A modular protein architecture for generalized RNA targeting: development and application to RNA

monitoring, control, and protein scaffolding

Daniel A. Martin-­�Alarcon, Katarzyna Adamala, Edward S. Boyden

A key goal of synthetic biology is to program cellular processes towards scientific and engineering goals. Recent advances in modular strategies for DNA editing have expanded our access to the eukaryotic genome, but our ability to target the transcriptome remains limited by the proteins available for RNA binding. Many RNA-­�binding proteins are known that bind to specific sequences, raising the question of whether a modular protein architecture could be generated with appropriately concatenated modules capable of targeting arbitrary RNA sequences. Here we present a system capable of such targeting, based on the human Pumilio homology domain (PumHD) protein. We developed a set of modular protein building blocks, one for each of the four RNA bases, which can be concatenated in chains to bind arbitrary single-­�stranded RNAs. We call our system Pumilio based assembly, or Pumby.

We validated the binding specificity of proteins based on Pumby modules to arbitrary RNA sequences, as well as their ability to detect particular transcripts in human cells. We further discovered that Pumby chains can report the translation state of specific open reading frames within those transcripts, enabling monitoring of protein production in live cells. We demonstrated the use of Pumby proteins to trigger RNA degradation and, conversely, to upregulate the translation of open reading frames, even those not preceded by ribosome binding sites. All these applications of Pumby proteins can be applied to unmodified native genes, without the need to introduce exogenous RNA target sequences.

Our system may open up many frontiers in the observation and control of RNA processes in living cells. We anticipate that the Pumby architecture could enable the orthogonal, simultaneous manipulation of multiple RNA targets within a cell. RNA manipulation could provide an additional layer of control over classical transcriptional genetic circuits. The availability of multiple orthogonal RNA-­�binding proteins may significantly augment the potential of mRNA as an intracellular scaffold for generating novel biomolecules. The Pumby architecture opens the door for many scientific inquiries and biotechnological applications of RNA in living systems.