Argonaute-Bound Small RNAs from Promoter-Proximal RNA Polymerase II

Zamudio, Jesse R.; Kelly, Timothy; Sharp, Phillip A.

doi:10.1016/j.cell.2014.01.041

Cited by 105 publications

(141 citation statements)

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“…Due to decreases in expression levels of AGO2 mRNA in old PBMCs implicate a change in miRNA abundance as observed in mouse embryonic fibroblasts lacking AGO2 (Zamudio Jesse, Kelly Timothy & Sharp Phillip, 2014). In order to test whether the reduction of AGO2 expression due to differences in m6A modification leads to the reduction of mature miRNA level during aging, we performed miRNA microarray analysis of the young and old PBMCs.…”

Section: Resultsmentioning

confidence: 97%

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Profiling of m6A RNA modifications identified an age‐associated regulation of AGO2 mRNA stability

et al. 2018

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SummaryGene expression is dynamically regulated in a variety of mammalian physiologies. During mammalian aging, there are changes that occur in protein expression that are highly controlled by the regulatory steps in transcription, post‐transcription, and post‐translation. Although there are global profiles of human transcripts during the aging processes available, the mechanism(s) by which transcripts are differentially expressed between young and old cohorts remains unclear. Here, we report on N6‐methyladenosine (m6A) RNA modification profiles of human peripheral blood mononuclear cells (PBMCs) from young and old cohorts. An m6A RNA profile identified a decrease in overall RNA methylation during the aging process as well as the predominant modification on proteincoding mRNAs. The m6A‐modified transcripts tend to be more highly expressed than nonmodified ones. Among the many methylated mRNAs, those of DROSHA and AGO2 were heavily methylated in young PBMCs which coincided with a decreased steady‐state level of AGO2 mRNA in the old PBMC cohort. Similarly, downregulation of AGO2 in proliferating human diploid fibroblasts (HDFs) also correlated with a decrease in AGO2 mRNA modifications and steady‐state levels. In addition, the overexpression of RNA methyltransferases stabilized AGO2 mRNA but not DROSHA and DICER1 mRNA in HDFs. Moreover, the abundance of miRNAs also changed in the young and old PBMCs which are possibly due to a correlation with AGO2 expression as observed in AGO2‐depleted HDFs. Taken together, we uncovered the role of mRNA methylation on the abundance of AGO2 mRNA resulting in the repression of miRNA expression during the process of human aging.

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Section: Resultsmentioning

confidence: 97%

“…This suggests that there are other key factors that modulate specific miRNA abundance. AGO2 is an ideal protein to modulate miRNA abundance in cellular senescence and aging since its association with mature miRNAs prevents them from degradation without affecting canonical miRNA processing (Zamudio Jesse et al., 2014). …”

Section: Discussionmentioning

confidence: 99%

Profiling of m6A RNA modifications identified an age‐associated regulation of AGO2 mRNA stability

et al. 2018

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“…It is unclear if similar dynamics affect termination by eukaryotic RNAPII, but RNA secondary structures have also been proposed to aid RNAPII termination (63,64), including effects of bacterial intrinsic terminators in yeast RNAPII (8) and the HIV-1 TAR hairpin on human RNAPII (65). Alterations of the TL in yeast RNAPIII also affect TE (66), and the presence of the TL in archaeal RNAP prevents aberrant termination (67).…”

Section: Resultsmentioning

confidence: 99%

Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact

Ray-Soni

Mooney

Landick

2017

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

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In bacteria, intrinsic termination signals cause disassembly of the highly stable elongating transcription complex (EC) over windows of two to three nucleotides after kilobases of RNA synthesis. Intrinsic termination is caused by the formation of a nascent RNA hairpin adjacent to a weak RNA−DNA hybrid within RNA polymerase (RNAP). Although the contributions of RNA and DNA sequences to termination are largely understood, the roles of conformational changes in RNAP are less well described. The polymorphous trigger loop (TL), which folds into the trigger helices to promote nucleotide addition, also is proposed to drive termination by folding into the trigger helices and contacting the terminator hairpin after invasion of the hairpin in the RNAP main cleft [Epshtein V, Cardinale CJ, Ruckenstein AE, Borukhov S, Nudler E (2007) Mol Cell 28:991-1001]. To investigate the contribution of the TL to intrinsic termination, we developed a kinetic assay that distinguishes effects of TL alterations on the rate at which ECs terminate from effects of the TL on the nucleotide addition rate that indirectly affect termination efficiency by altering the time window in which termination can occur. We confirmed that the TL stimulates termination rate, but found that stabilizing either the folded or unfolded TL conformation decreased termination rate. We propose that conformational fluctuations of the TL (TL dynamics), not TL-hairpin contact, aid termination by increasing EC conformational diversity and thus access to favorable termination pathways. We also report that the TL and the TL sequence insertion (SI3) increase overall termination efficiency by stimulating pausing, which increases the flux of ECs into the termination pathway.T ranscription termination is an essential process in all organisms that releases the RNA chain and DNA template from transcribing RNA polymerase (RNAP). Termination prevents unwanted gene expression (1), recycles RNAP for new initiation events (2), and prevents genome-destabilizing collisions with the replication machinery (3). In bacteria, intrinsic termination of the elongating transcription complex (EC) can occur without accessory factors in response to a signal in the DNA and newly synthesized RNA. Intrinsic termination occurs over a 2-to 3-nt window of EC destabilization (4) and is caused by the formation of a GC-rich RNA hairpin in the RNA exit channel of RNAP, immediately upstream of a 3′-terminal, ∼8-nt U-rich RNA tract (U-tract; Fig. 1A). Precise termination of the highly stable EC requires rapid entry into a termination pathway before continued nucleotide addition moves the EC beyond the region of destabilization (4-6).A thermodynamic termination model posits that the relative free energy barriers to termination versus elongation determine the fraction of ECs that terminate; thus, termination efficiency (TE) is kinetically determined by competing rates of nucleotide addition and termination (5, 7) (Fig. 1B). The EC is stabilized by polar and van der Waals contacts between RNAP and (i) the 9-to ...

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“…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). Despite these reports demonstrating a role for RNAi in regulating the chromatin structure of vertebrates, similar studies have shown that the loss of the RNAi machinery impacts chromatin structure indirectly through miRNA biogenesis and post-transcriptional gene regulation (14).…”

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confidence: 99%

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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Argonaute-Bound Small RNAs from Promoter-Proximal RNA Polymerase II

Cited by 105 publications

References 63 publications

Profiling of m6A RNA modifications identified an age‐associated regulation of AGO2 mRNA stability

Profiling of m6A RNA modifications identified an age‐associated regulation of AGO2 mRNA stability

Trigger loop dynamics can explain stimulation of intrinsic termination by bacterial RNA polymerase without terminator hairpin contact

Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions

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