Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in <i>cis</i>

Woolnough, Jessica L.; Atwood, Blake L.; Giles, Keith E.

doi:10.1128/mcb.00076-15

Cited by 29 publications

(35 citation statements)

References 89 publications

(112 reference statements)

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“…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%

“…The amplicons indicate the region of the rRNA gene that was amplified: promoter ϭ Ϫ57, ETS1 ϭ 851, IGS ϭ 37997. The primer sequences were all previously reported (11). measurement of the specific cause and effect of each AGO2-miRNA-rRNA interaction as well as the sum total of all such interactions.…”

Section: Discussionmentioning

confidence: 99%

“…Although the ChIP-seq data were determined within K562 cells, we have focused on publicly available RNA-seq data from HEK293T cells. Our previous work indicated that the interactions between AGO2 and tRNA were conserved between the two cell lines (11).…”

Section: Ago2mentioning

confidence: 96%

“…Each of the 3 mature rRNA species is indicated by a blue box (18S, 5.8S, and 28S). All ChIP-seq data were reanalyzed from a previously published AGO2 study (11) and are available within the Gene Expression Omnibus: GSE68813. The horizontal gray bars above each track represent regions called as peaks using MACS version 1.4, false discovery rate Ͻ 10% (see "Experimental Procedures") (20).…”

Section: Ago2mentioning

confidence: 99%

“…This correlated with an increase in transcription levels and a loss in physical condensation. Recent work has indicated that human AGO proteins can regulate splicing through modulating chromatin structure (9,10) and can promote gene repression in cis by localizing to nascent tRNA (11). Thus, RNAi-mediated control of gene expression exists also in vertebrate cells but functions in mechanisms distinct from those within the cytoplasm (12,13).…”

mentioning

confidence: 99%

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Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Atwood

Woolnough

Lefèvre

et al. 2016

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Ago2mentioning

confidence: 96%

Section: Ago2mentioning

confidence: 99%

mentioning

confidence: 99%

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Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Atwood

Woolnough

Lefèvre

et al. 2016

Journal of Biological Chemistry

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From tRNA to miRNA: RNA‐folding contributes to correct entry into noncoding RNA pathways

Hasler

Meister

2016

FEBS Letters

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Edited by Wilhelm JustAlthough RNA are synthesized as single-stranded molecules, most of them are characterized by extensive secondary structures. Two RNA that require distinct folding for biogenesis and function are miRNA and tRNA. While miRNA are processed from hairpin-containing precursors, tRNA are folded into characteristic L-shaped structures. In addition, tRNA and their precursors are a rich source of RNA fragments. Recent findings suggest that their production might be determined by structural characteristics of the tRNA substrates. Importantly, correct folding of pre-tRNA is assisted by the Lupus autoantigen La, an RNA chaperone. In this context, La interacts with pre-tRNA to prevent alternative foldings leading to mis-channeling into the miRNA pathway. Thus, RNA chaperones also function as gatekeepers for correct RNA pathway selection.Keywords: Lupus autoantigen La; miRNA; RNA chaperone; tRNA fragments RNA molecules are characterized by secondary structures that are important for their individual functions. Such structures can be restricted to short sequence stretches or can involve distant regions of large RNA molecules as known for the complex folding of rRNA, for example [1]. Shorter RNA, such as tRNA, small nuclear RNA (snRNA), or small nucleolar RNA (snoRNA), often adopt specific structures providing RNA-or protein-binding platforms or scaffolds for ribonucleoprotein particle (RNP) formation. Other RNA including miRNA or short interfering RNA (siRNA) use distinct folding strategies of precursor molecules, which allow efficient processing to their mature and functional forms [2]. Therefore, adopting the correct structure is vital for any RNA molecule and processes have evolved that support RNA folding and the stability of RNA structures [3]. Biogenesis of tRNAOne class of RNA that requires a very specific folding to fulfill its functions is tRNA. In addition, tRNA are highly modified at various nucleotides and these alterations can also dramatically affect the secondary structure (reviewed in [4]). In eukaryotes, tRNA are transcribed by RNA polymerase III (pol III) as precursor molecules that contain a 5 0 extension and a 3 0 trailer terminating with a stretch of U residues. The 5 0 extension (often referred to as 5 0 leader) is removed by RNase P. The human RNase P is a RNP complex composed of 10 protein subunits and the noncoding RNA H1 (or RPPH1) [5], which is transcribed by pol III as well. RNA H1 is the catalytic subunit and therefore RNase P was among the first ribozymes that has been discovered [6].The canonical processing of the 3 0 end of pre-tRNA requires the endonucleolytic cleavage activity of a Abbreviations ANG, angiogenin; LARP, La-related protein; RBP, RNA-binding protein; RISC, RNA-induced silencing complex; RNP, ribonucleoprotein particle; siRNA, short interfering RNA; snoRNA, small nucleolar RNA; snRNA, small nuclear RNA; tRF, tRNA fragment; TSEN, tRNA-splicing endonuclease.

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Aggregation of Mod5 is affected by tRNA binding with implications for tRNA gene‐mediated silencing

et al. 2017

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Mod5 is a multifunctional protein that modifies a subset of tRNAs in the cytoplasm and is also required for an RNA-mediated form of transcriptional silencing. Previous in vivo studies have shown that the nuclear silencing function of Mod5 does not require that the causative tRNA gene encode a Mod5 substrate, though Mod5 is still required. However, previous data have not directly tested whether Mod5 can directly bind substrate and nonsubstrate RNAs. We herein demonstrate that Mod5 directly binds to both substrate and nonsubstrate RNAs, including a highly structured, non-tRNA sequence (5S-rRNA), consistent with previous in vivo data. Furthermore, we show that some RNAs drastically change the aggregation behavior of Mod5 with implications for tRNA-gene mediated silencing.

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Argonaute 2 Binds Directly to tRNA Genes and Promotes Gene Repression in cis

Cited by 29 publications

References 89 publications

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

From tRNA to miRNA: RNA‐folding contributes to correct entry into noncoding RNA pathways

Aggregation of Mod5 is affected by tRNA binding with implications for tRNA gene‐mediated silencing

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