New technology provides a "golden key" to unlock the mystery of genes

New technology provides a "golden key" to unlock the mystery of genes

Scientists create a new method for RNA-binding protein target research, LACE-seq

New technology provides a "golden key" to unlock the mystery of genes New technology provides a "golden key" to unlock the mystery of genes.

In eukaryotic cells, RNA can be "single shadow", or it can be "combined" with protein
.

The latter is called RNA-binding protein, and it has powerful gene regulation capabilities

What protein does RNA bind to? How will it affect human development and disease? These are all unsolved problems
.

On June 10th, Xue Yuanchao, a researcher at the Institute of Biophysics, Chinese Academy of Sciences, and his collaborators developed a new technology LACE-seq that can identify RNA-binding protein targets in trace cells, achieving single-base resolution and single-cell resolution for the first time.
The precise identification of the binding sites of RNA-binding proteins at the level opens the door for studying the functional mechanisms of RNA-binding proteins in embryonic development and reproductive diseases
.

Related research results were published in "Nature-Cell Biology"

Find tools

Find tools

The human genome encodes a total of about 1500 RNA binding proteins, which often regulate the processing, positioning, translation, and stability of various RNA molecules in cells by binding specific motifs or structural elements on RNA molecules
.

Studies have shown that RNA-binding proteins play a key regulatory role in almost all physiological processes such as early reproduction, ontogeny, cell differentiation, proliferation and apoptosis
.

At the same time, mutations in RNA binding proteins can cause a variety of genetic diseases

Therefore, accurate identification of the binding target and precise binding position of RNA-binding protein is a prerequisite for understanding its physiological and pathological regulatory mechanisms
.

Xue Yuanchao introduced that the currently commonly used methods for identifying RNA-binding protein targets mainly include RIP-seq and CLIP-seq
.

Because these two methods rely on the use of specific antibodies to enrich RNA-binding proteins and the RNA they bind, and require millions of cells, this severely limits the application of these methods in rare cell types and clinical puncture samples

For example, due to the lack of experimental methods that can study RNA-binding protein targets at the micro-cell or even single-cell level, people still do not know the molecular mechanism of RNA-binding protein and its complexes during early reproduction
.

"Currently, most researches on RNA-binding proteins are carried out in cell lines that can be expanded in vitro in large numbers, such as stem cells and somatic cells.
However, research on small samples such as early reproduction and clinical puncture is still a blank
.

" Xue Yuanchao told.

Open the door

Open the door

Su Ruibao, who has been engaged in RNA-related technology research and development at BGI for two years, joined Xue Yuanchao's team and became Xue Yuanchao's second doctoral student, laying the foundation for the team to develop the LACE-seq method
.

In order to establish this method, Su Ruibao carefully optimized every experimental condition
.

After the establishment of the LACE-seq method, the team focused on the target of the RNA-binding protein Ago2/endo-siRNA silencing complex in mouse MII egg cells, and analyzed the Ago2/endo-siRNA complex in the egg cell to regulate mRNA translation And a series of new mechanisms and new laws, such as the suppression of transposon-mediated generation of chimera transcripts

In application, the team used the created LACE-seq method to map the specific targets and binding positions of RNA-binding proteins such as Ddx4, Ptbp1, Ago2, and Mili in egg cells for the first time, and was the first to discover that the Ago2/endo-siRNA silencing complex was in egg cells.
It inhibits the translation of mRNA in a way of non-perfect complementary pairing, and proves that the protein level changes of Bub3, Chk1, Nuf2, etc.
of endo-siRNA targets may be related to the phenotype after Ago2 knockout
.

In addition, it was also proved that the Ago2/endo-siRNA complex can cleave the chimeric RNA initiated by the retrotransposon promoter, and this mechanism ensures the integrity of the transcriptome in the egg cell
.

LACE-seq technology has a series of advantages such as short experimental operation time, high signal-to-noise ratio, more effective data, and lower cost, which effectively solves the problems in this field and the shortcomings of existing experimental methods
.

These advantages make it possible to study the regulation and pathogenic mechanism of RNA binding proteins in rare and trace cells, such as early germ cells, cancer stem cells, and clinical puncture samples

The LACE-seq technology linearly amplifies the termination signal of the reverse transcriptase at the RNA-binding protein binding site, and for the first time realizes the precise identification of the RNA-binding protein binding site at the single-base resolution and single-cell level
.

It can be said that the LACE-seq technology has opened the door to the study of new laws regulating gene expression during early reproduction and embryonic development of RNA-binding proteins and their complexes
.

Create a map

Create a map

RNA binding protein, abbreviated as RBP in English
.

According to related studies, about 5-10% of human proteins can bind to RNA
.

But so far, researchers have not conducted a census of all RNA-binding proteins, and the number of known RBPs is still an estimate
.

RBP is related to the mystery of human genes, and its function is important.
The establishment of the "family" map of RBP can be more helpful for mining "gold mines"
.

The United States launched a large-scale research project in 2013 to use the enhanced UV cross-linking and immunoprecipitation method eCLIP to systematically decode the regulatory mechanism of RBP.
At present, 150 RBP binding maps have been successfully drawn
.

However, our country has no relevant RBP map research plan so far
.

"Next, we will cooperate with domestic counterparts, hoping to quickly promote the application of the LACE-seq method, especially in the research of reproduction and related diseases.
At the same time, we hope to establish a fully automated LACE-seq platform to annotate RBP on a large scale.
Functional mechanism, and initiated the RBP map plan with Chinese characteristics
.
" Xue Yuanchao said
.
(Source: China Science News Han Yangmei)

The LACE-seq method can accurately identify the binding target of PTBP1 in a small amount of cells (photo courtesy of the research group)

Related paper information: https://doi.
org/10.
1038/s41596-021-00524-2

https://doi.
org/10.
1038/s41596-021-00524-2