Distribution of cell type-specific lincRNAs in primary sensory neurons and their function in itch transmission

On December 16, EMBO Reports, an international academic journal, published the latest collaborative research paper by Bao Lan Group of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, in the form of Resource, "Somatosensory neurons express specific sets of lincRNAs, and lincRNA CLAP promotes itch sensation in mice"
。 This study systematically analyzed and constructed the expression profile of long intergenic noncoding RNA (lincRNA) specific between cell types in primary sensory neurons through single-cell sequencing, and revealed the involvement of cell type-specific expression of lincRNA CLAP in regulating itch function and its molecular mechanism
.

Bao Lan's research group has long studied
the function and molecular mechanism of non-coding RNA in neuronal development.
Previous research work in the research group found that microRNAs (miRNAs) enriched in axons of primary sensory neurons participate in axon extension by regulating local translation of mRNA in axons (Wang et al.
, Cell Reports, 2015; Wang & Bao, Journal of Molecular Cell Biology, 2017）
。 Recent research by the research group has found that axon-enriched long-stranded non-coding RNA (lncRNA) ALAE regulates axonal local translation and axon growth by competing for the interaction of the RNA-binding protein KHSRP with Gap43 mRNA (Wei et al.
, Cell Reports, 2021),_m6 of lncRNA Dubr A dynamic modification is involved in neuronal migration and axon development by stabilizing the RNA-binding protein YTHDF1/3 mediating mRNA translation (Huang et al.
, Cell Reports, 2022), and these studies further reveal that noncoding RNAs with specific distribution of neurons play an important regulatory role
in nervous system development.

Primary sensory neurons are pseudounipolar neurons, and their somatosomes aggregate to form the Dorsal root ganglion (DRG).

DRG neurons transmit peripheral stimuli through the spinal cord to the brain, transmitting pain, heat, cold, itching, mechanical and proprioceptive sensations
.
There are different types of neurons that are highly specialized in primary sensory neurons, and the previous research of researcher Zhang Xu's team in the cooperative laboratory divided DRG neurons into 10 large categories and 14 subcategories through single-cell sequencing, and revealed that different types of neurons participate in sensory transmission of different peripheral stimuli (Li et a.
, Cell Research, 2016; Wang et al.
, Cell Research, 2021）
。 Although the classification and function of primary sensory neurons are well understood, the molecular regulatory mechanisms specific to neuron type are far from being well understood
.

lincRNAs are a class of noncoding RNAs
transcribed from intergenic regions that code, lack protein translation capabilities, and are more than 200 nucleotides long.
Current analysis of multiomic sequencing data suggests that lincRNA in the nervous system has higher tissue specificity than mRNA and may play an important role
.
Therefore, it is of great significance
to explore the distribution, function and mechanism of lincRNA in different types of DRG neurons to solve the molecular mechanism of sensory transmission induced by different peripheral stimuli.

In this study, the expression profile
of lincRNA of single neurons was constructed by integrating and analyzing the single-cell sequencing data of primary sensory neurons of Smart-seq2 and 10x Genomics.
200 cell type-specific and highly expressed lincRNAs were screened and discovered by calculating the Specificity measure (SPM) method, and lincRNAs of different cell types were further verified
by single-cell PCR, in situ hybridization and flow cytometry sorting 。 At the same time, it was also found that cell type-specific lincRNAs were highly correlated with neighboring genes on their genomes, and because the lincRNAs localized in the nucleus could participate in regulating the expression of neighboring genes, suggesting that cell type-specific lincRNAs may have cis-regulatory effects
.

Subsequently, through the evolutionary conserved analysis of cell type-specific lincRNA, it was found that lncRNA 9130409J20Rik was highly expressed in neurons expressing somatostatin (Sst⁺), and the conserved nature was highest
in multiple species.
Using gene co-expression analysis, lncRNA 9130409J20Rik is highly correlated with itch function, so we named it CLAP (Cell-type-specificLincRNAAssociated withPruritus).

。 Furthermore, neuronal calcium imaging, intrathecal injection of siRNA and the behavioral detection of itch after shRNA carried by AAV virus, and RNA-seq experiments were used to find that knocking down CLAP significantly inhibited the activation of histamine on Sst+ neurons, reduced the itch-scratching response of mice to histamine-induced itching, and led to significant downregulation
of itch-related genes in Sst⁺ neurons 。 Finally, through high-throughput co-immunoprecipitation sequencing database (CLIP-seq) analysis, RNA co-immunoprecipitation and intrathecal injection of siRNA in mice, it was found that the RNA-binding protein MSI2 binds to CCAP and participates in regulating the expression
of CLAP to itch-related genes.

In summary, single-cell sequencing was used to establish the lincRNA expression profile of the specific distribution of primary sensory neurons, revealing the regulatory function and molecular mechanism
of cell type-specific expression lincRNA in itch transmission.
This study not only helps to deeply understand the role and mechanism of different types of specific distribution of lincRNAs in neurons in different somatosensory information transmission, but also provides a theoretical basis
for the pathogenesis and clinical diagnosis and treatment of sensation-related diseases such as pain and chronic itching.

Wang Bin, researcher of Guangdong Academy of Intelligence, doctoral student of Bao Lan's research group of the Center of Excellence of Molecular Cells, Jiang Bowen, and Li Guowei, a doctoral student in Yang Li's team at the Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, are co-first authors
of this paper.
Researcher Bao Lan, researcher Yang Li and researcher Wang Bin are co-corresponding authors
.
This work was strongly supported
by researcher Zhang Xu from Shanghai Advanced Research Institute of Chinese Academy of Sciences/Guangdong Institute of Intelligent Science and Technology, researcher Li Changlin from Guangdong Institute of Intelligent Science and Technology, researcher Han Qingjian from the Institute of Brain Science of Fudan University, and researcher Xu Zhenzhong from Zhejiang University School of Medicine.
The work was supported
by the National Foundation of China, the Guangdong High-level Innovation Research Institute Program, and the Chinese Academy of Medical Sciences Project.

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Single-cell sequencing to identify primary sensory neuronal cell type-specific lincRNAs

Single-cell sequencing was used to construct a cell type-specific lincRNA expression profile of primary sensory neurons, and the functional and molecular mechanisms of lincRNA CCAP in regulating itch were discovered