Molecular Cell: Famous Israeli scholars decipher the rules of RNA editing

As an important RNA editing enzyme, ADAR1 catalyzes A-to-I RNA editing at thousands of sites on the human transcriptome
.

Although people have studied ADAR1 from multiple angles, the rules for its substrate selection are still poorly understood

This paper entitled "Deciphering the principles of the RNA editing code via large-scale systematic probing" was recently published in the journal "Molecular Cell".
Application
.

As we all know, RNA molecules are similar in structure to DNA molecules and are composed of four different bases, but unlike DNA, they have a limited life span and are mostly single-stranded
.

Some RNA molecules in double-stranded form are sometimes modified by ADAR1 to deaminate adenosine (A) into inosine (I), which may be used to distinguish the double-stranded RNA of the cell itself from the double-stranded RNA of the virus Come

From the perspective of physiology, bioengineering and treatment, ADAR1 has aroused great interest among scientists
.

However, they still don't understand the basic rules behind A-to-I RNA editing, such as which sites in the double-stranded RNA are edited, and to what level

Systematic screening of ADAR1 substrates

In order to systematically reveal the determinants of A-to-I editing, the researchers designed two double-stranded hairpin structure reporter sequences: B2 and mNG constructs
.

Later, they designed an oligonucleotide library on this basis, containing approximately 2,000 different RNA variants, and commissioned Twist Bioscience to synthesize it (Figure 1)

Figure 1.

Sequence combination of oligonucleotide library

Twist Oligonucleotide Pool is a highly diverse collection of single-stranded oligonucleotides (ssDNA) synthesized using its proprietary silicon-based DNA preparation technology
.

 

▪ Oligonucleotides up to 300 nt, suitable for more application conditions
▪ Unparalleled uniformity
▪ Extremely high precision and accuracy
▪ Very low error rate, no more than 1:2000 bp
▪ In the same primer pool, pair specific There is no limit to the number of oligonucleotides

Directed and symmetrical RNA editing

The researchers observed a significant increase in editing levels at 35 bp upstream and 30 bp downstream of structural damage (Figure 2)
.

The 3 bp mismatch greatly increases the -35 and +30 editing levels

Figure 2.

Induction of editing at 35 bp upstream and 30 bp downstream of structural disruption

The researchers showed great interest in the 5-bp shift between 35 bp upstream and 30 bp downstream
.

They hypothesized two models and verified them through experiments

Improve the efficiency of RNA targeted editing

To further test the applicability of this editing rule, they recruited endogenous ADAR1 for programmable RNA editing
.

They designed a 151-nt mRNA targeting the SMAD4 gene and designed a 4-nt mismatch 35 bp from the targeted adenosine

Figure 3.
A-to-I editing model mediated by ADAR1

In general, the researchers proposed a directional, symmetric, periodic, and cyclic RNA editing model (Figure 3)
.
The ADAR1 enzyme first recognizes the slight structural damage and edits its upstream 35 bp
.
Then, it starts editing the opposite chain, with a 5-bp offset
.
In this step, they proposed three possible editing mechanisms
.
After that, ADAR1 recognizes the destruction of the new structure introduced by the editing event, and edits the upstream 35 bp again, to and fro
.
When the edited site is no longer in the double-stranded RNA structure, editing is terminated
.

This research deciphers the basic rules of RNA editing and deepens our understanding of RNA modification
.
RNA molecules have a short lifespan, and the changes they introduce will be short-lived.
Therefore, RNA editing is safer than editing that permanently changes DNA
.
In the future, it is expected to be used to correct some common mutations in genetic diseases, including cystic fibrosis
.

Original Search

Uzonyi A, Nir R, Shliefer O, Stern-Ginossar N, Antebi Y, Stelzer Y, Levanon EY, Schwartz S.
Deciphering the principles of the RNA editing code via large-scale systematic probing.
Mol Cell.
2021 Jun 3; 81( 11): 2374-2387.
e3.
doi: 10.
1016/j.
molcel.
2021.
03.
024.