DGCR8-dependent efficient pri-miRNA processing of human pri-miR-9-2

Masahiro Nogami,Kazumasa Miyamoto,Yoshika Hayakawa-Yano,Atsushi Nakanishi,Masato Yano,Hideyuki Okano

doi:10.1016/j.jbc.2021.100409

Abstract

Microprocessor complex, including DiGeorge syndrome critical region gene 8 (DGCR8) and DROSHA, recognizes and cleaves primary transcripts of microRNAs (pri-miRNAs) in the maturation of canonical miRNAs. The study of DGCR8 haploinsufficiency reveals that the efficiency of this activity varies for different miRNA species. It is thought that this variation might be associated with the risk of schizophrenia with 22q11 deletion syndrome caused by disruption of the DGCR8 gene. However, the underlying mechanism for varying action of DGCR8 with each miRNA remains largely unknown. Here, we used in vivo monitoring to measure the efficiency of DGCR8-dependent microprocessor activity in cultured cells. We confirmed that this system recapitulates the microprocessor activity of endogenous pri-miRNA with expression of a ratiometric fluorescence reporter. Using this system, we detected mir-9-2 as one of the most efficient targets. We also identified a novel DGCR8-responsive RNA element, which is highly conserved among mammalian species and could be regulated at the epi-transcriptome (RNA modification) level. This unique feature between DGCR8 and pri-miR-9-2 processing may suggest a link to the risk of schizophrenia.

Highlights

Several RNA-binding proteins, including DiGeorge syndrome critical region gene 8 (DGCR8), function in pri-miRNA processing and are involved in neuronal development and diseases such as DiGeorge syndrome [1, 13, 14]
Our findings show that the pri-miR-9-2 reporter had the most efficient processing of all pri-miRNA reporters investigated in this study and that the DGCR8responsive RNA element (DRE) identified in pri-miR-9-2 might be a unique RNA element for potentiating DGCR8-dependent processing
We found that pri-miR-9 is the transcript most efficiently processed out of other miRNA species by the canonical microprocessor complex

Summary

RESEARCH ARTICLE

Masahiro Nogami1,2,* , Kazumasa Miyamoto, Yoshika Hayakawa-Yano, Atsushi Nakanishi, Masato Yano4,6,* , and Hideyuki Okano From the 1Innovative Biology Laboratories, Neuroscience Drug Discovery Unit, Research, 2Shonan Incubation Laboratories, and 3Drug Safety Research Laboratories, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan; 4Division of Neurobiology and Anatomy, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan; 5Regenerative Medicine Unit, Takeda Pharmaceutical Company Limited, Fujisawa, Kanagawa, Japan; and 6Department of Physiology, School of Medicine, Keio University, Tokyo, Japan

Edited by Ronald Wek

Results

Discussion

Vector construction and plasmid preparation

Cell culture and transfection

Western blotting with the Wes system