In vivo discovery of RNA proximal proteins via proximity-dependent biotinylation

Xianzhi Lin; Marcos A S Fonseca; Joshua J Breunig; Rosario I Corona; Kate Lawrenson

doi:10.1080/15476286.2021.1917215

In vivo discovery of RNA proximal proteins via proximity-dependent biotinylation

RNA Biol. 2021 Dec;18(12):2203-2217. doi: 10.1080/15476286.2021.1917215. Epub 2021 May 19.

Authors

Xianzhi Lin^{1

2}, Marcos A S Fonseca^{1

2}, Joshua J Breunig^{3

4

5

6}, Rosario I Corona^{1

2}, Kate Lawrenson^{1

2

7}

Affiliations

¹ Cedars-Sinai Medical Center, Women's Cancer Research Program at Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA.
² Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
³ Cedars-Sinai Medical Center, Board of Governors Regenerative Medicine Institute, Los Angeles, CA, USA.
⁴ Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA.
⁵ Cedars-Sinai Medical Center, Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA.
⁶ Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.
⁷ Cedars-Sinai Medical Center, Center for Bioinformatics and Functional Genomics, Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA.

Abstract

RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins in vivo via proximity-based biotinylation. RPL applied to U1 identified proteins involved in both U1 canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5'-3' proximity and unexplored subcellular localizations of poly(A)⁺ RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA-protein interactions important for health and disease.

Keywords: RNA binding proteins (RBPs); RNA proximal proteins; RNA proximity labelling (RPL); RNA-centric method; RRNA–proteininteractions; Type VI Crispr-Cas (Cas13); U1 snRNA RBPS; engineered soybean ascorbate peroxidase APEX2; noncoding RNAs (ncRNAs); poly(A) tail; proximity-dependent biotinylation.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Ascorbate Peroxidases / genetics*
Biotinylation
CRISPR-Associated Proteins / genetics*
CRISPR-Cas Systems
HEK293 Cells
Humans
Poly A
RNA / chemistry
RNA / metabolism*
RNA, Guide, CRISPR-Cas Systems / genetics
RNA, Small Nuclear / genetics
RNA-Binding Proteins / metabolism*
Recombinant Fusion Proteins / genetics
Staining and Labeling

Substances

CRISPR-Associated Proteins
RNA, Guide, CRISPR-Cas Systems
RNA, Small Nuclear
RNA-Binding Proteins
Recombinant Fusion Proteins
U1 small nuclear RNA
Poly A
RNA
Ascorbate Peroxidases

Abstract

Publication types

MeSH terms

Substances

Grants and funding