RanBP9 controls the oligomeric state of CTLH complex assemblies.

The CTLH (C-terminal to lissencephaly-1 homology motif) complex is a multisubunit RING E3 ligase with poorly defined substrate specificity and flexible subunit composition. Two key subunits, muskelin and Wdr26, specify two alternative CTLH complexes that differ in quaternary structure, thereby allowing the E3 ligase to presumably target different substrates. With the aid of different biophysical and biochemical techniques, we characterized CTLH complex assembly pathways, focusing not only on Wdr26 and muskelin but also on RanBP9, Twa1, and Armc8ß subunits, which are critical to establish the scaffold of this E3 ligase. We demonstrate that the ability of muskelin to tetramerize and the assembly of Wdr26 into dimers define mutually exclusive oligomerization modules that compete with nanomolar affinity for RanBP9 binding. The remaining scaffolding subunits, Armc8ß and Twa1, strongly interact with each other and with RanBP9, again with nanomolar affinity. Our data demonstrate that RanBP9 organizes subunit assembly and prevents higher order oligomerization of dimeric Wdr26 and the Armc8ß-Twa1 heterodimer through its tight binding. Combined, our studies define alternative assembly pathways of the CTLH complex and elucidate the role of RanBP9 in governing differential oligomeric assemblies, thereby advancing our mechanistic understanding of CTLH complex architectures.

PROTAC-mediated degradation reveals a non-catalytic function of AURORA-A kinase.

The mitotic kinase AURORA-A is essential for cell cycle progression and is considered a priority cancer target. Although the catalytic activity of AURORA-A is essential for its mitotic function, recent reports indicate an additional non-catalytic function, which is difficult to target by conventional small molecules. We therefore developed a series of chemical degraders (PROTACs) by connecting a clinical kinase inhibitor of AURORA-A to E3 ligase-binding molecules (for example, thalidomide). One degrader induced rapid, durable and highly specific degradation of AURORA-A. In addition, we found that the degrader complex was stabilized by cooperative binding between AURORA-A and CEREBLON. Degrader-mediated AURORA-A depletion caused an S-phase defect, which is not the cell cycle effect observed upon kinase inhibition, supporting an important non-catalytic function of AURORA-A during DNA replication. AURORA-A degradation induced rampant apoptosis in cancer cell lines and thus represents a versatile starting point for developing new therapeutics to counter AURORA-A function in cancer.

Reconstitution of CPSF active in polyadenylation: recognition of the polyadenylation signal by WDR33.

Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four (CPSF160, CPSF30, hFip1, and WDR33) are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73, and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV cross-linked to such RNAs, as can CPSF30. Transcriptome-wide identification of WDR33 targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) showed that WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity. Thus, our data indicate that the large CPSF subunit participating in recognition of the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.

Reconstitution of mammalian cleavage factor II involved in 3' processing of mRNA precursors.

Cleavage factor II (CF II) is a poorly characterized component of the multiprotein complex catalyzing 3' cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5' kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.

Crystal structure of a covalently linked Aurora-A-MYCN complex.

Formation of the Aurora-A-MYCN complex increases levels of the oncogenic transcription factor MYCN in neuroblastoma cells by abrogating its degradation through the ubiquitin proteasome system. While some small-molecule inhibitors of Aurora-A were shown to destabilize MYCN, clinical trials have not been satisfactory to date. MYCN itself is considered to be `undruggable' due to its large intrinsically disordered regions. Targeting the Aurora-A-MYCN complex rather than Aurora-A or MYCN alone will open new possibilities for drug development and screening campaigns. To overcome the challenges that a ternary system composed of Aurora-A, MYCN and a small molecule entails, a covalently cross-linked construct of the Aurora-A-MYCN complex was designed, expressed and characterized, thus enabling screening and design campaigns to identify selective binders.

Sequential Processing of Cold Gas Sprayed Alloys by Milling and Deep Rolling.

Cold gas spraying (CS) is a solid-state material deposition process which, in addition to the flexible repair of individual component areas, also enables the build-up of larger samples. The layers are created on a substrate by the impact-induced bonding of highly accelerated micrometer particles. Since melting does not occur, the material composition can be varied flexibly and independently of material-specific melting points. In this work, the influence of the described forming process on subsequent machining by milling and deep rolling is investigated. The process forces measured during milling and the surface topography after milling and deep rolling were influenced by the material composition and the CS-related properties, e.g., high material hardness or particle bonding. In contrast to prior assumptions, deep rolling was shown to have no influence on the determined hardness depth profile for the investigated materials. Future work will focus on additional analyses, such as the determination of half-widths, to obtain further insight on the material behavior.