Abstract
RNA silencing processes are guided by small RNAs known as siRNAs and microRNAs (miRNAs) . They reside in ribonucleoprotein complexes, which guide the cleavage of complementary mRNAs or affect stability and translation of partial complementary mRNAs . Argonaute (Ago) proteins are at the heart of silencing effector complexes and bind the single-stranded siRNA and miRNA . Our biochemical analysis revealed that Ago2 is present in a pre-miRNA processing complex that is able to transfer the miRNA into a target-mRNA cleaving complex. To gain insight into the function and composition of RNA silencing complexes, we purified Ago1- and Ago2-containing complexes from human cells. Several known Ago1- and/or Ago2-associated proteins including Dicer were identified, but also two novel factors, the putative RNA helicase MOV10, and the RNA recognition motif (RRM)-containing protein TNRC6B/KIAA1093. The new proteins localize, similar to Ago proteins, to mRNA-degrading cytoplasmic P bodies, and they are functionally required to mediate miRNA-guided mRNA cleavage.
Publication types
-
Comparative Study
-
Research Support, N.I.H., Extramural
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Argonaute Proteins
-
Blotting, Northern
-
Blotting, Western
-
Cell Line
-
Eukaryotic Initiation Factor-2
-
Eukaryotic Initiation Factors / metabolism
-
Fluorescent Antibody Technique
-
Humans
-
Intracellular Signaling Peptides and Proteins / genetics*
-
Intracellular Signaling Peptides and Proteins / metabolism
-
Mass Spectrometry
-
MicroRNAs / metabolism
-
Oligonucleotides
-
Peptide Initiation Factors / metabolism
-
RNA Helicases / genetics*
-
RNA Helicases / metabolism
-
RNA, Messenger / metabolism*
-
RNA, Small Interfering / genetics
-
RNA-Induced Silencing Complex / genetics
-
RNA-Induced Silencing Complex / metabolism*
-
Reverse Transcriptase Polymerase Chain Reaction
-
Ribonuclease III / metabolism
Substances
-
AGO1 protein, human
-
AGO2 protein, human
-
Argonaute Proteins
-
Eukaryotic Initiation Factor-2
-
Eukaryotic Initiation Factors
-
Intracellular Signaling Peptides and Proteins
-
MicroRNAs
-
Oligonucleotides
-
Peptide Initiation Factors
-
RNA, Messenger
-
RNA, Small Interfering
-
RNA-Induced Silencing Complex
-
Ribonuclease III
-
RNA Helicases