Two RNA Folds from One Sequence: A Ribozyme with Versatile Substrate Processing Abilities.

We report the design of a single RNA sequence capable of adopting one of two ribozyme folds and catalyzing the cleavage and/or ligation of the respective substrates. The RNA is able to change its conformation in response to its environment, hence it is called chameleon ribozyme (CHR). Efficient RNA cleavage of two different substrates as well as RNA ligation by CHR is demonstrated in separate experiments and in a one pot reaction. Our study shows that sequence variants of the hairpin ribozyme intersect with the hammerhead ribozyme and that rather short RNA molecules can have comprehensive conformational flexibility, which is an important feature for the emergence of new functional folds in early evolution.

A Hairpin Ribozyme Derived Spliceozyme.

The vast majority of RNA splicing in today's organisms is achieved by the highly regulated and precise removal of introns from pre-mRNAs via the spliceosome. Here we present a model of how RNA splicing may have occurred in earlier life forms. We have designed a hairpin ribozyme derived spliceozyme that mediates two RNA cleavages and one ligation event at specific positions and thus cuts a segment (intron) out of a parent RNA and ligates the remaining fragments (exons). The cut-out intron then performs a downstream function, acting as a positive regulator of the activity of a bipartite DNAzyme. This simple scenario shows how small RNAs can perform complex RNA processing dynamics, involving the generation of new phenotypes by restructuring segments of given RNA species, as well as delivering small RNAs that may play a functional role in downstream processes.

CAG-Repeat RNA Hairpin Folding and Recruitment to Nuclear Speckles with a Pivotal Role of ATP as a Cosolute.

A hallmark of Huntington's disease (HD) is a prolonged polyglutamine sequence in the huntingtin protein and, correspondingly, an expanded cytosine, adenine, and guanine (CAG) triplet repeat region in the mRNA. A majority of studies investigating disease pathology were concerned with toxic huntingtin protein, but the mRNA moved into focus due to its recruitment to RNA foci and emerging novel therapeutic approaches targeting the mRNA. A hallmark of CAG-RNA is that it forms a stable hairpin in vitro which seems to be crucial for specific protein interactions. Using in-cell folding experiments, we show that the CAG-RNA is largely destabilized in cells compared to dilute buffer solutions but remains folded in the cytoplasm and nucleus. Surprisingly, we found the same folding stability in the nucleoplasm and in nuclear speckles under physiological conditions suggesting that CAG-RNA does not undergo a conformational transition upon recruitment to the nuclear speckles. We found that the metabolite adenosine triphosphate (ATP) plays a crucial role in promoting unfolding, enabling its recruitment to nuclear speckles and preserving its mobility. Using in vitro experiments and molecular dynamics simulations, we found that the ATP effects can be attributed to a direct interaction of ATP with the nucleobases of the CAG-RNA rather than ATP acting as "a fuel" for helicase activity. ATP-driven changes in CAG-RNA homeostasis could be disease-relevant since mitochondrial function is affected in HD disease progression leading to a decline in cellular ATP levels.

RNA self-splicing by engineered hairpin ribozyme variants.

Small RNAs capable of self-cleavage and ligation might have been the precursors for the much more complex self-splicing group I and II introns in an early RNA world. Here, we demonstrate the activity of engineered hairpin ribozyme variants, which as self-splicing introns are removed from their parent RNA. In the process, two cleavage reactions are supported at the two intron-exon junctions, followed by ligation of the two generated exon fragments. As a result, the hairpin ribozyme, here acting as the self-splicing intron, is cut out. Two self-splicing hairpin ribozyme variants were investigated, one designed by hand, the other by a computer-aided approach. Both variants perform self-splicing, generating a cut-out intron and ligated exons.