Global profiling of RNA-binding protein target sites by LACE-seq

Ruibao Su; Li-Hua Fan; Changchang Cao; Lei Wang; Zongchang Du; Zhaokui Cai; Ying-Chun Ouyang; Yue Wang; Qian Zhou; Ligang Wu; Nan Zhang; Xiaoxiao Zhu; Wen-Long Lei; Hailian Zhao; Yong Tian; Shunmin He; Catherine C L Wong; Qing-Yuan Sun; Yuanchao Xue

doi:10.1038/s41556-021-00696-9

Global profiling of RNA-binding protein target sites by LACE-seq

Nat Cell Biol. 2021 Jun;23(6):664-675. doi: 10.1038/s41556-021-00696-9. Epub 2021 Jun 9.

Authors

Ruibao Su^#^{1

2}, Li-Hua Fan^#^{3

4}, Changchang Cao^#¹, Lei Wang^{1

5}, Zongchang Du^{1

2}, Zhaokui Cai^{1

2}, Ying-Chun Ouyang⁴, Yue Wang^{2

4}, Qian Zhou^{2

4}, Ligang Wu⁶, Nan Zhang⁷, Xiaoxiao Zhu^{1

2}, Wen-Long Lei⁴, Hailian Zhao^{1

2}, Yong Tian^{1

2}, Shunmin He¹, Catherine C L Wong^{8

9

10

11}, Qing-Yuan Sun^{12

13}, Yuanchao Xue^{14

15}

Affiliations

¹ Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
² University of Chinese Academy of Sciences, Beijing, China.
³ Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China.
⁴ State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
⁵ College of Life Sciences, Xinyang Normal University, Xinyang, China.
⁶ State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China.
⁷ Center for Precision Medicine Multi-Omics Research, Peking University Health Science Center, Beijing, China.
⁸ Center for Precision Medicine Multi-Omics Research, Peking University Health Science Center, Beijing, China. [email protected].
⁹ School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China. [email protected].
¹⁰ Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, China. [email protected].
¹¹ Peking University First Hospital, Beijing, China. [email protected].
¹² Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China. [email protected].
¹³ State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China. [email protected].
¹⁴ Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China. [email protected].
¹⁵ University of Chinese Academy of Sciences, Beijing, China. [email protected].

^# Contributed equally.

PMID: 34108658
DOI: 10.1038/s41556-021-00696-9

Abstract

RNA-binding proteins (RBPs) have essential functions during germline and early embryo development. However, current methods are unable to identify the in vivo targets of a RBP in these low-abundance cells. Here, by coupling RBP-mediated reverse transcription termination with linear amplification of complementary DNA ends and sequencing, we present the LACE-seq method for identifying RBP-regulated RNA networks at or near the single-oocyte level. We determined the binding sites and regulatory mechanisms for several RBPs, including Argonaute 2 (Ago2), Mili, Ddx4 and Ptbp1, in mature mouse oocytes. Unexpectedly, transcriptomics and proteomics analysis of Ago2^-/- oocytes revealed that Ago2 interacts with endogenous small interfering RNAs (endo-siRNAs) to repress mRNA translation globally. Furthermore, the Ago2 and endo-siRNA complexes fine-tune the transcriptome by slicing long terminal repeat retrotransposon-derived chimeric transcripts. The precise mapping of RBP-binding sites in low-input cells opens the door to studying the roles of RBPs in embryonic development and reproductive diseases.

Publication types

Comparative Study
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Argonaute Proteins / genetics
Argonaute Proteins / metabolism
Binding Sites
DEAD-box RNA Helicases / genetics
DEAD-box RNA Helicases / metabolism
Female
Gene Expression Profiling*
Gene Expression Regulation, Developmental
HeLa Cells
Heterogeneous-Nuclear Ribonucleoproteins / genetics
Heterogeneous-Nuclear Ribonucleoproteins / metabolism
High-Throughput Nucleotide Sequencing*
Humans
K562 Cells
Mice
Mice, Inbred C57BL
Mice, Inbred ICR
Mice, Knockout
Oocytes / metabolism*
Polypyrimidine Tract-Binding Protein / genetics
Polypyrimidine Tract-Binding Protein / metabolism
Protein Binding
RNA, Messenger / genetics
RNA, Messenger / metabolism
RNA, Small Interfering / genetics
RNA, Small Interfering / metabolism
RNA-Binding Proteins / genetics
RNA-Binding Proteins / metabolism*
RNA-Seq
Transcriptome

Substances

Ago2 protein, mouse
Argonaute Proteins
Heterogeneous-Nuclear Ribonucleoproteins
Piwil2 protein, mouse
Ptbp1 protein, mouse
RNA, Messenger
RNA, Small Interfering
RNA-Binding Proteins
Polypyrimidine Tract-Binding Protein
Ddx4 protein, mouse
DEAD-box RNA Helicases