A novel monoclonal antibody against human Argonaute proteins reveals unexpected characteristics of miRNAs in human blood cells

Abstract

Argonaute (Ago) proteins bind to microRNA (miRNAs) and short interfering RNAs (siRNAs) and form the core components of effector complexes that mediate miRNA and siRNA function. Currently, there is a paucity of reliable antibodies against mammalian Ago proteins, thus precluding studies of endogenous Ago proteins from tissues. Here we report the development of 2A8, a novel anti-Ago monoclonal antibody that recognizes human and mouse Ago proteins and efficiently immunoprecipitates miRNAs. We report the characterization of 2A8 and its use to clone miRNAs from human brain and from preparations of human polymorphonuclear leukocytes (neutrophils), which revealed a prevalent miRNA with unusual features.

FIGURE 1.

Properties of 2A8, a novel anti-Ago monoclonal antibody. (a) 2A8 recognizes Ago proteins on Western blot. Cell lysate from human HeLa cells was resolved on a 4%–12% NuPAGE gel, blotted and probed with 2A8. Molecular mass markers (in kilodaltons) are shown on the right. hAgos are shown with arrow. Asterisk indicates cross-reaction with radixin. (b) 2A8 immunoprecipitates Ago proteins. 2A8 immunoprecipitates from human 293 cells were resolved on a 4%–12% NuPAGE gel, blotted, and probed with 2A8 ascites. hAgos are shown with arrow. Asterisk indicates cross-reaction with radixin. (c) 2A8 coimmunoprecipitates miRNAs. Immunoprecipitations (IP) from HeLa cells were performed with 2A8 or nonimmune mouse serum (IgG) or Sp2/0 (both negative controls). RNA was isolated from the immunoprecipitates, 3′-end-labeled with [5′-³²P]-pCp and T4 RNA ligase, resolved by electrophoresis on a 20% denaturing polyacrylamide gel, and visualized by autoradiography. Nucleotide sizes of the radiolabeled marker (M) are shown on the left. (d) Identification of immunoprecipitated protein with mass spectrometry. Immunoprecipitations were performed from human Raji cells or from human brain with 2A8 or nonimmune mouse serum (NMS). Immunoprecipitates were resolved on a 4%–12% NuPAGE gel, stained with Coomasie blue, and the P95 and P70 bands (boxed) were excised from the gel, subjected to trypsin digestion and mass spectrometry. (e) 2A8 recognizes all human Ago proteins. 293 cells were transfected with FLAG-tagged Ago1–4 proteins or with empty vector (Mock), followed by immunoprecipitations with anti-FLAG antibody. Immunoprecipitates were probed with 2A8 on Western blot. (f) 2A8 immunoprecipitates are active in miRNA-directed target RNA cleavage. Schematic of the RNA target (let7-TP) and the base-pairing with let-7a (blue); the [5′-³²P] of pCp is shown in red. Cleavage site is indicated with red lightning bolt. Immunoprecipitations were performed with 2A8 or nonimmune mouse serum (NMS) and the beads were incubated with 3′-end-radiolabeled let7-TP RNA target. The products of the reactions were analyzed on 20% denaturing polyacrylamide gel. Nucleotide sizes of the radiolabeled marker (M) are shown on the right. (g,h) Immunocytochemistry with 2A8 was performed in formalin-fixed and paraffin-embedded (FFPE) human brain tissue (g) or MN-1, mouse motor neuron-like cells (h), and revealed predominantly cytoplasmic staining. In human FFPE brain, neurons were prominently stained (g). Arrow in h indicates a growth cone-like process of MN-1 cells, which is also labeled. The bar in (g) is either 50 μm (g) or 30 μm (h).

FIGURE 2.

Cloning of miR-451 from immunopurified Agos from human neutrophils. (a) Sequence and secondary structure of human pre-miR-451. Mature miR-451 is shown in red. Nucleotide positions of the 3′-end of miR-451 are indicated. Percentages of clones that ended in specified nucleotide are shown in parentheses. Cloned miR-451 from human blood varied in length from 20 to 26 nt; 1% of clones were 26 nt long. (b) Genomic position of miR-451 gene in different species.