Protein complexes containing Prp19 play a central role during catalytic activation of the spliceosome, and Prp19 and its related proteins are major components of the spliceosome's catalytic core RNP. To learn more about the spatial organization of the human Prp19 (hPrp19)/CDC5L complex, which is comprised of hPrp19, CDC5L, PRL1, AD002, SPF27, CTNNBL1, and HSP73, we purified native hPrp19/CDC5L complexes from HeLa cells stably expressing FLAG-tagged AD002 or SPF27. Stoichiometric analyses indicated that, like Saccharomyces cerevisiae NTC (nineteen complex), the human Prp19/CDC5L complex contains four copies of hPrp19. Salt treatment identified a stable core comprised of CDC5L, hPrp19, PRL1, and SPF27. Proteinprotein interaction studies revealed that SPF27 directly interacts with each component of the hPrp19/CDC5L complex core and also elucidated several additional, previously unknown interactions between hPrp19/CDC5L complex components. Limited proteolysis of the hPrp19/CDC5L complex revealed a protease-resistant complex comprised of SPF27, the C terminus of CDC5L, and the N termini of PRL1 and hPrp19. Under the electron microscope, purified hPrp19/CDC5L complexes exhibit an elongated, asymmetric shape with a maximum dimension of ϳ20 nm. Our findings not only elucidate the molecular organization of the hPrp19/CDC5L complex but also provide insights into potential protein-protein interactions at the core of the catalytically active spliceosome.Pre-mRNA splicing, the two consecutive transesterification reactions leading to intron removal and exon ligation, is catalyzed by the spliceosome, a highly dynamic, multiplemegadalton molecular machine (41). The major subunits of the spliceosome are the U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs). Each snRNP consists of an RNA moeity, the snRNA, and a set of particle-specific proteins, plus seven Sm proteins (or Lsm proteins in the case of U6) that are found in all of the spliceosomal snRNPs. In addition, the spliceosome is comprised of numerous non-snRNP proteins, some of which are preassembled into stable heteromeric complexes.Spliceosome assembly occurs in a stepwise and highly dynamic manner (41). Initially, the U1 snRNP binds the 5Ј splice site, followed by the ATP-dependent recognition of the pre-mRNA's branch point sequence (BPS) by the U2 snRNP, forming the prespliceosome or A complex. The assembly of snRNPs on the pre-mRNA is completed by the addition of the U4/U6-U5 trisnRNP, generating the precatalytic B complex, which is still catalytically inactive. In order to catalyze the first step of splicing, the spliceosome must undergo dramatic compositional and structural remodeling events, culminating in the destabilization of the U1 and U4 snRNPs and the formation of the catalytically activated spliceosome (B* complex). The first transesterification reaction then occurs, generating the C complex, which in turn catalyzes the second step of splicing. After catalysis, the postspliceosomal complex dissociates, releasing the mRNA and the U2, U5...

Many cellular processes are driven by protein complexes. Although the identification of protein components in such complexes has become almost a routine matter, accurate determination of their stoichiometry within a protein complex is still a challenge. We have established a method to determine the stoichiometries of protein complexes using absolute quantification (AQUA) with the help of synthetic standard peptides in combination with multiple reaction monitoring (MRM). Our approach is exemplified by the analysis of the human spliceosomal hPrp19/CDC5L complex, which consists of seven individual proteins and plays a crucial role in the assembly of the fully catalytically active spliceosome during pre-mRNA splicing. We evaluated several conditions for complete hydrolysis of the protein complex and found that the denaturing conditions under which hydrolysis is performed are absolutely crucial for accurately determining protein stoichiometries within this complex. In addition, we tested the suitability of different AQUA peptides and further compared different MS techniques to read out the relative signal intensities that were then used in absolute quantification. Our analyses revealed that dependent on the denaturing conditions different stoichiometries within the complex were obtained. The most consistent results were obtained by enzymatic hydrolysis in the presence of acetonitrile in combination with MRM.

Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5 splice sites (5ss). This interaction did not require branch point recognition and was sensitive to 5ss mutations, suggesting that 48K interacts with the 5ss during the first steps of prespliceosome assembly in a sequence-dependent manner. RNA interference-induced knockdown of 48K in HeLa cells led to reduced cell growth and the inhibition of U12-type splicing, as well as the activation of cryptic, U2-type splice sites, suggesting that 48K plays a critical role in U12-type intron recognition. 48K knockdown also led to reduced levels of U11/U12 di-snRNP, indicating that 48K contributes to the stability and/or formation of this complex. In addition to making contact with the 5ss, 48K interacts with the U11-59K protein, a protein at the interface of the U11/U12 di-snRNP. These studies provide important insights into the protein-mediated recognition of the U12-type 5ss, as well as functionally important interactions within the U11/U12 di-snRNP.Most metazoans and some unicellular eukaryotes contain two distinct spliceosomes (reference 28 and references therein). The majority of introns are excised by the major, U2-dependent spliceosome, while a subset (Ͻ0.5%) with highly conserved 5Ј splice sites (5Јss) and branch point sequences (BPS) are removed by the minor, U12-dependent spliceosome (for a review, see reference 24). Both spliceosomes consist of five small nuclear ribonucleoprotein particles (snRNPs) and numerous non-snRNP proteins. The two spliceosomes share the U5 snRNP, while the remaining four snRNPs in each spliceosome are distinct but functionally analogous, with U11, U12, U4atac, and U6atac of the minor spliceosome being the counterparts of U1, U2, U4, and U6 in the major spliceosome, respectively (12,14,16,34,35,42).Intron recognition is achieved via multiple, dynamic RNAand protein-mediated interactions. RNA-RNA interactions in the two spliceosomes are highly analogous. In the major spliceosome, the first assembly step (generating the E complex) involves the recognition of the 5Јss by U1, while the nonsnRNP protein factors SF1, U2AF65, and U2AF35 bind to the BPS, the polypyrimidine tract, and the 3Јss, respectively (for a review, see reference 26). During this stage, U1 base pairs with the pre-mRNA's 5Јss, while U1-associated proteins facilitate 5Јss recognition or stabilize the U1-5Јss complex (for a review, see reference 37). During prespliceosome (A complex) formation, U2 associates stably with the BPS (17), while non-snRNP proteins bridge U1 and U2 snRNPs (40, 41). The U4/U6/U5 tri-snRNP then joins the spliceosome (generating the B complex), which triggers the displacement of U1 from the 5Јss by U6 and additional rearrangements that lead to catalytic core formation (for a review, see references 22 and 33).The overall assembly pathway of the minor...

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.

Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.

An mRNA encoding the esterase from Alicyclobacillus acidocaldarius with catalytically essential serine codon (ACG) replaced by an amber (UAG) codon was used to study the suppression in in vitro translation system. Suppression of UAG by tRNA Ser(CUA) was monitored by determination of the full-length and active esterase. It was shown that commonly used increase of suppressor tRNA concentration inhibits protein production and therefore limits suppression. In situ deactivation of release factor by specific antibodies leads to efficient suppression already at low suppressor tRNA concentration and allows an in vitro synthesis of fully active enzyme in high yield undistinguishable from wild-type protein.

Humanized hapten-binding IgGs were designed with an accessible cysteine close to their binding pockets, for specific covalent payload attachment. Individual analyses of known structures of digoxigenin (Dig)- and fluorescein (Fluo) binding antibodies and a new structure of a biotin (Biot)-binder, revealed a “universal” coupling position (52+2) in proximity to binding pockets but without contributing to hapten interactions. Payloads that carry a free thiol are positioned on the antibody and covalently linked to it via disulfides. Covalent coupling is achieved and driven toward complete (95–100%) payload occupancy by spontaneous redox shuffling between antibody and payload. Attachment at the universal position works with different haptens, antibodies, and payloads. Examples are the haptens Fluo, Dig, and Biot combined with various fluorescent or peptidic payloads. Disulfide-bonded covalent antibody-payload complexes do not dissociate in vitro and in vivo. Coupling requires the designed cysteine and matching payload thiol because payload or antibody without the Cys/thiol are not linked (<5% nonspecific coupling). Hapten-mediated positioning is necessary as hapten-thiol-payload is only coupled to antibodies that bind matching haptens. Covalent complexes are more stable in vivo than noncovalent counterparts because digoxigeninylated or biotinylated fluorescent payloads without disulfide-linkage are cleared more rapidly in mice (approximately 50% reduced 48 hour serum levels) compared with their covalently linked counterparts. The coupling technology is applicable to many haptens and hapten binding antibodies (confirmed by automated analyses of the structures of 140 additional hapten binding antibodies) and can be applied to modulate the pharmacokinetics of small compounds or peptides. It is also suitable to link payloads in a reduction-releasable manner to tumor- or tissue-targeting delivery vehicles.—Dengl, S., Hoffmann, E., Grote, M., Wagner, C., Mundigl, O., Georges, G., Thorey, I., Stubenrauch, K.-G., Bujotzek, A., Josel, H.-P., Dziadek, S., Benz, J., Brinkmann, U. Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies.

TriFabs are IgG-shaped bispecific antibodies (bsAbs) composed of two regular Fab arms fused via flexible linker peptides to one asymmetric third Fab-sized binding module. This third module replaces the IgG Fc region and is composed of the variable region of the heavy chain (VH) fused to CH3 with “knob”-mutations, and the variable region of the light chain (VL) fused to CH3 with matching “holes”. The hinge region does not contain disulfides to facilitate antigen access to the third binding site. To compensate for the loss of hinge-disulfides between heavy chains, CH3 knob-hole heterodimers are linked by S354C-Y349C disulphides, and VH and VL of the stem region may be linked via VH44C-VL100C disulphides. TriFabs which bind one antigen bivalent in the same manner as IgGs and the second antigen monovalent “in between” these Fabs can be applied to simultaneously engage two antigens, or for targeted delivery of small and large (fluorescent or cytotoxic) payloads.