DUSP11 - An RNA phosphatase that regulates host and viral non-coding RNAs in mammalian cells

Abstract

Dual-specificity phosphatase 11 (DUSP11) is a conserved protein tyrosine phosphatase (PTP) in metazoans. The cellular substrates and physiologic activities of DUSP11 remain largely unknown. In nematodes, DUSP11 is required for normal development and RNA interference against endogenous RNAs (endo-RNAi) via molecular mechanisms that are not well understood. However, mammals lack analogous endo-RNAi pathways and consequently, a role for DUSP11 in mammalian RNA silencing was unanticipated. Recent work from our laboratory demonstrated that DUSP11 activity alters the silencing potential of noncanonical viral miRNAs in mammalian cells. Our studies further uncovered direct cellular substrates of DUSP11 and suggest that DUSP11 is part of regulatory pathway that controls the abundance of select triphosphorylated noncoding RNAs. Here, we highlight recent findings and present new data that advance understanding of mammalian DUSP11 during gene silencing and discuss the emerging biological activities of DUSP11 in mammalian cells.

Keywords: Argonaute; BLV; DUSP11; PIR-1; RNAi; VA RNA; dicer; miRNA; miRNA biogenesis; triphosphate.

Figure 1.

Canonical and noncanonical host and viral miRNA biogenesis pathways. Schematic diagrams of the canonical and noncanonical host and viral miRNA biogenesis in animals. Dashed red lines indicate noncanonical processing mechanisms. Enzymes in red indicate noncanonical miRNA biogenesis enzymes or mechanisms used by those enzymes to generate miRNA RNA species.

Figure 2.

Model of DUSP11-dependent Argonaute association of BLV and AdV 5p miRNAs. The BLV and Adenovirus miRNA precursors are transcribed by RNAP III and initially contain a 5′ triphosphate. In the absence of DUSP11-mediated dephosphorylation, the 5′ triphosphorylated precursors are processed by Dicer to yield mature 5p:3p duplex miRNAs. Through a 5′-monophosphate-dependent strand selection mechanism that is coupled to Dicer processing, the triphosphorylated 5p miRNAs are excluded from being stably loaded into Argonaute proteins, resulting in preferential loading of the 3p strand. The dashed line indicates that 5′ triphosphorylated 5p miRNAs may have a reduced stable association with Argonaute proteins, resulting in their increased turnover. However, miRNA precursors that are dephosphorylated by DUSP11 produce 5′ monophosphorylated 5p miRNAs that are more efficiently loaded and/or more stably associate with Argonaute proteins.

Figure 3.

DUSP11 motifs/domains involved in localization and RNA phosphatase activity in cells. (A) Schematic diagram of DUSP11 (not to scale) illustrating the relative location of the arginine-rich motifs, putative NLS, catalytic domain, and proline-rich motif. Below are the DUSP11 mutants that we generated, which are described in the text. We cloned the constructs into pcDNA3.1+ and tagged the N-terminus via PCR-based engineering with a 3xFLAG epitope. (B) Nuclear/cytosolic fractionation of A549 and HEK293T cells to determine localization of DUSP11. Cells from one well of 12-well format plate were trypsinized, washed with phosphate-buffered solution (PBS), resuspended in 50-ul of CSKT buffer [10mM PIPES (pH 6.8), 100mM NaCl, 300mM sucrose, 3mM MgCl₂, 1mM EDTA, 1mM DTT, 0.5% (vol/vol) tritonX-100, protease inhibitors (Roche)], and placed on ice for 10 minutes. Nuclei were then pelleted by centrifugation at 5000xg for 5 minutes. The supernatant [cytosolic fraction (Cyto)] was removed (50-ul) and added to 50-ul of SDS lysis buffer (1% SDS; 2% 2-mercaptoethanol). The nuclei were then washed in 50-ul of CSKT buffer and centrifuged at 5000xg for 5 minutes. The nuclei were then resuspended in 50-ul of water followed by addition of 50-ul of SDS buffer to make the nuclear fraction (Nuc). A whole cell lysate (WCL) was collected in parallel by trypsinization and washing of the cells, which were then resuspended in 50-ul of water, followed by addition of 50-ul of SDS lysis buffer. Samples were boiled for 10 minutes followed by vortexing for 30 seconds. Equal volumes (10-ul) of fractions were then fractionated via 12.5% SDS-PAGE. Protein was then transferred to nitrocellulose membrane (Bio-Rad). Anti-DUSP11 polyclonal rabbit antibody (1:2000 dilution; Proteintech, catalog no. 10204–2-AP), anti-α-Tubulin monoclonal mouse antibody (1:10,000 dilution; Sigma-Aldrich, catalog no. T6199), and anti-Histone H3 (D1H2) rabbit antibody (Cell signaling; mAb #4499) in phosphate-buffered saline (PBS) with 0.1% Tween20 (PBST) and 5% BSA were used to blot for DUSP11, α-Tubulin (cytosolic localized control), and Histone H3 (nuclear localized control). After washing with PBST, membranes were blotted with IRDye 800CW and IRDye 680LT secondary antibodies (1:10,000 dilution; LI-COR) in PBST with 5% BSA. Blots were washed 4 times with PBST and then scanned on an Odyssey CLx infrared imaging system (LI-COR). (C) Nuclear/cytosolic fractionation as described in (B) of HEK293T cells (12-well format) transfected with 200-ng/well of the 3xFLAG-tagged DUSP11 constructs from (A). The anti-FLAG M2 mouse monoclonal antibody (Sigma-Aldrich) was used to stain for the 3xFLAG-tagged DUSP11 proteins. (D) Analysis of the BLV-B2 5p:3p miRNA ratios with co-expression of the DUSP11 constructs from (A). DUSP11-null HEK293T cells (24-well format; 70% confluent) were co-transfected with 5-ng/well of the EBER1 transfection control expression vector (pEBV RIJ), 400-ng/well of pBLV-B2, and 100-ng/well of pDUSP11, pDUSP11-C152, or the DUSP11 construct shown in (A). Northern blot analysis was then performed as described in refs.  and . The membrane was probed with the BLV-miR-B2–5p probe, stripped and re-probed with the BLV-miR-B2–3p probe, and stripped and re-probed with the EBER1 probe as a loading/transfection control. Below is immunoblot analysis that was performed on duplicate lysates to confirm and quantitate DUSP11 expression from each construct using the anti-FLAG M2 mouse monoclonal antibody (Sigma-Aldrich). (E) Bar graph of the relative band density from 4 independent RNA gel blot analyses, as shown in D (except for DUSP11-Δ13–26, in which 3 replicates were performed). Bars represent the average band density ratio (5p/3p) +/− SD of the BLV-B2 miRNAs. The ratio with co-expression of each DUSP11 construct was normalized to the C152S catalytic mutant. Asterisks represent statistical significance (p<0.05) in comparison to wild type DUSP11 (WT). P-values were determined using student's t-test.

Figure 4.

Model for putative roles of DUSP11 in mammalian cells. Illustration of the model for DUSP11s role in mammalian cells. DUSP11 dephosphorylates exogenous or endogenous 5′-triphosphorylated RNAs. This activity results in 5′ monophosphorylated RNAs, which can alter the function/activity of the RNA (for example loading into RISC) or permit degradation of the RNA by nucleases. This process may also reduce activation of PRRs, which recognize 5′ triphosphorylated RNAs and trigger the innate immune response.