B complexes revealed a high degree of structural similarity with human B complexes, indicating that higher-order interactions are also largely conserved. A comparison of Drosophila spliceosomes formed on a short versus long intron revealed only small differences in protein composition but, nonetheless, clear structural differences under the electron microscope. Finally, the characterization of affinity-purified Drosophila mRNPs indicated that exon junction complex proteins are recruited in a splicing-dependent manner during C complex formation. These studies provide insights into the evolutionarily conserved composition and structure of the metazoan spliceosome, as well as its compositional dynamics during catalytic activation.

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.