Nuclear localization of Argonaute 2 is affected by cell density and may relieve repression by microRNAs.

Argonaute protein is associated with post-transcriptional control of cytoplasmic gene expression through miRNA-induced silencing complexes (miRISC). Specific cellular and environmental conditions can trigger AGO protein to accumulate in the nucleus. Localization of AGO is central to understanding miRNA action, yet the consequences of AGO being in the nucleus are undefined. We show nuclear enrichment of AGO2 in HCT116 cells grown in two-dimensional culture to high density, HCT116 cells grown in three-dimensional tumor spheroid culture, and human colon tumors. The shift in localization of AGO2 from cytoplasm to nucleus de-represses cytoplasmic AGO2-eCLIP targets that were candidates for canonical regulation by miRISC. Constitutive nuclear localization of AGO2 using an engineered nuclear localization signal increases cell migration. Critical RNAi factors also affect the localization of AGO2. Knocking out an enzyme essential for miRNA biogenesis, DROSHA, depletes mature miRNAs and restricts AGO2 localization to the cytoplasm, while knocking out the miRISC scaffolding protein, TNRC6, results in nuclear localization of AGO2. These data suggest that AGO2 localization and miRNA activity can be regulated depending on environmental conditions, expression of mature miRNAs, and expression of miRISC cofactors. Localization and expression of core miRISC protein machinery should be considered when investigating the roles of miRNAs.

RNAi in cell nuclei: potential for a new layer of biological regulation and a new strategy for therapeutic discovery.

RNA interference is almost always associated with post-transcriptional silencing in the cytoplasm. MicroRNAs (miRNAs) and critical RNAi protein factors like argonaute (AGO) and trinucleotide repeat binding containing 6 protein (TNRC6), however, are also found in cell nuclei, suggesting that nuclear miRNAs may be targets for gene regulation. Designed small duplex RNAs (dsRNAs) can modulate nuclear processes such as transcription and splicing, suggesting that they can also provide leads for therapeutic discovery. The goal of this Perspective is to provide the background on nuclear RNAi necessary to guide discussions on whether nuclear RNAi can play a role in therapeutic development programs.

Consequences of depleting TNRC6, AGO, and DROSHA proteins on expression of microRNAs.

The potential for microRNAs (miRNAs) to regulate gene expression remains incompletely understood. DROSHA initiates the biogenesis of miRNAs while variants of Argonaute (AGO) and trinucleotide repeat containing six (TNRC6) family proteins form complexes with miRNAs to facilitate RNA recognition and gene regulation. Here we investigate the fate of miRNAs in the absence of these critical RNAi protein factors. Knockout of DROSHA expression reduces levels of some miRNAs annotated in miRBase but not others. The identity of miRNAs with reduced expression matches the identity of miRNAs previously identified by experimental approaches. The MirGeneDB resource offers the closest alignment with experimental results. In contrast, the loss of TNRC6 proteins had much smaller effects on miRNA levels. Knocking out AGO proteins, which directly contact the mature miRNA, decreased expression of the miRNAs most strongly associated with AGO2 as determined from enhanced crosslinking immunoprecipitation (AGO2-eCLIP). Evaluation of miRNA binding to endogenously expressed AGO proteins revealed that miRNA:AGO association was similar for AGO1, AGO2, AGO3, and AGO4. Our data emphasize the need to evaluate annotated miRNAs based on approximate cellular abundance, DROSHA-dependence, and physical association with AGO when forming hypotheses related to their function.

Argonaute binding within human nuclear RNA and its impact on alternative splicing.

Mammalian RNA interference (RNAi) is often linked to the regulation of gene expression in the cytoplasm. Synthetic RNAs, however, can also act through the RNAi pathway to regulate transcription and splicing. While nuclear regulation by synthetic RNAs can be robust, a critical unanswered question is whether endogenous functions for nuclear RNAi exist in mammalian cells. Using enhanced crosslinking immunoprecipitation (eCLIP) in combination with RNA sequencing (RNA-seq) and multiple AGO knockout cell lines, we mapped AGO2 protein binding sites within nuclear RNA. The strongest AGO2 binding sites were mapped to micro RNAs (miRNAs). The most abundant miRNAs were distributed similarly between the cytoplasm and nucleus, providing no evidence for mechanisms that facilitate localization of miRNAs in one compartment versus the other. Beyond miRNAs, most statistically significant AGO2 binding was within introns. Splicing changes were confirmed by RT-PCR and recapitulated by synthetic miRNA mimics complementary to the sites of AGO2 binding. These data support the hypothesis that miRNAs can control gene splicing. While nuclear RNAi proteins have the potential to be natural regulatory mechanisms, careful study will be necessary to identify critical RNA drivers of normal physiology and disease.

Argonaute binding within 3'-untranslated regions poorly predicts gene repression.

Despite two decades of study, the full scope of RNAi in mammalian cells has remained obscure. Here we combine: (i) Knockout of argonaute (AGO) variants; (ii) RNA sequencing analysis of gene expression changes and (iii) Enhanced Crosslinking Immunoprecipitation Sequencing (eCLIP-seq) using anti-AGO2 antibody to identify potential microRNA (miRNA) binding sites. We find that knocking out AGO1, AGO2 and AGO3 together are necessary to achieve full impact on steady state levels of mRNA. eCLIP-seq located AGO2 protein associations within 3'-untranslated regions. The standard mechanism of miRNA action would suggest that these associations should repress gene expression. Contrary to this expectation, associations between AGO and RNA are poorly correlated with gene repression in wild-type versus knockout cells. Many clusters are associated with increased steady state levels of mRNA in wild-type versus knock out cells, including the strongest cluster within the MYC 3'-UTR. Our results suggest that assumptions about miRNA action should be re-examined.

Nuclear Localization of Argonaute is affected by Cell Density and May Relieve Repression by microRNAs.

Argonaute protein is associated with post-transcriptional control of cytoplasmic gene expression through miRNA-induced silencing complexes (miRISC). Specific cellular and environmental conditions can trigger AGO protein to accumulate in the nucleus. Localization of AGO is central to understanding miRNA action, yet the consequences of AGO being in the nucleus are undefined. We show nuclear enrichment of AGO2 in HCT116 cells grown in two-dimensional culture to high density, HCT116 cells grown in three-dimensional tumor spheroid culture, and human colon tumors. The shift in localization of AGO2 from cytoplasm to nucleus de-represses cytoplasmic AGO2-eCLIP targets that were candidates for canonical regulation by miRISC. Constitutive nuclear localization of AGO2 using an engineered nuclear localization signal increases cell migration. Critical RNAi factors also affect the localization of AGO2. Knocking out an enzyme essential for miRNA biogenesis, DROSHA, depletes mature miRNAs and restricts AGO2 localization to the cytoplasm, while knocking out the miRISC scaffolding protein, TNRC6, results in nuclear localization of AGO2. These data suggest that AGO2 localization and miRNA activity can be regulated depending on environmental conditions, expression of mature miRNAs, and expression of miRISC cofactors. Localization and expression of core miRISC protein machinery should be considered when investigating the roles of miRNAs.

Human GW182 Paralogs Are the Central Organizers for RNA-Mediated Control of Transcription.

In the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription.