Expanded RNA-binding activities of mammalian Argonaute 2

Tan, Grace; Garchow, Barry G.; Liu, Xuhang; Yeung, Jennifer; Morris, John P.; Cuellar, Trinna; McManus, Michael T.; Kiriakidou, Marianthi

doi:10.1093/nar/gkp812

Cited by 120 publications

(141 citation statements)

References 47 publications

Supporting

Mentioning

132

Contrasting

Order By: Relevance

“…AGO2 has been shown to interact with long RNA (Ͼ24 nucleotides) directly (11,37). However, the primary mechanism by which AGO2 is recruited to specific loci is through base-pairing between a miRNA and target.…”

Section: Discussionmentioning

confidence: 99%

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Atwood

Woolnough

Lefèvre

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

The primary role of the RNAi machinery is to promote mRNA degradation within the cytoplasm in a microRNA-dependent manner. However, both Dicer and the Argonaute protein family have expanded roles in gene regulation within the nucleus. To further our understanding of this role, we have identified chromatin binding sites for AGO2 throughout the 45S region of the human rRNA gene. The location of these sites was mirrored by the positions of AGO2 cross-linking sites identified via PAR-CLIP-seq. AGO2 binding to the rRNA within the nucleus was confirmed by RNA immunoprecipitation and quantitative-PCR. To explore a possible mechanism by which AGO2 could be recruited to the rRNA, we identified 1174 regions within the 45S rRNA transcript that have the ability to form a perfect duplex with position 2-6 (seed sequence) of each microRNA expressed in HEK293T cells. Of these potential AGO2 binding sites, 479 occurred within experimentally verified AGO2-rRNA crosslinking sites. The ability of AGO2 to cross-link to rRNA was almost completely lost in a DICER knock-out cell line. The transfection of miR-92a-2-3p into the noDICE cell line facilitated AGO2 cross-linking at a region of the rRNA that has a perfect seed match at positions 3-8, including a single G-U base pair. Knockdown of AGO2 within HEK293T cells causes a slight, but statistically significant increase in the overall rRNA synthesis rate but did not impact the ratio of processing intermediates or the recruitment of the Pol I transcription factor UBTF.The RNAi machinery has many functions in the eukaryotic cell, and aspects of the RNAi molecular mechanism are highly conserved between yeast and humans (1). Essentially, a small RNA is bound by a member of the Argonaute family of proteins and contributes sequence specificity to a larger protein complex. In the cytoplasm, the RNAi machinery uses Watson-Crick base pairing to target the RNA-induced silencing complex to a specific mRNA and facilitate its degradation. A related process is well established in the nucleus of Schizosaccharomyces pombe, where instead of targeting cytoplasmic mRNAs for destruction, a small RNA targets the RNA-induced transcriptional silencing complex to the pericentromeric regions of each chromosome and facilitates the generation of heterochromatin (2, 3).Work in a chicken-human hybrid cell line supports the possibility that the RNAi machinery is responsible for centromeric chromatin structure in vertebrates as well (4). Indeed, when Dicer is conditionally inactivated, transcription of ␣-satellite DNA from human chromosome 21 increases. Furthermore, the loss of Dicer results in a loss of siRNAs originating from these repeat regions, a delocalization of HP1, and disruption of mitosis (4). The RNAi machinery is also implicated in the creation and/or maintenance of heterochromatin at various sites throughout the genome, in addition to the centromeric regions. Transfection of a siRNA homologous to the EF1a promoter in human cells silences the endogenous gene (5). In a related study, human Argonaute 1 (A...

show abstract

Section: Discussionmentioning

confidence: 99%

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Atwood

Woolnough

Lefèvre

et al. 2016

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…Thus, simple diffusion is unlikely to explain the effective maturation of selective miRNAs in the XPO5 knockout cells. We do not exclude the possibility that some of the nuclear retained pre-miRNAs are slowly processed by nuclear DICER, given that DICER is detected in the nucleus albeit at a lower level than that in the cytoplasm (32,33). Moreover, some primiRNAs may escape nuclear processing and get processed by the cytoplasmic Microprocessor.…”

Section: Parentalmentioning

confidence: 92%

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

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 ...

show abstract

“…The MID and the PAZ domains recognize the 5 ′ phosphate and the 3 ′ hydroxyl at the ends of a miRNA, respectively. Because miRNAs bind to Ago proteins with very high affinity (Lima et al 2009;Tan et al 2009), coimmunoprecipitation of miRNAs from cell lysate by anti-Ago antibodies provides a good assessment of miRNA association with Ago.…”

Section: Introductionmentioning

confidence: 99%

3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells

Guo¹,

Steitz

2014

RNA

View full text Add to dashboard Cite

Synthetic 3′ -biotin-tagged microRNAs (miRNAs) have often been used to select interacting messenger RNA (mRNA) and noncoding RNA (ncRNA) targets. Here, we examined the extent of association of 3 ′ -end biotinylated miR-27 with Argonaute (Ago) proteins in transfected human cells using a coimmunoprecipitation assay followed by Northern blot analysis. We report that biotinylated miR-27 does not efficiently associate with Ago compared to unmodified miR-27. These results suggest that 3 ′ -end biotin-modified miRNAs are questionable monitors of miRNA function in cells.

show abstract

Expanded RNA-binding activities of mammalian Argonaute 2

Cited by 120 publications

References 47 publications

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

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

3′-Biotin-tagged microRNA-27 does not associate with Argonaute proteins in cells

Contact Info

Product

Resources

About