# Effective RNA Regulation by Combination of Multiple Programmable RNA-Binding Proteins
## 3 Results and Discussion
### Effective Regression of Reporter Genes with Not-Repetitive Sequences by Multiple TTP-PUF Proteins  
It is unusual that four or more times sequences exist at the regulatory region of single transcripts. Instead of targeting repeat sequences at the 3\({}^{\prime}\) UTR, the synergistic effect of multiple TTP-PUFs was shown through the targeting of multiple RNA sequences at the 3\({}^{\prime}\) UTR. As a model of transcripts, a reporter vector with a 1 \(\times\) (a-b-c-d) sequence at the 3\({}^{\prime}\) UTR of the firefly luciferase gene was designed, in which only one binding sequence for each PUF (A)-(D) existed at the 3\({}^{\prime}\) UTR (Figure 3a). In contrast to the 4-6-fold repression of the luciferase genes with 4 \(\times\) binding sequences, each TTP-PUF (A), (C), or (D) showed only about a 1.5-fold repression (Figure 4, lanes 3,5,6), and TTP-PUF (B) alone did not repress luciferase activity (Figure 4, lane 4), maybe because of the low TTP-PUF (B) expression compared to other TTP-PUF proteins (Figure S1c).  
When NIH3T3 cells were co-transfected with all of the expression vectors (TTP-PUF (A)-(D); 25 ng each) and the 1 \(\times\) (a-b-c-d) reporter vector, significant repression (2.5-fold) of luciferase activity was observed, even though the amount of each expression vector (25 ng each) was one-fourth of that when used alone (100 ng) (Figure 4, lane 2). The mixture of four TTP-PUF expression vectors had little effect on the reporter lacking a PUF-binding sequence (Figure S3). Furthermore, when the cells were transfected with 25 ng of one of the four TTP-PUF vectors, the same amount used for the mixture, none of them showed significant repression of the 1 \(\times\) (a-b-c-d) reporter (Figure 4, lanes 7-10). These results indicated that the luciferase activity of the 1 \(\times\) (a-b-c-d) reporter vector was repressed by the sequence-specific binding of TTP-PUF proteins to their binding sites at the 3\({}^{\prime}\) UTR. Additionally, the combination of four TTP-PUF proteins, targeting different RNA sequences, caused effective repression of luciferase activity compared to the use of a single TTP-PUF protein. Differences in repression ratios were observed between reporters with 4 \(\times\) binding sequences by the corresponding single TTP-PUF (4-6-fold) (Figure 3b) and the 1 \(\times\) (a-b-c-d) reporter by the mixture of TTP-PUF (A)-(D) (2.5-fold) (Figure 4). One plausible reason for the difference is avidity effects for the  
Figure 4: Combination of TTP-PUF (A)–(D) with 1 \(\times\) (a-b-c-d) reporter efficiently repressed the luciferase reporter activities at the 3\({}^{\prime}\) UTR of the luciferase mRNA. NIH3T3 cells were co-transfected with 1 \(\times\) (a-b-c-d) reporter (1 ng) and 100 ng (total) of the following expression vectors. Lane 1, control vector; lane 2, mixture of TTP-PUF (A), (B), (C), and (D) (25 ng each); lanes 3–6, 100 ng of TTP-PUF (A) (lane 3), (B) (lane 4), (C) (lane 5), (D) (lane 6); lanes 7–10, 75 ng of control vector and 25 ng of TTP-PUF (A) (lane 7), (B) (lane 8), (C) (lane 9), (D) (lane 10). Data are presented as mean \(\pm\) SD. (***, \(p<\) 0.005; **, \(p<\) 0.01; *, \(p<\) 0.05; n.s. = not significant).  
\(4\times\) proximity binding sites. The overall RNA structure of the PUF binding sequences can affect the binding of TTP-PUFs. Further optimization will increase the synergistic effects of multiple functional PUF proteins.  
So far, synergistic gene activation or repression was shown by a combination of multiple artificial transcription regulators based on transcription activator-like effectors (TALEs) and CRISPR-dCas9 [32; 33]. The results using a combination of multiple RNA binding proteins are in accordance with these reports targeting DNA.