Structural basis for guide RNA selection by the RESC1-RESC2 complex.

Kinetoplastid parasites, such as trypanosomes or leishmania, rely on RNA-templated RNA editing to mature mitochondrial cryptic pre-mRNAs into functional protein-coding transcripts. Processive pan-editing of multiple editing blocks within a single transcript is dependent on the 20-subunit RNA editing substrate binding complex (RESC) that serves as a platform to orchestrate the interactions between pre-mRNA, guide RNAs (gRNAs), the catalytic RNA editing complex (RECC), and a set of RNA helicases. Due to the lack of molecular structures and biochemical studies with purified components, neither the spacio-temporal interplay of these factors nor the selection mechanism for the different RNA components is understood. Here we report the cryo-EM structure of Trypanosoma brucei RESC1-RESC2, a central hub module of the RESC complex. The structure reveals that RESC1 and RESC2 form an obligatory domain-swapped dimer. Although the tertiary structures of both subunits closely resemble each other, only RESC2 selectively binds 5'-triphosphate-nucleosides, a defining characteristic of gRNAs. We therefore propose RESC2 as the protective 5'-end binding site for gRNAs within the RESC complex. Overall, our structure provides a starting point for the study of the assembly and function of larger RNA-bound kinetoplast RNA editing modules and might aid in the design of anti-parasite drugs.

Strategy to replace animal-derived ECM by a modular and highly defined matrix.

Many extracellular matrices (ECM) used for modern cell culture are derived from animals. An alternative approach is the recombinant production of individual matrix protein components. A further development of this strategy uses a constant core protein polymer that is modifiable with functional domains of various ECM proteins. This way, a single, highly defined ECM system could be used for a large variety of cell types. Self-assembling protein domains from human muscle sarcomeres, termed here ZT material (ZT), have been shown to be suitable for this modular approach of generating ECMs. We explored in a proof-of-concept study, whether ZT, modified with the fibronectin 10 domain (ZTFn10) is able to substitute bovine serum-derived fibronectin as coating for neural crest cell (NCC)-based toxicity testing. Human NCC were generated from pluripotent stem cells and used in the automated version of a NCC migration assay (cMINC). ZTFn10, but not the unmodified core material (ZT), allowed for a high migration activity. The classical cMINC setup, with bovine fibronectin coating, was used as positive control, and detailed analysis of NCC migration by time-lapse recording indicated that the novel ECM fully matched the bioactivity of the traditional ECM. A final set of experiments showed that various positive controls of the cMINC assay (PCB180, LiCl, cytochalasin D) showed nearly identical inhibition curves on the traditional and the novel ECM. Thus, the cMINC, and possibly other bioassays, can be performed with a ZT-based ECM instead of traditional animal-derived protein coatings.

The ZT Biopolymer: A Self-Assembling Protein Scaffold for Stem Cell Applications.

The development of cell culture systems for the naturalistic propagation, self-renewal and differentiation of cells ex vivo is a high goal of molecular engineering. Despite significant success in recent years, the high cost of up-scaling cultures, the need for xeno-free culture conditions, and the degree of mimicry of the natural extracellular matrix attainable in vitro using designer substrates continue to pose obstacles to the translation of cell-based technologies. In this regard, the ZT biopolymer is a protein-based, stable, scalable, and economical cell substrate of high promise. ZT is based on the naturally occurring assembly of two human proteins: titin-Z1Z2 and telethonin. These protein building blocks are robust scaffolds that can be conveniently functionalized with full-length proteins and bioactive peptidic motifs by genetic manipulation, prior to self-assembly. The polymer is, thereby, fully encodable. Functionalized versions of the ZT polymer have been shown to successfully sustain the long-term culturing of human embryonic stem cells (hESCs), human induced pluripotent stem cells (hiPSCs), and murine mesenchymal stromal cells (mMSCs). Pluripotency of hESCs and hiPSCs was retained for the longest period assayed (4 months). Results point to the large potential of the ZT system for the creation of a modular, pluri-functional biomaterial for cell-based applications.