Isaac Newton based his first Rule of Reasoning in Philosophy on the teleological principle of purposeful design and assertion that “Nature is pleased with simplicity.”  Such simplicity is often illustrated in Nature’s elegant solution to control gene expression with a modular molecule. In many key gene expression steps, such as RNA splicing, the regulatory proteins usually comprise an RNA binding module to recognize target and a functional module to activate or inhibit the biochemical process. We have adopted this simple design principle to engineer protein factors that can manipulate RNA metabolism such as the alternative splicing or RNA degradation.

We take advantage of an unique RNA binding domain, PUF domain, which have 8 repeats to recognize 8-nt RNA target, with each PUF repeat recognizing the edge of an RNA base (Figure 1 A).  Three amino acids in each PUF repeat determine what RNA base it will specifically bind, and we have solved the RNA binding code for all the four bases.  Therefore the specificity of a PUF domain can be reprogrammed by changing each repeat, and we can produce various “designer PUF” with customized specificity.

Engineered splicing factors (ESFs):

A large number of naturally occurring splicing factors have modular configuration: they have a separable RNA binding module to recognize RNA target and a functional domain to activate or inhibit splicing. We have developed engineered splicing factors (ESFs) by fusing a PUF domain with Arg/Ser-rich (RS) domains of SRSF1 or the glycine-rich (Gly) domain of hnRNP A1 (Fig. 1B). The resulting ESFs can function either as splicing activators (PUF-RS) or inhibitors (PUF-Gly), and can specifically control different types of alternative splicing.

 Figure 1. Design principles for engineered splicing fcators

 

Engineered splicing factors against cancer

Using this general approach, a PUF-Gly ESF was generated to shift splicing of the Bcl-x pre-mRNA from the anti-apoptotic long isoform (Bcl-xL) to the pro-apoptotic short isoform (Bcl-xS). The altered splice isoform distribution was sufficient to sensitize several cancer cell lines to multiple anti-cancer drugs, thus can sensitize cancer cells to multiple chemotherapy drugs.

 

Engineered RNA endonucleases with designable specificity

Specific cleavage of RNAs is critical for in vitro manipulation of RNA and for in vivo gene silencing. However, a simple enzyme that cleaves RNA in a sequence-specific manner has not been found in Nature despite extensive investigations. Most RNA endonucleases either have limited sequence specificity, or recognize their targets by specific structures or through guide RNA that pairs with target. By fusing a PUF domain with a non-specific endoribonuclease domain (PIN domain of Smg6p), We developed artificial site-specific RNA endonucleases (ASREs) that specifically recognize RNA substrates and efficiently cleave near the binding sites both in vitro and in cultured cells. Since PUF domains recognize their targets through an 8-nt sequence, comparable length to the seed match of siRNA, engineered ASREs may serve as an RNA silencing tool complementary to RNAi, which will be effective in organisms or cellular compartments where RNAi machinery is not present.