TRIM65 regulates microRNA activity by ubiquitinationof TNRC6Shitao Li1,2, Lingyan Wang1, Bishi Fu1, Michael A. Berman, Alos Diallo, and Martin E. Dorf2

Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115

Edited by Aaron Ciechanover, Technion–Israel Institute of Technology, Haifa, Israel, and approved April 3, 2014 (received for review December 4, 2013)

MicroRNAs (miRNAs) are small evolutionarily conserved regulatoryRNAs that modulate mRNA stability and translation in a wide rangeof cell types. MiRNAs are involved in a broad array of biologicalprocesses, including cellular proliferation, differentiation, and apo-ptosis. To identify previously unidentified regulators of miRNA, weinitiated a systematic discovery-type proteomic analysis of the miRNApathway interactome in human cells. Six of 66 genes identified inour proteomic screen were capable of regulating lethal-7a (let-7a)miRNA reporter activity. Tripartite motif 65 (TRIM65) was identi-fied as a repressor of miRNA activity. Detailed analysis indicatesthat TRIM65 interacts and colocalizes with trinucleotide repeatcontaining six (TNRC6) proteins in processing body-like structures.Ubiquitination assays demonstrate that TRIM65 is an ubiquitin E3ligase for TNRC6 proteins. The combination of overexpression andknockdown studies establishes that TRIM65 relieves miRNA-drivensuppression of mRNA expression through ubiquitination and subse-quent degradation of TNRC6.

RNA-induced silencing complex | protein interaction networks |GW proteins

MicroRNAs (miRNAs) are small noncoding RNAs thatregulate the translation and stability of mRNA in animals and

plants (1, 2). The canonical biogenesis of miRNAs starts witha hairpin-like primary miRNA (primiRNA), typically a product ofRNA polymerase II (3, 4). In the nucleus, the DROSHA/DGCR8microprocessor complex recognizes and cleaves the primiRNAhairpin, which leads to release of a precursor miRNA (premiRNA)hairpin that is ∼55–70 nt in length (5–7). The premiRNA is exportedto the cytoplasm by a complex of Exportin 5 and RAN-GTP (8, 9).In the cytoplasm, the premiRNA terminal loop is cleaved by DICERin collaboration with TARBP2, yielding ∼22-nt RNA duplexes. Onestrand of the duplex is preferentially incorporated into the RNA-induced silencing complex (RISC), where the miRNA and mRNAinteract (3). In the RISC, miRNA targets mRNA for translationalrepression, deadenylation, or degradation (10–12).The RISC minimally consists of two core protein compo-

nents, Argonaute (AGO) and trinucleotide repeat containingsix (TNRC6; also known as GW182) proteins or their paralogs,which are key factors for function of the RISC. These proteinslocalize in specialized cytoplasmic foci known as mRNA pro-cessing bodies (P-bodies) (13, 14). P-bodies also contain effectormolecules that facilitate mRNA degradation, including decappingenzymes (DCP1 and DCP2) required for miRNA-mediated genesilencing (15) and the CCR4–NOT deadenylase complex, whichremoves poly(A) from messages destabilized by miRNA (15).The core proteins that participate in miRNA biogenesis and

regulation have been identified (6, 16–20), but the global orga-nization and coordination of this system are incompletely un-derstood. To explore the miRNA protein–protein interactionnetwork systematically, we initiated a global proteomic analysis ofthe miRNA pathway interactome (MPI) in human cells. Analysisof 40 MPI-associated baits revealed connections with 363 pro-teins, forming a framework of 499 unique protein interactions.RNAi screening of 66 previously unidentified proteins associatedwith the MPI identified tripartite motif 65 (TRIM65) and fiveother regulators of miRNA activity. TRIM65 is an ubiquitin E3

ligase that colocalizes with TNRC6 paralogs and triggers protea-some-dependent degradation of TNRC6 proteins. TRIM65 rep-resents a previously unidentified member of the miRNA pathway,which negatively regulates the miRNA-guided mRNA silencingmachinery.

ResultsMapping the MPI.A proteomic search strategy was used to identifyprotein components putatively associated with the miRNA pro-cessing machinery. Forty proteins with known or suspected in-volvement in canonical miRNA biogenesis and regulation werechosen as baits. Each bait was tagged with the FLAG epitope andstably expressed in HEK 293 cells. Affinity purification by elutionfrom anti-FLAG beads coupled with MS (AP-MS) was used toidentify proteins linked to the MPI (21). Controls for excludingnonspecific binding proteins (NSBPs) include eluates fromFLAG-GFP–transfected and nontransfected HEK 293 cells.Protein complexes were retrieved from two independent bio-logical experiments. Furthermore, each affinity-purified samplewas divided into two aliquots, which were analyzed by liquidchromatography-tandem MS on different days. In total, 80 com-plexes were purified and 160 samples were examined.For data processing, we compared AP-MS data from the MPI

with our laboratory database containing 158 FLAG-tagged proteincomplexes from stably transfected HEK 293 cells, including 55proteins involved in innate immunity (21). We identify high-confidence interacting proteins (HCIPs) by calculation of z-scoresbased on total spectral counts (protein abundance). In addi-tion, we consider prey occurrence (the uniqueness of a protein in

Significance

MicroRNAs (miRNAs) comprise a large family of small RNAmolecules that regulate gene expression in diverse biologicalpathways of both plants and animals. We used a combinationof proteomic and functional analyses of proteins associatedwith the miRNA pathway to identify the ubiquitin E3 ligasetripartite motif 65 (TRIM65) and five other genes implicated inregulation of miRNA activity in human cells. Biochemical anal-ysis established that TRIM65 forms stable complexes with tri-nucleotide repeat containing six (TNRC6) proteins and thatthese molecules colocalize in processing body-like structures.Gain of function and RNAi analyses reveal that TRIM65 regu-lates miRNA-driven suppression of mRNA translation by tar-geting TNRC6 proteins for ubiquitination and degradation. Thus,TRIM65 represents a new negative regulator of miRNA activity.

Author contributions: S.L., L.W., B.F., and M.E.D. designed research; S.L., L.W., B.F., andM.A.B. performed research; S.L., L.W., B.F., A.D., and M.E.D. analyzed data; and S.L. andM.E.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1S.L., L.W., and B.F. contributed equally to this work.2To whom correspondence may be addressed. E-mail: shitao_li@hms.harvard.edu ordorf@hms.harvard.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1322545111/-/DCSupplemental.

6970–6975 | PNAS | May 13, 2014 | vol. 111 | no. 19 www.pnas.org/cgi/doi/10.1073/pnas.1322545111

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

021

our database) and reproducibility in our algorithm. We set highstringency thresholds: The z-score is set at ≥3 when prey oc-currence is <5%, the z-score is set at ≥10 when prey occurrenceis ≥ 5%, and the cutoff for reproducibility is fixed at ≥50%(appearance in at least two of four MS runs for the same bait).We identified 363 HCIPs making a protein interactome of 499unique interactions and forming major clusters corresponding tothe core components, DROSHA, DICER, and AGO (Fig. 1Aand Dataset S1). In comparison to the Biological General Re-pository for Interaction Datasets (BioGRID), IntAct, and SearchTool for the Retrieval of Interacting Genes/Proteins (STRING)protein interaction databases and curated literature, we found 55known interactions and 444 interactions that were not present inthese databases (Dataset S2). Thus, this proteomic analysis expandsthe number of candidate proteins linked to the miRNA pathway.

Validation and RNAi Analysis of the MPI. Coimmunoprecipitation(co-IP) was used to confirm previously unreported protein in-teractions. Bait and HCIPs were tagged with different epitopesand coexpressed in HEK 293 cells. In total, 93% (50 of 54) ofpreviously unknown interactions that were tested by co-IP werevalidated (Fig. S1 A–H and Dataset S2). Proteomic analysisestablished 16 reciprocal protein interactions. Thus, combiningpreviously established, co-IP, and reciprocal interactions, 24%(121 of 499) of protein interactions have been validated in theMPI (Fig. S2A and Dataset S2).RNAi knockdown was used to screen the effects of HCIP

on let-7a miRNA function. HeLa cells were transfected with a

plasmid expressing Renilla luciferase mRNA with a 3′ UTRcontaining two copies of a let-7a miRNA binding site, along witha firefly luciferase control (22). Using this let-7a reporter, wesystematically examined the function of 66 HCIPs that lackedknown roles in miRNA-mediated translational repression. RNAidepletion (four short interfering RNAs per gene) was used toscreen functional activity. We focused on HCIPs with multipleinteractions in the MPI or with enzymatic activity. Z-scores ≥2or ≤−2 were considered significant hits. Genes with two or moresignificant hits were selected as candidate regulators of the miRNApathway. In total, depletion of six identified genes (GTPBP4,NOC3L, POP1, PRKRIR, PRMT1, and PURA) increasesreporter activity, whereas knockdown of only one gene (TRIM65)reduces reporter activity (Fig. 1B).To validate the screening results, a side-by-side comparison

was performed with siRNAs against AGO2 as a positive controland the seven candidate genes identified in the primary screen.With the exception of PRMT1, triplicate experiments confirmedtwo or more significant RNAi hits for six genes (NOC3L, POP1,PRKRIR, PURA, TRIM65, and GTBP4) that modulate let-7areporter activity in HeLa cells (Fig. 1C). RNAi activity inthe reporter assays correlated with their efficiency in inhibitingprotein or RNA expression (Fig. S2 B–G). Knockdown of fourgenes (NOC3L, POP1, PURA, and TRIM65) also regulated let-7a reporter activity in A549 cells (Fig. S2H). Although depletionof GTPBP4 and PRKRIR increased let-7a reporter levels inHeLa cells, it did not significantly regulate reporter activity inA549 cells (Fig. S2H). In summary, a sampling of HCIPs (66 of

A siRNA

TRIM65TCF25RALY

B

RALYPASKPPANZBTB24SNRPB2LSG1KRI1ATXN2FBXO3ELAVL2MRTO4DHX57DHX57RBM28KRR1TSR1KLHL12SART3UPF1RBM34ZNF346BXDC2NMD3PURBPURBBYSLNKRFRAE1LTV1ERHC1ORF35HP1BP3SRP54FTSJ3DNA2PNRC1PNRC1KLHL29CCDC124NOP16ICT1MEPCELARP7NOB1SRRM1GLTSCR2AATFPOP7

New interactionsKnown interactions

Preys

B it POP7HECTD1PWP1TCOF1ZCCHC8SNRPA1SDCCAG1SRPK1GPRASP2RNF219PTCD1DDX50

C

3

3.5

4

4.5

se a

ctiv

ity

***

**

**

* **

Baits

Z-score

DDX50PHAXEBNA1BP2NOC3LGTPBP4PRKRIRPURAPRMT1POP1

0

0.5

1

1.5

2

2.5

Rel

ativ

e lu

cife

ras

* * **

*

** *

**

-3 3RNAi control AGO2 GTPBP4 NOC3L POP1 PRKRIR PRMT1 PURA TRIM650

Fig. 1. Overview and RNAi analysis of MPI. (A) Baitsand HCIPs are shown as squares and circles, re-spectively. The red line indicates a previously knowninteraction, and the green line indicates a previouslyunidentified interaction. Identification of HCIPs is pro-vided in Dataset S1. (B) Heat map screening of siRNAson a let-7a reporter (let7 A) in HeLa cells. Sixty-six HCIPRNAi oligos (four siRNAs per gene) were transfectedinto HeLa cells along with a let-7a reporter andcontrol firefly luciferase. Log transformation ofnormalized values, (Renilla luciferase value)/(fireflyluciferase value), was calculated for z-score analysis. Forstatistical analysis, the data were combined with ourlaboratory database containing results from 483 othersiRNAs tested for let-7a reporter activity in HeLa cells.(C) To validate the screening assay, HeLa cells weretreated with the indicated siRNA along with let-7a re-porter and control firefly luciferase. RNAi efficiency ispresented in Fig. S2 B–G. Each bar represents a dif-ferent siRNA sequence. Data depict the mean ± SDof triplicate samples. *P < 0.05.

Li et al. PNAS | May 13, 2014 | vol. 111 | no. 19 | 6971

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

021

363) identified six genes capable of regulating miRNA activity,two of which are cell type-dependent.

TRIM65 Interaction and Colocalization with TNRC6. The E3 ubiquitinligase TRIM65 associates with TNRC6B in the MPI. Therefore,we examined the interactions of TRIM65 with the family ofTNRC6 proteins (TNRC6A, TNRC6B, and TNRC6C). Endoge-nous TNRC6A bound to transiently (Fig. S3A) and stably expressedFLAG-TRIM65, and the interaction was enhanced by MG132proteasome inhibitor (Fig. 2A). Given the ability of TNRC6 to bindRNA, we examined the effect of RNase treatment on TNRC6–TRIM65 interactions. Depletion of RNA had little effect onTNRC6–TRIM65 associations, indicating the interaction is notbridged by RNA (Fig. 2A). TRIM65 also bound ectopically ex-pressed TNRC6B and TNRC6C when coexpressed in HEK 293

cells (Fig. S3B). To determine if TRIM65 was also associatedwith other RISC components, we looked for co-IP betweenTRIM65 and AGO1 or AGO2 proteins. TRIM65 selectivelyinteracts with TNRC6 and fails to coimmunoprecipitate withAGO proteins (Fig. S3 A and C). These data suggest directprotein interactions between TRIM65 and TNRC6 proteins.The tripartite N-terminal portion of TRIM65 contains the

RING (Really Interesting New Gene), BBOX (B-Box–type zincfinger), and CC (Coiled Coil) domains characteristic of TRIMfamily members. TRIM65 also contains a SPRY (SPla domain ofthe ryanodine receptor) domain within the C-terminal segment(Fig. 2B). TNRC6A and TNRC6B were cotransfected with GFPor a GFP-tagged TRIM65 domain. Domain mapping experimentsdemonstrate that the CC domain is sufficient for TNRC6 associ-ation (Fig. 2B and Fig. S3D). To investigate the relationshipamong AGO, TNRC6, and TRIM65, we fractionated cell lysatesfrom cells stably expressing FLAG-TRIM65 by centrifugationover a 15–55% sucrose gradient. Consistent with a previousreports (19, 23), AGO and TNRC6 sedimented across a broadrange of densities, which are referred to as complexes 1–3 (Fig.S4A). FLAG-TRIM65 is primarily located in complex 1, a low-sucrose density fraction. Digestion of sucrose gradient fractionswith a serine/threonine phosphatase, calf-intestinal alkaline phos-phatase, suggests the presence of phosphorylated TRIM65 (Fig.S4B). In addition to TRIM65 monomers, more slowly migratingforms were observed by SDS/PAGE under reducing conditions.The TRIM65 oligomers cosediment with the monomers. Co-IPsuggests that TNRC6A interacts with both TRIM65 monomersand oligomers (Fig. 2A and Fig. S4C). In contrast, FXR1 andPABP1 did not sediment with TRIM65 but were found in complexes3 and 2/3, respectively (Fig. S4A). Coexpression of HA- and FLAG-tagged TRIM65 demonstrates the capacity for TRIM65 self-asso-ciation (Fig. S4D). The data suggest multiple AGO–TNRC6 sub-complexes, at least one of which contains TRIM65 and TNRC6A.TNRC6 family proteins are enriched in cytoplasmic foci

known as P-bodies (14, 24). When expressed alone, GFP-TRIM65 formed speckles in the cytoplasm (Fig. S5A). A highdegree of overlap was observed between GFP-TRIM65 andendogenous TNRC6A foci (Fig. S5B). Endogenous TRIM65 isalso found in punctate cytosolic structures that partially colo-calize with endogenous TNRC6A in HeLa and HEK 293 cells(Fig. 2C and Fig. S5C). After treatment with MG132, these P-body–like structures appear enlarged and concentrated (Fig. 2C and Fig.S5C). Taken together, TRIM65 interacts with TNRC6 and colo-calizes with TNRC6 in P-body–like structures.

TRIM65 Regulates TNRC6 Ubiquitination and Stability. We hypothe-sized that TNRC6 proteins were substrates of the TRIM65 E3ubiquitin ligase. In vitro ubiquitination assays demonstrate bacterial-derived TRIM65-GST effectively delivers ubiquitin to TNRC6A. Incontrast, the TRIM65 ligase mutant containing a disrupted RINGdomain failed to induce ubiquitination (Fig. 3A). To examine therole of TRIM65 in the ubiquitination of endogenous TNRC6A,TRIM65 and its ligase-defective mutant were transfected into HeLacells treated with the MG132 proteasome inhibitor. As reportedpreviously (25), TNRC6A was constitutively ubiquitinated, whichmay be due to endogenous TRIM65 activity (Fig. S6A). Over-expression of TRIM65 enhanced the in vivo ubiquitination ofTNRC6A, whereas the TRIM65 mutant had no substantial effecton TNRC6A ubiquitination (Fig. S6A).We next investigated the effect of TRIM65 overexpression on

the stability of TNRC6 proteins. Consistently, TRIM65 inducedendogenous TNRC6A protein degradation in HeLa cells whenproteasome inhibitor was absent. In contrast, TNRC6A proteinlevels showed no substantial change when cells were transfectedwith the TRIM65 ligase mutant (Fig. 3B). TNRC6B or TNRC6Cprotein levels were also reduced when coexpressed with TRIM65in HEK 293 cells (Fig. S6B). The effects on TNRC6 were

BCC SPRY

A

150

250

IP: anti FLAG

RNase - - - + + MG132 - - + - +

TRIM65-FLAG con

MW(kDa)

TNRC6A-FLAG

CC SPRY

12 90 139 227 313 506

50

37

MW(kDa)

250

IP: anti-FLAGWB: anti-TNRC6A

IP: anti-FLAGWB: anti-FLAG

50

150IP: anti-FLAGWB: anti-GFP

25

150

IP: anti-FLAGWB: anti-FLAG

75

100

CMergeTRIM65TNRC6A DAPI

150

250

Lysate (2% input)WB: anti-TNRC6A

Lysate (2% input)WB: anti-GFP

25

37

50

*

10 m

-MG

132

+MG

132

10

Fig. 2. TRIM65 interacts and colocalizes with TNRC6. (A) HEK 293 cells stablyexpressing TRIM65-FLAG were treated with 10 μM protease inhibitor MG132or DMSO for 4 h. Cell lysates were collected and treated with 100 μg/mLRNase A. Immunoprecipitation (IP) and immunoblotting were performedusing the indicated antibodies. con, control; WB, Western blot. (B) GFP andvarious TRIM65 domains fused with GFP were transfected with TNRC6A-FLAG into HEK 293 cells. Cell lysates were coimmunoprecipitated with anti-FLAG antibody and blotted with the indicated reagents. The asterisk indi-cates GFP protein size. (C) HeLa cells were first treated with 10 μMMG132 orDMSO for 4 h and then fixed before incubation with anti-TNRC6A and anti-TRIM65 antibodies, followed by detection with secondary antibodies. DAPIwas used to detect nuclei. Arrows highlight colocalization.

6972 | www.pnas.org/cgi/doi/10.1073/pnas.1322545111 Li et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

021

specific, because the levels of other proteins in the RISC ma-chinery were not affected (Fig. 3B). To corroborate the role ofTRIM65 in the ubiquitination of endogenous TNRC6A, we usedsiRNA to knock down TRIM65. TNRC6A ubiquitination wasreduced after silencing TRIM65 (Fig. 3C). To assess the role ofthe proteasome in TNRC6A degradation, cells were transfectedwith control vector or TRIM65 and MG132 was added for vari-ous times. Accumulation of endogenous TNRC6A was observedafter addition of MG132 in the presence or absence of exogenousTRIM65 (Fig. S6C). Next, cells were treated with the translationinhibitor cycloheximide to examine the role of TRIM65 in TNRC6Astability further. After TRIM65 knockdown, TNRC6A t1/2 wasprolonged (Fig. 3D). The effect of TRIM65 knockdown on TNRC6protein levels appears specific, because the levels of other RISCcomponents are unchanged (Fig. S6D). To determine the effects ofTRIM65 protein on TNRC6A and AGO2 mRNA expression, weexamined relative mRNA expression levels after TRIM65 over-expression or depletion. Neither overexpression nor knockdown ofTRIM65 regulated TNRC6A and AGO2 mRNA expression (Fig.S6E). Taken together, the data indicate TRIM65 is a cognateubiquitin E3 ligase that selectively targets TNRC6 for ubiquiti-nation and subsequent protein degradation.

TRIM65 Relieves miRNA-MediatedmRNA Repression.We next examinedthe effect of overexpression of TRIM65 on let-7a miRNA reporteractivity. Ectopic expression of wild-type TRIM65, but not the mu-tant, increased reporter activity (Fig. 4A). Because TNRC6 degra-dation is associated with increased miRNA reporter activity, wereasoned that overexpression of TNRC6 would have the oppositeeffect. As predicted, overexpression of TNRC6A and TNRC6Bdecreased reporter activity (Fig. 4A). We next assessed the effect ofTRIM65 depletion on endogenous miRNA-targeted mRNA ex-pression. Three siRNAs were used to silence TRIM65 (Fig. S2E).Two miR-21–targeted genes, PDCD4 and PTEN (26, 27), and one

let-7–targeted gene, IMP1 (28), were examined after knock-down of TRIM65. Depletion of TRIM65 resulted in increasedTNRC6A protein levels and concomitant reduction of PDCD4,PTEN, and IMP1 protein levels (Fig. 4B). Real-time PCRquantification was used to monitor transcription rates afterActinomycin D treatment. The data suggest mRNA t1/2 is notaltered by silencing TRIM65 (Fig. S7 A–D). Conversely, knock-down of TNRC6A and/or TNRC6B leads to increases of PDCD4,PTEN, and IMP1 expression (Fig. 4C and Fig. S7E). Collectively,the data suggest that TRIM65 ubiquitination of TNRC6 results inTNRC6 degradation, and consequently relieves miRNA-mediatedtranslational repression.

DiscussionHere, we provide an extensive map describing a physical andfunctional protein interaction network associated with miRNAbiogenesis and regulation. AP-MS was used to assemble a net-work of 499 unique interactions between 40 baits and 363 HCIPs.AP-MS has been widely used to map protein interaction networksof mammalian signaling pathways. As with any screening ap-proach, the results must be interpreted with care and validatedwith more rigid experiments. About 11% of proteins (40 of 363)in the MPI harbor RNA-binding domains. Therefore, some pro-tein interactions may depend on RNA to bridge or stabilizeprotein complexes (29). Conversely, some protein interactionsmay go undetected by AP-MS because of low levels of prey ex-pression or cell-type dependence. Interactions may also beomitted due to the high stringency of our algorithm, which is de-signed to help exclude NSBPs. Although ribosomal proteins areknown to be involved in the miRNA pathway (30), they are fil-tered out from the MPI because of pervasive associations withRNA-binding proteins. Thus, the MPI does not represent a com-plete interaction network. However, it is important to emphasizethat 89% of the MPI interactions were not previously reported,

A

150

250

TRIM65-FLAG - + - - + -TRIM65mut-FLAG - - + - - +

B

HA-Ub + + + + + +HIS-TNRC6A + + + + + +

GST-TRIM65 + + - + + mutUBCH5A + - + + + +

E1 - + + + + +

ATP + + + - + +

150

75100

150

5050

150

WB: anti-TNRC6A

WB: anti-AGO1

WB: anti-DICER

WB: anti-PABP1

75

WB: anti-FXR1150

WB: anti-TNRC6A

HA Ub

75

250

150

WB: anti-HA

75

100

250

75

100

250

RNAi

C D

150

250

CHX (h)

WB: anti-TNRC6A

0 2 4 6 8

ontro

l RN

Ai

emai

ning

control RNAiTRIM65 RNAi

100

120

WB: anti-FLAG WB: anti- -Actin70

WB: anti-GST

150

50

75

25050

50

75

IP: anti-TNRC6AWB: anti-Ub

IP: anti-TNRC6AWB: anti-TNRC6A

LysateWB: anti-Ub

LysateWB: anti- -actin

50

50

150

250

CTR

IM65

RN

Ai

WB: anti- -actin

WB: anti-TNRC6A

WB: anti- -actin

Per

cent

age

of T

NRC

6A re

0 2 4 6 8Chase time (h)

20

40

60

80

Fig. 3. TRIM65 regulates ubiquitination and stabilityof TNRC6. (A) In vitro ubiquitination of TNRC6A byTRIM65 plus E1, E2 (UBCH5A, also known as UBE2D1),ATP, and HA-Ub. GST-tagged TRIM65 and mutantTRIM65 were purified from bacteria. His-taggedTNRC6A protein was purified from HEK 293 cellsthrough denatured washings. After an in vitro ubiq-uitination reaction, the mixture was incubated withnickel resins. The resins were washed under dena-tured conditions, and eluates were blotted with anti-HAantibody. The arrow indicates approximate TNRC6Aposition. (B) TRIM65 and the ligase mutant were trans-fected into HeLa cells. Cells were collected and blottedwith the indicated antibodies. (C) Control and TRIM65siRNA (RNAi sequence #4, see Supporting Information)were transfected into HeLa cells. Knockdown efficiencyis shown in Fig. S2E. Cells were treated with 10 μMMG132 for 4 h before harvest. Cell lysates were immu-noprecipitated with anti-TNRC6A antibody and immu-noblotted as indicated. (D) TRIM65 siRNA and controlsiRNA were transfected into HEK 293 cells. After 48 h,cells were equally aliquoted into 24-well plates andtreated with 50 μg/mL cycloheximide (CHX). Whole-celllysates were harvested at the indicated times. Quanti-tative analysis of Western blots by densitometry isshown in the graph. Values aremeans± SD of data fromthree separate experiments.

Li et al. PNAS | May 13, 2014 | vol. 111 | no. 19 | 6973

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

021

including six genes with demonstrated functional activity in reg-ulating miRNA activity. Thus, the MPI represents a resource forthe study of molecular mechanisms controlling miRNA biogenesisor regulation and offers opportunities to integrate the miRNAsilencing machinery with other cellular processes.TNRC6 proteins are critical cofactors of AGO proteins and serve

as key components of the RISC silencing machinery. The molecularmechanisms by which TNRC6 proteins contribute to translationalrepression remain incompletely understood. Furthermore, little is

known about how TNRC6 proteins are regulated. It has beenshown that TNRC6A undergoes ubiquitination and inhibition ofHSP90 activity diminishes TNRC6A levels (25, 31), but the linkbetween TNRC6A ubiquitination and proteasome-mediateddegradation has not been established. We demonstrate thatTRIM65 regulates TNRC6 activity at the posttranscriptionallevel by targeting TNRC6 proteins for ubiquitination and sub-sequent degradation. We conclude that TRIM65 is an ubiquitinE3 ligase for TNRC6 based on several lines of evidence: TRIM65associates with TNRC6 through its CC domain; TRIM65 cosedi-ments and colocalizes with TNRC6 proteins in P-body–like struc-tures; TRIM65 is able to ubiquitinate TNRC6 in vitro and in vivo;overexpression of TRIM65, but not its ligase-deficient mutant, leadsto TNRC6 degradation and let-7a reporter induction; and depletionof TRIM65 stabilizes and increases TNRC6A protein levels, andconsequently enhances miRNA-mediated suppression of endoge-nous gene expression. Overall, TRIM65 acts as an E3 ligase forTNRC6 ubiquitination and degradation.TNRC6 proteins participate in the downstream effector steps

controlling miRNA-mediated gene silencing (32, 33). DespiteTNRC6 redundancy, depleting TNRC6A, TNRC6B, or TNRC6Cpartially relieves the silencing of miRNA targets in human cells (11,13, 14, 19, 34, 35). TNRC6 proteins bridge and interact with RISCcomponents through distinct domains. Their aminoterminal do-main binds to AGO proteins, whereas a bipartite silencing domainin the TNRC6 middle and carboxyl-terminal regions interacts withPABP1. TNRC6 presumably promotes translational repression andmRNA degradation by coupling AGO proteins with the PABP1and CCR4/NOT deadenylase complexes. TRIM65 degradation ofTNRC6 proteins does not result in noticeable degradation of othercomponents in the RISC. Although we, as well as others, havenoted constitutive ubiquitination of TNRC6 (25), the physiologi-cally relevant cues that regulate TNRC6 protein stability andTRIM65 E3 ligase activity remain enigmatic.Here, we demonstrate a central role for the E3 ligase TRIM65

in regulating miRNA activity. Apart genome-wide associationstudies demonstrating that dementia-associated, age-relatedwhite matter lesions of the brain are linked to a locus nearTRIM65 (36, 37), little has been reported about TRIM65 func-tion. To our knowledge, this is the first report demonstratingTRIM65 E3 ligase activity. TRIM65 targeted TNRC6 proteinsfor ubiquitin-dependent degradation, releasing miRNA-medi-ated mRNA repression. However, such activity must be carefullyregulated to permit physiological miRNA function. The molec-ular mechanisms underlying TRIM65 E3 ligase function are in-completely understood. The roles of TRIM65 phosphorylationor oligomerization in regulating TNRC6 functional activity re-main under further investigation, because such modificationshave the potential for positively or negatively regulating themiRNA pathway. An understanding of the signals and mecha-nisms that control TNRC6 homeostasis and the interrelationshipwith TRIM65 E3 ligase activity is needed for improved un-derstanding of miRNA in health and disease.TRIM proteins represent one of the oldest and largest families

of ubiquitin E3 ligases. In mammals, TRIM32 ubiquitinatesPIASy, leading to its relocalization to the P-body (38). TRIM32also directly associates with AGO proteins and activates miRNAs(39). The Caenorhabditis elegans TRIM-NHL protein (NHL-2)localizes to the core RISC in the P-body and functions as a cofactorto enhance miRNA-mediated gene silencing (40). In contrast to theabove positive regulators, TRIM71 (also known as Lin41) associateswith and ubiquitinates AGO proteins (41). However, the functionalconsequences of TRIM71 ubiquitination of AGO are unclear (41–44). Other TRIM proteins that interact with AGOs to regulatemiRNAs include the Drosophila TRIM members Mei-P26 andBRAT (45). We now identify an additional TRIM protein criticalfor regulating miRNA function. Given the fundamental impor-tance of miRNA-guided regulation of gene expression in nearly all

A *

1.5

2

2.5po

rter a

ctiv

ity

0

0.5

1

Rel

ativ

e le

t7A

rep

* *

B

250

50

50

50

150

WB: anti-TNRC6A

WB: anti-PDCD4

WB: anti-PTEN

TRIM65 RNAicon #2 #3 #4 con #2 #3 #4

TRIM65 RNAi

50WB: anti- -actinWB: anti-IMP1

250

RNAi RNAiC

50

50

50

37

150

WB: anti-TNRC6A

WB: anti-PTEN

WB: anti-PDCD4

WB: anti-IMP1

WB: anti-Actin

Fig. 4. TRIM65 relieves miRNA repression. (A) TNRC6A, TNRC6B, TRIM65,and its mutant were transfected into HeLa cells along with let-7a Renillaluciferase reporter and control firefly luciferase. Data depict the mean ± SDof triplicate samples. *P < 0.05. (B) Knockdown of TRIM65 represses en-dogenous miRNA targets at the protein level. Control and three validatedTRIM65 siRNAs (Fig. S2E) were transfected into HeLa cells. TNRC6A, PTEN,IMP1, PDCD4, and actin protein levels were examined by Western blot. (C)Depletion of TNRC6A and/or TNRC6B relieved the suppression of miRNA-targeted gene expression. Control and TNRC6A and/or TNRC6B siRNAs weretransfected into HeLa cells. TNRC6A, PTEN, IMP1, PDCD4, and actin proteinlevels were examined by Western blot. The efficiency of TNRC6B RNAi is il-lustrated in Fig. S7E.

6974 | www.pnas.org/cgi/doi/10.1073/pnas.1322545111 Li et al.

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

021

cellular pathways, it is not surprising that multiple layers ofmiRNA control exist. TRIM65 serves as a rheostat for modulatingTNRC6 stability, and thereby provides a mechanism for regulatingmiRNA biology.

Materials and MethodsCell Lines and Constructs. HEK 293, HeLa, and A549 cells were purchased fromtheAmerican Type Culture Collection. To establish stable cell lines, bait cDNAswere transfected into HEK 293 cells using Lipofectamine 2000 (Invitrogen).Two days after transfection, cells were treatedwith hygromycin or puromycinfor 14 d. Single colonies were picked and expanded in six-well plates.Westernblotting was used to select clones for AP-MS. The DROSHA stable HEK 293 cellline was a gift from R. Gregory (Children’s Hospital, Boston).

Human TRIM65 was cloned from T98G glioblastoma cells (American TypeCulture Collection). The first and second Cys (Cys12 and Cys15) in the TRIM65-RING domain were mutated to Ala to destroy E3 ligase activity. AGO2-HA-FLAG,TNRC6A-HA-FLAG, TNRC6B-HA-FLAG, TNRC6C-HA-FLAG, and DGCR8-HA-FLAGwere kind gifts from T. Tuschl (The Rockefeller University, New York, NY). Thelet-7a reporter (let7 A) and DICER plasmids were generously provided byP. Sharp (Massachusetts Institute of Technology, Cambridge, MA) and P. Provost(Université Laval, Quebec City, Canada), respectively. Affinity-purified Flageluates revealed the dominant presence of bait protein by MS analysis.

Affinity Protein Purification. A total of 2 × 108 cells were lysed in 10 mL of lysisTAP buffer [50 mM Tris·HCl (pH 7.5), 10 mM MgCl2, 100 mM NaCl, 0.5%Nonidet P-40, 10% (vol/vol) glycerol, phosphatase inhibitors, and protease

inhibitors]. Supernatants were collected and precleared with 50 μL of pro-tein A/G resin. After shaking for 1 h at 4 °C, resin was removed by centri-fugation. Cell lysates were added to 20 μL of anti-FLAG M2 resin (Sigma) andincubated on a shaker. After 12 h, anti-FLAG M2 resin was washed threetimes (total of 15 min) with 10 mL of TAP buffer. After removing the washbuffer, the resin was transferred to a spin column (Sigma) and incubatedwith 40 μL of 3× FLAG peptide (Sigma) for 1 h at 4 °C on a shaker. Eluateswere collected by centrifugation and stored at −80 °C.

RNAi Perturbation. Dharmacon siRNA oligos were transfected using Lipo-fectamine 2000 according to the manufacturer’s protocol.

Statistics. The experimental data were grouped by prey, followed by a cal-culation of mean total spectral count and SD for all bait–prey associations.The results were used to derive a z-score for each bait–prey interaction. Thez-scores were then used to derive a P value for each interaction using thepnorm function of R, which calculates the P value by integrating over the areaof the normal distribution from negative infinity to the z-score.

ACKNOWLEDGMENTS. We thank Drs. Phillip Sharp (Massachusetts Instituteof Technology), Thomas Tuschl (The Rockefeller University), Narry Kim (SeoulNational University), and Patrick Provost (Université Laval) for constructs;Richard Gregory (Children’s Hospital, Boston) for the DROSHA HEK 293 cellline; and the Taplin Mass Spectrometry Facility (Harvard University) for serv-ices. S.L. is a John and Virginia Kaneb fellow. This work was supported byNational Institutes of Health Grants AI089829 and AI099860.

1. Ebert MS, Sharp PA (2012) Roles for microRNAs in conferring robustness to biologicalprocesses. Cell 149(3):515–524.

2. Bartel DP (2009) MicroRNAs: Target recognition and regulatory functions. Cell 136(2):215–233.

3. Czech B, Hannon GJ (2011) Small RNA sorting: Matchmaking for Argonautes. Nat RevGenet 12(1):19–31.

4. Ghildiyal M, Zamore PD (2009) Small silencing RNAs: An expanding universe. Nat RevGenet 10(2):94–108.

5. Han J, et al. (2006) Molecular basis for the recognition of primary microRNAs by theDrosha-DGCR8 complex. Cell 125(5):887–901.

6. Gregory RI, et al. (2004) The Microprocessor complex mediates the genesis of mi-croRNAs. Nature 432(7014):235–240.

7. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ (2004) Processing of primarymicroRNAs by the Microprocessor complex. Nature 432(7014):231–235.

8. Lund E, Güttinger S, Calado A, Dahlberg JE, Kutay U (2004) Nuclear export ofmicroRNA precursors. Science 303(5654):95–98.

9. Yi R, Qin Y, Macara IG, Cullen BR (2003) Exportin-5 mediates the nuclear export ofpre-microRNAs and short hairpin RNAs. Genes Dev 17(24):3011–3016.

10. Karginov FV, et al. (2010) Diverse endonucleolytic cleavage sites in the mammaliantranscriptome depend upon microRNAs, Drosha, and additional nucleases. Mol Cell38(6):781–788.

11. Meister G, et al. (2005) Identification of novel argonaute-associated proteins. CurrBiol 15(23):2149–2155.

12. Shin C, et al. (2010) Expanding the microRNA targeting code: Functional sites withcentered pairing. Mol Cell 38(6):789–802.

13. Jakymiw A, et al. (2005) Disruption of GW bodies impairs mammalian RNA in-terference. Nat Cell Biol 7(12):1267–1274.

14. Liu J, et al. (2005) A role for the P-body component GW182 in microRNA function. NatCell Biol 7(12):1261–1266.

15. Behm-Ansmant I, et al. (2006) mRNA degradation by miRNAs and GW182 requiresboth CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes. Genes Dev 20(14):1885–1898.

16. Bernstein E, Caudy AA, Hammond SM, Hannon GJ (2001) Role for a bidentate ribo-nuclease in the initiation step of RNA interference. Nature 409(6818):363–366.

17. Chendrimada TP, et al. (2007) MicroRNA silencing through RISC recruitment of eIF6.Nature 447(7146):823–828.

18. Haase AD, et al. (2005) TRBP, a regulator of cellular PKR and HIV-1 virus expression,interacts with Dicer and functions in RNA silencing. EMBO Rep 6(10):961–967.

19. Landthaler M, et al. (2008) Molecular characterization of human Argonaute-containingribonucleoprotein complexes and their bound target mRNAs. RNA 14(12):2580–2596.

20. Lee Y, et al. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature425(6956):415–419.

21. Li S, Wang L, Berman M, Kong YY, Dorf ME (2011) Mapping a dynamic innate im-munity protein interaction network regulating type I interferon production. Immu-nity 35(3):426–440.

22. Doench JG, Sharp PA (2004) Specificity of microRNA target selection in translationalrepression. Genes Dev 18(5):504–511.

23. Höck J, et al. (2007) Proteomic and functional analysis of Argonaute-containingmRNA-protein complexes in human cells. EMBO Rep 8(11):1052–1060.

24. Eystathioy T, et al. (2002) A phosphorylated cytoplasmic autoantigen, GW182, asso-ciates with a unique population of human mRNAs within novel cytoplasmic speckles.Mol Biol Cell 13(4):1338–1351.

25. Gibbings DJ, Ciaudo C, Erhardt M, Voinnet O (2009) Multivesicular bodies associate

with components of miRNA effector complexes and modulate miRNA activity. Nat

Cell Biol 11(9):1143–1149.26. Lewis BP, Shih IH, Jones-Rhoades MW, Bartel DP, Burge CB (2003) Prediction of

mammalian microRNA targets. Cell 115(7):787–798.27. Talotta F, et al. (2009) An autoregulatory loop mediated by miR-21 and PDCD4

controls the AP-1 activity in RAS transformation. Oncogene 28(1):73–84.28. Boyerinas B, et al. (2008) Identification of let-7-regulated oncofetal genes. Cancer Res

68(8):2587–2591.29. Frohn A, et al. (2012) Dicer-dependent and -independent Argonaute2 protein in-

teraction networks in mammalian cells. Mol Cell Proteomics 11(11):1442–1456.30. Janas MM, et al. (2012) Reduced expression of ribosomal proteins relieves microRNA-

mediated repression. Mol Cell 46(2):171–186.31. Johnston M, Geoffroy MC, Sobala A, Hay R, Hutvagner G (2010) HSP90 protein sta-

bilizes unloaded argonaute complexes and microscopic P-bodies in human cells. Mol

Biol Cell 21(9):1462–1469.32. Eulalio A, Tritschler F, Izaurralde E (2009) The GW182 protein family in animal cells:

New insights into domains required for miRNA-mediated gene silencing. RNA 15(8):

1433–1442.33. Miyoshi K, Okada TN, Siomi H, Siomi MC (2009) Characterization of the miRNA-RISC

loading complex and miRNA-RISC formed in the Drosophila miRNA pathway. RNA

15(7):1282–1291.34. Chu CY, Rana TM (2006) Translation repression in human cells by microRNA-induced

gene silencing requires RCK/p54. PLoS Biol 4(7):e210.35. Zipprich JT, Bhattacharyya S, Mathys H, Filipowicz W (2009) Importance of the

C-terminal domain of the human GW182 protein TNRC6C for translational repression.

RNA 15(5):781–793.36. Fornage M, et al. (2011) Genome-wide association studies of cerebral white matter

lesion burden: The CHARGE consortium. Ann Neurol 69(6):928–939.37. Freudenberger P, Schmidt R, Schmidt H (2012) Genetics of age-related white matter

lesions from linkage to genome wide association studies. J Neurol Sci 322(1-2):82–86.38. Albor A, et al. (2006) The interaction of Piasy with Trim32, an E3-ubiquitin ligase

mutated in limb-girdle muscular dystrophy type 2H, promotes Piasy degradation and

regulates UVB-induced keratinocyte apoptosis through NFkappaB. J Biol Chem

281(35):25850–25866.39. Schwamborn JC, Berezikov E, Knoblich JA (2009) The TRIM-NHL protein TRIM32 activates

microRNAs and prevents self-renewal in mouse neural progenitors. Cell 136(5):913–925.40. Hammell CM, Lubin I, Boag PR, Blackwell TK, Ambros V (2009) nhl-2 Modulates

microRNA activity in Caenorhabditis elegans. Cell 136(5):926–938.41. Rybak A, et al. (2009) The let-7 target gene mouse lin-41 is a stem cell specific E3

ubiquitin ligase for the miRNA pathway protein Ago2. Nat Cell Biol 11(12):1411–1420.42. Chang HM, et al. (2012) Trim71 cooperates with microRNAs to repress Cdkn1a ex-

pression and promote embryonic stem cell proliferation. Nat Commun 3:923.43. Chen J, Lai F, Niswander L (2012) The ubiquitin ligase mLin41 temporally promotes

neural progenitor cell maintenance through FGF signaling. Genes Dev 26(8):803–815.44. Loedige I, Gaidatzis D, Sack R, Meister G, Filipowicz W (2013) The mammalian TRIM-NHL

protein TRIM71/LIN-41 is a repressor of mRNA function. Nucleic Acids Res 41(1):518–532.45. Neumüller RA, et al. (2008) Mei-P26 regulates microRNAs and cell growth in the

Drosophila ovarian stem cell lineage. Nature 454(7201):241–245.

Li et al. PNAS | May 13, 2014 | vol. 111 | no. 19 | 6975

BIOCH

EMISTR

Y

Dow

nloa

ded

by g

uest

on

Mar

ch 2

1, 2

021