Mono-Uridylation of Pre-MicroRNA as a Key Step in the Biogenesis of Group II let-7 MicroRNAs

Heo, Inha; Ha, Minju; Lim, Ju Young; Yoon, Mi Jeong; Park, Jong Eun; Kwon, Soyoung; Chang, Hyeshik; Kim, V. Narry

doi:10.1016/j.cell.2012.09.022

Cited by 282 publications

(364 citation statements)

References 73 publications

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“…On the other hand, additional processing steps or a modified biogenesis pathway is used for other noncanonical miRNAs (groups 2 through 6). The production of group 2 miRNAs requires monouridylation of premiRNAs for their efficient processing by DICER, because these pre-miRNAs have a short (1 nt) 3′ overhang (39). Most vertebrate let-7 members and miR-105 belong to group 2.…”

Section: Discussionmentioning

confidence: 99%

Re-evaluation of the roles of DROSHA , Exportin 5 , and DICER in microRNA biogenesis

Kim

2016

Proc. Natl. Acad. Sci. U.S.A.

402

327

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Biogenesis of canonical microRNAs (miRNAs) involves multiple steps: nuclear processing of primary miRNA (pri-miRNA) by DROSHA, nuclear export of precursor miRNA (pre-miRNA) by Exportin 5 (XPO5), and cytoplasmic processing of pre-miRNA by DICER. To gain a deeper understanding of the contribution of each of these maturation steps, we deleted DROSHA, XPO5, and DICER in the same human cell line, and analyzed their effects on miRNA biogenesis. Canonical miRNA production was completely abolished in DROSHA-deleted cells, whereas we detected a few DROSHA-independent miRNAs including three previously unidentified noncanonical miRNAs (miR-7706, miR-3615, and miR-1254). In contrast to DROSHA knockout, many canonical miRNAs were still detected without DICER albeit at markedly reduced levels. In the absence of DICER, pre-miRNAs are loaded directly onto AGO and trimmed at the 3′ end, yielding miRNAs from the 5′ strand (5p miRNAs). Interestingly, in XPO5 knockout cells, most miRNAs are affected only modestly, suggesting that XPO5 is necessary but not critical for miRNA maturation. Our study demonstrates an essential role of DROSHA and an important contribution of DICER in the canonical miRNA pathway, and reveals that the function of XPO5 can be complemented by alternative mechanisms. Thus, this study allows us to understand differential contributions of key biogenesis factors, and provides with valuable resources for miRNA research.icroRNA (miRNA) biogenesis begins with the synthesis of primary miRNA (pri-miRNA) by RNA polymerase II (1). The stem-loop structure embedded in pri-miRNA is cleaved by the Microprocessor complex composed of DROSHA and DGCR8 (2-6). The released hairpin, called precursor miRNA (pre-miRNA), is exported to the cytoplasm by Exportin 5 (XPO5) in a Ran-GTPdependent manner (7-9). In the cytoplasm, the pre-miRNA is further processed by DICER, producing a duplex RNA of ∼22 nt with its 3′ ends having a two nucleotide overhang (10-13). The duplex is loaded onto the ARGONAUTE (AGO) proteins, and one strand of the duplex remains as mature miRNA, whereas the other strand is discarded from AGO (11,14). The strand selection is dictated mainly by the relative thermodynamic stability of the two ends of the duplex: the strand whose 5′ terminal nucleotides are less stable is selected as mature miRNA (15,16). miRNAs originating from the 5′ and 3′ strands of pre-miRNA are referred to as the 5p and 3p miRNAs, respectively. Mammals have four closely related AGO proteins (AGO1-4) that interact with deadenylation factors and translational machinery to induce mRNA degradation and translational repression.Although the aforementioned canonical pathway accounts for the production of most miRNAs (1), it has also been shown that there exist alternative (noncanonical) pathways for miRNA biogenesis, which bypass a part of the biogenesis steps mentioned above. Mirtrons are one of the first miRNA groups described as noncanonical miRNAs, which do not require DROSHA for their production (17-19). Because mirtrons are located inside short ...

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Section: Discussionmentioning

confidence: 99%

Re-evaluation of the roles of DROSHA , Exportin 5 , and DICER in microRNA biogenesis

Kim

2016

Proc. Natl. Acad. Sci. U.S.A.

402

327

View full text Add to dashboard Cite

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“…99 Sequencing data revealed that the loop of pre-miRNA let-7 has a tetra-nucleotide sequence required for Lin28 binding and consequently Lin28/TUT4 uridylation. 109 Furthermore, knockdown of TUT4 and Lin28 in cancer cells decreased the level of stem cell markers, suggesting that they are required for the maintenance of a tumoral stem cell phenotype (Fig. 3).…”

mentioning

confidence: 99%

miRNA biogenesis: Biological impact in the development of cancer

Romero-Córdoba

Salido-Guadarrama

Rodríguez-Dorantes

et al. 2014

Cancer Biology & Therapy

214

138

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“…This modification impairs the stability of prelet-7, resulting in reduced levels of let-7. In addition, monouridylation has been shown to be required for the processing of some miRNA precursors (12). Deep sequencing analysis reveals that precursor uridylation is a widespread phenomenon occurring in many miRNA families in animals (13).…”

mentioning

confidence: 99%

Methylation protects microRNAs from an AGO1-associated activity that uridylates 5′ RNA fragments generated by AGO1 cleavage

Ren

Xie

Zhang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

108

122

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In plants, methylation catalyzed by HEN1 (small RNA methyl transferase) prevents microRNAs (miRNAs) from degradation triggered by uridylation. How methylation antagonizes uridylation of miRNAs in vivo is not well understood. In addition, 5′ RNA fragments (5′ fragments) produced by miRNA-mediated RNA cleavage can be uridylated in plants and animals. However, the biological significance of this modification is unknown, and enzymes uridylating 5′ fragments remain to be identified. Here, we report that in Arabidopsis, HEN1 suppressor 1 (HESO1, a miRNA nucleotidyl transferase) uridylates 5′ fragments to trigger their degradation. We also show that Argonaute 1 (AGO1), the effector protein of miRNAs, interacts with HESO1 through its Piwi/Argonaute/Zwille and PIWI domains, which bind the 3′ end of miRNA and cleave the target mRNAs, respectively. Furthermore, HESO1 is able to uridylate AGO1-bound miRNAs in vitro. miRNA uridylation in vivo requires a functional AGO1 in hen1, in which miRNA methylation is impaired, demonstrating that HESO1 can recognize its substrates in the AGO1 complex. On the basis of these results, we propose that methylation is required to protect miRNAs from AGO1-associated HESO1 activity that normally uridylates 5′ fragments. S mall interfering RNAs (siRNAs) and microRNAs (miRNAs), ∼20-25 nucleotides (nt) in size, are important regulators of gene expression. miRNAs and siRNAs are derived from imperfect hairpin transcripts and perfect long double-stranded RNAs, respectively (1, 2). miRNAs and siRNAs are then associated with Argonaute (AGO) proteins to repress gene expression through target cleavage and/or translational inhibition (3). The cleavage of target mRNAs usually occurs at a position opposite the tenth and eleventh nucleotides of miRNAs, resulting in a 5′ RNA fragment (5′ fragment) and a 3′ fragment (4). In Arabidopsis, the major effector protein for miRNA-mediated gene silencing is AGO1, which possesses the endonuclease activity required for target cleavage (5-7). In Drosophila, the exosome removes the 5′ fragments through its 3′-to-5′ exoribonuclease activity (8). How 5′ fragments are degraded in higher plants remains unknown. It has been shown that the 5′ fragments are subject to untemplated uridine addition at their 3′ termini (uridylation) in both animals and plants (9). However, the biological significance of this modification remains unknown because of a lack of knowledge of the enzymes targeting 5′ fragments for uridylation.Uridylation plays important roles in regulating miRNA biogenesis. In animals, TUT4, a terminal uridyl transferase, is recruited by Lin-28 (an RNA binding protein) to the let-7 precursor (prelet-7), resulting in uridylation of prelet-7 (10, 11). This modification impairs the stability of prelet-7, resulting in reduced levels of let-7. In addition, monouridylation has been shown to be required for the processing of some miRNA precursors (12). Deep sequencing analysis reveals that precursor uridylation is a widespread phenomenon occurring in many miRNA families in ani...

show abstract

Mono-Uridylation of Pre-MicroRNA as a Key Step in the Biogenesis of Group II let-7 MicroRNAs

Cited by 282 publications

References 73 publications

Re-evaluation of the roles of DROSHA , Exportin 5 , and DICER in microRNA biogenesis

Re-evaluation of the roles of DROSHA , Exportin 5 , and DICER in microRNA biogenesis

miRNA biogenesis: Biological impact in the development of cancer

Methylation protects microRNAs from an AGO1-associated activity that uridylates 5′ RNA fragments generated by AGO1 cleavage

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