MicroRNAs (miRNAs) are 19-22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer(-)/(-) mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3' untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ~68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences cross-linked to Ago2 in both the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.
Experimental Support 2 for Functional miRNA-Target Interaction
miRNA:Target
----
Validation Method
Conditions
Liver
Location of target site
3'UTR
Tools used in this research
TargetScan
,
miRTarCLIP
,
Piranha
Original Description (Extracted from the article)
...
HITS-CLIP data was present in ERR266281. RNA binding protein: AGO2. Condition:A_Liver partial hapatectomy 48h
HITS-CLIP data was present in ERR266286. RNA binding protein: AGO2. Condition:A_Liver partial hapatectomy 1h
HITS-CLIP data was present in ERR266287. RNA binding protein: AGO2. Condition:B_Liver partial hapatectomy 36h
HITS-CLIP data was present in ERR266292. RNA binding protein: AGO2. Condition:B_Liver partial hapatectomy 48h
HITS-CLIP data was present in ERR266293. RNA binding protein: AGO2. Condition:A_Liver partial hapatectomy 36h
HITS-CLIP data was present in ERR266295. RNA binding protein: AGO2. Condition:B_Liver partial hapatectomy 1h
HITS-CLIP data was present in ERR266298. RNA binding protein: AGO2. Condition:A_Untreated
...
- Schug J; McKenna LB; Walton G; Hand N; et al., 2013,
BMC genomics.
BACKGROUND: Validation of physiologic miRNA targets has been met with significant challenges. We employed HITS-CLIP to identify which miRNAs participate in liver regeneration, and to identify their target mRNAs. RESULTS: miRNA recruitment to the RISC is highly dynamic, changing more than five-fold for several miRNAs. miRNA recruitment to the RISC did not correlate with changes in overall miRNA expression for these dynamically recruited miRNAs, emphasizing the necessity to determine miRNA recruitment to the RISC in order to fully assess the impact of miRNA regulation. We incorporated RNA-seq quantification of total mRNA to identify expression-weighted Ago footprints, and developed a microRNA regulatory element (MRE) prediction algorithm that represents a greater than 20-fold refinement over computational methods alone. These high confidence MREs were used to generate candidate 'competing endogenous RNA' (ceRNA) networks. CONCLUSION: HITS-CLIP analysis provide novel insights into global miRNA:mRNA relationships in the regenerating liver.
Experimental Support 3 for Functional miRNA-Target Interaction
miRNA:Target
----
Validation Method
Conditions
C2C12
Location of target site
3'UTR
Tools used in this research
TargetScan
,
miRTarCLIP
,
Piranha
Original Description (Extracted from the article)
...
HITS-CLIP data was present in GSM1385342. RNA binding protein: 聽AGO2. Condition:C2C12_GM_Ago2_CLIP-Seq_myoblast
HITS-CLIP data was present in GSM1385343. RNA binding protein: 聽AGO2. Condition:C2C12_DM_Ago2_CLIP-Seq_myotubes
...
- Zhang X; Zuo X; Yang B; Li Z; Xue Y; Zhou et al., 2014,
Cell.
- Zhang X; Zuo X; Yang B; Li Z; Xue Y; Zhou et al.
- Cell, 2014
MicroRNAs are well known to mediate translational repression and mRNA degradation in the cytoplasm. Various microRNAs have also been detected in membrane-compartmentalized organelles, but the functional significance has remained elusive. Here, we report that miR-1, a microRNA specifically induced during myogenesis, efficiently enters the mitochondria where it unexpectedly stimulates, rather than represses, the translation of specific mitochondrial genome-encoded transcripts. We show that this positive effect requires specific miR:mRNA base-pairing and Ago2, but not its functional partner GW182, which is excluded from the mitochondria. We provide evidence for the direct action of Ago2 in mitochondrial translation by crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq), functional rescue with mitochondria-targeted Ago2, and selective inhibition of the microRNA machinery in the cytoplasm. These findings unveil a positive function of microRNA in mitochondrial translation and suggest a highly coordinated myogenic program via miR-1-mediated translational stimulation in the mitochondria and repression in the cytoplasm.
