Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing

Kwon, Soyoung; Baek, Sungmin; Choi, Yeon; Yang, Jian; Lee, Young suk; Woo, Jae Sung; Kim, V. Narry

doi:10.1016/j.molcel.2018.11.005

Cited by 76 publications

(110 citation statements)

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“…This strengthens the interaction between DROSHA and the junction, and thus enhances its cleavage efficiency and accuracy 13,21 . In addition, DROSHA has a double-stranded RNA binding domain (dsRBD), which it uses to interact with an mGHG motif located in the lower stem of the pri-miRNA to precisely find the correct cleavage sites 18,22 . DGCR8 in a dimer form also plays an essential role in ensuring that DROSHA cleaves accurately~22 nt from the apical junction.…”

mentioning

confidence: 99%

Mismatched and wobble base pairs govern primary microRNA processing by human Microprocessor

Nguyễn

et al. 2020

Nat Commun

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MicroRNAs (miRNAs) are small RNAs that regulate gene expression. miRNAs are produced from primary miRNAs (pri-miRNAs), which are cleaved by Microprocessor. Microprocessor, therefore, plays a crucial role in determining the efficiency and precision of miRNA production, and thus the function of the final miRNA product. Here, we conducted highthroughput enzymatic assays to investigate the catalytic mechanism of Microprocessor cleaving randomized pri-miRNAs. We identified multiple mismatches and wobble base pairs in the upper stem of pri-miRNAs, which influence the efficiency and accuracy of their processing. The existence of these RNA elements helps to explain the alternative cleavage of Microprocessor for some human pri-miRNAs. We also demonstrated that miRNA biogenesis can be altered via modification of the RNA elements by RNA-editing events or single nucleotide polymorphisms (SNPs). These findings improve our understanding of pri-miRNA processing mechanisms and provide a foundation for interpreting differential miRNA expression due to RNA modifications and SNPs.

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mentioning

confidence: 99%

Mismatched and wobble base pairs govern primary microRNA processing by human Microprocessor

Nguyễn

et al. 2020

Nat Commun

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“…Previous studies have employed screening of libraries of miRNA sequences to identify features that improved Drosha and Dicer cleavage. This has led to the identification of primary sequence motifs including CNNC, GHG, and GUG, present in flanking regions and loops, respectively, that improve Drosha binding and cleavage (Auyeung et al 2013;Fang and Bartel 2015;Kwon et al 2019). We wanted to identify features that improve the entire biogenesis pathway, and avoid Ago2 slicing of the stem, to maximize target knockdown potential.…”

Section: Resultsmentioning

confidence: 99%

“…However, the overall goal of our study is to model single-copy shRNA potency, and high copy shRNA expression might favor different structural features. Previous studies have used libraries of artificial miRNA sequences to probe Drosha and Dicer processing motifs (Auyeung et al 2013;Fang and Bartel 2015;Kwon et al 2019). These studies were designed to capture and identify the library RNAs without the background of endogenous miRNAs, allowing complete saturation of the screen.…”

Section: Discussionmentioning

confidence: 99%

A functional screen for optimization of short hairpin RNA biogenesis and RISC loading

Sons¹,

Kaufmann²,

Hammond³

2020

Preprint

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Gene silencing via short hairpin mediated RNAi (shRNA) is a valuable experimental tool and has promise as a therapeutic strategy. Several shRNA platforms make use of the loop and flanking sequences from the endogenous microRNA (miRNAs) miR-30a or other miRNAs to provide an RNA structure for efficient and accurate biogenesis of the RNA trigger. However, the stem regions of these shRNAs are typically designed as perfect duplex structures which is an uncommon feature for endogenous miRNA precursors. A limitation of these designs is that shRNAs with perfect duplex stems undergo extensive stem cleavage analogous to the Dicer independent miRNA miR-451, destroying the shRNA trigger sequence that is present in the 3P arm. We employed an unbiased screen of > 9000 shRNA structures to identify features that prevent stem cleavage and promote canonical biogenesis and loading into the effector complex RISC. We find that a central stem bulge or kink reduces central stem cleavage and improves accuracy of Dicer processing. Furthermore, 9 -10 GC nucleotides in the guide strand improves shRNA efficiency. These design rules enable more effective shRNA tools and are compatible with existing sets of optimized guide/target sequences.

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“…Some studies have shown that deviations from the expected cleavage site between the basal junction and the apical junction of a pri-miRNA can result in alternative cleavage by Drosha 27 . Furthermore, structural defects affect imprecise Drosha cleavage in the lower stem, altering the overall helical structure of the pri-miRNA, as well as disrupting the interaction between Drosha and the sequence motif of the lower stem 28 . RNA-binding proteins such as hnRNPA1 control Drosha cleavage by changing the secondary structure of the pri-miRNA, which might be one of the reasons why such proteins regulate alternative cleavage by Drosha 29 .…”

Section: Mirna Heterogeneitymentioning

confidence: 99%

Pathogenic diversity of RNA variants and RNA variation-associated factors in cancer development

Yang

Nam

2020

Exp Mol Med

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Recently, with the development of RNA sequencing technologies such as next-generation sequencing (NGS) for RNA, numerous variations of alternatively processed RNAs made by alternative splicing, RNA editing, alternative maturation of microRNA (miRNA), RNA methylation, and alternative polyadenylation have been uncovered. Furthermore, abnormally processed RNAs can cause a variety of diseases, including obesity, diabetes, Alzheimer's disease, and cancer. Especially in cancer development, aberrant RNAs caused by deregulated RNA modifiers or regulators are related to progression. Accumulating evidence has reported that aberrant RNAs promote carcinogenesis in many cancers, including liver cancer, leukemia, melanoma, lung cancer, breast cancer, and other cancers, in which abnormal RNA processing occurs in normal cells. Therefore, it is necessary to understand the precise roles and mechanisms of disease-related RNA processing in various cancers for the development of therapeutic interventions. In this review, the underlying mechanisms of variations in the RNA life cycle and the biological impacts of RNA variations on carcinogenesis will be discussed, and therapeutic strategies for the treatment of tumor malignancies will be provided. We also discuss emerging roles of RNA regulators in hepatocellular carcinogenesis.

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Molecular Basis for the Single-Nucleotide Precision of Primary microRNA Processing

Abstract: Highlights d The mGHG motif can dictate the cleavage site with singlenucleotide precision d Processing site of primary microRNA is determined by DROSHA rather than DGCR8 d The dsRBD of DROSHA recognizes the mGHG motif

Cited by 76 publications

References 50 publications

Mismatched and wobble base pairs govern primary microRNA processing by human Microprocessor

Mismatched and wobble base pairs govern primary microRNA processing by human Microprocessor

A functional screen for optimization of short hairpin RNA biogenesis and RISC loading

Pathogenic diversity of RNA variants and RNA variation-associated factors in cancer development

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