Human Argonaute 2 Is Tethered to Ribosomal RNA through MicroRNA Interactions.
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 cross-linking 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.
From the UAB Stem Cell Institute, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35209 and.
Blake L
BL
Atwood
From the UAB Stem Cell Institute, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35209 and.
Jessica L
JL
Woolnough
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland.
Gaelle M
GM
Lefevre
From the UAB Stem Cell Institute, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35209 and.
Mariana
M
Saint Just Ribeiro
Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, Maryland.
Gary
G
Felsenfeld
From the UAB Stem Cell Institute, Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35209 and kegiles@uab.edu.
Keith E
KE
Giles
D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D020014:K562 Cells; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D035463:Pol1 Transcription Initiation Complex Proteins / Q000235:genetics / Q000378:metabolism*; D012318:RNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000096:biosynthesis* / Q000235:genetics; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism
C466422:AGO2 protein, human; D060565:Argonaute Proteins; C541874:MIRN92 microRNA, human; D035683:MicroRNAs; D035463:Pol1 Transcription Initiation Complex Proteins; D012335:RNA, Ribosomal; C088179:RNA, ribosomal, 45S; C063844:transcription factor UBF; D012318:RNA Polymerase I; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases
Argonaute; ChIP-sequencing (ChIP-seq); Dicer; miRNA; microRNA (miRNA); par-clip; rRNA; ribosomal ribonucleic acid (rRNA) (ribosomal RNA)