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. 2014 Feb 11;111(6):2373-8.
doi: 10.1073/pnas.1314851111. Epub 2014 Jan 27.

Selective mRNA sequestration by OLIGOURIDYLATE-BINDING PROTEIN 1 contributes to translational control during hypoxia in Arabidopsis

Reed Sorenson  1 , Julia Bailey-Serres
Affiliations

Selective mRNA sequestration by OLIGOURIDYLATE-BINDING PROTEIN 1 contributes to translational control during hypoxia in Arabidopsis

Reed Sorenson et al. Proc Natl Acad Sci U S A. .

Abstract

Low oxygen stress dynamically regulates the translation of cellular mRNAs as a means of energy conservation in seedlings of Arabidopsis thaliana. Most of the highly hypoxia-induced mRNAs are recruited to polysomes and actively translated, whereas other cellular mRNAs become translationally inactive and are either targeted for stabilization or degradation. Here we identify the involvement of OLIGOURIDYLATE BINDING PROTEIN 1 (UBP1), a triple RNA Recognition Motif protein, in dynamic and reversible aggregation of translationally repressed mRNAs during hypoxia. Mutation or down-regulation of UBP1C interferes with seedling establishment and reduces survival of low oxygen stress. By use of messenger ribonucleoprotein (mRNP) immunopurification, we show that UBP1C constitutively binds a subpopulation of mRNAs characterized by uracil-rich 3'-untranslated regions under normoxic conditions. During hypoxia, UBP1C association with non-uracil-rich mRNAs is enhanced concomitant with its aggregation into microscopically visible cytoplasmic foci, referred to as UBP1 stress granules (SGs). This UBP1C-mRNA association occurs as global levels of protein synthesis decline. Upon reoxygenation, rapid UBP1 SG disaggregation coincides with the return of the stabilized mRNAs to polysomes. The mRNAs that are highly induced and translated during hypoxia largely circumvent UBP1C sequestration. Thus, UBP1 is established as a component of dynamically assembled cytoplasmic mRNPs that sequester mRNAs that are poorly translated during a transient low energy stress.

Keywords: RNA-binding; TIA-1; posttranscriptional; ribosome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
UBP1C contributes to survival of oxygen deprivation. Stratified seeds grown in the presence of 1% (wt/vol) sucrose on vertically oriented plates under a diurnal light cycle for 10 d and deprived of oxygen for up to 13 h. After 6 d of recovery, seedlings with green apical shoots were scored as survivors. Genotypes evaluated were Col-0, ubp1c-1, upb1c-amiR-1, and two independent ubp1c-1 35S:FLAG-UBP1C (ubp1c-1 FLAG-UBP1C) lines. (A) Photograph of representative untreated (mock) and oxygen-deprived (13 h) seedlings following recovery (day 16). (B) Percentage survival ±SEM of seedlings (10 seedlings per plate; n = 12–14 replicates). Bars with same letter are not significantly different (adjusted P value <0.05, one-way ANOVA, Tukey honest significant difference adjustment).
Fig. 2.
Fig. 2.
UBP1C-GFP dynamically and reversibly aggregates into cytoplasmic granules containing poly(A)+ RNA and PAB in response to hypoxia. Time course of UBP1C-GFP cytoplasmic granule formation in response to hypoxia in leaves of ubp1c-1 35S:UBP1C-GFP-2. Tissue was submerged under a coverslip and imaged after 8 (A), 25 (B), and 45 min (C) (Movie S1). Images are 3D reconstructions from a 10-image z stack. GFP and chlorophyll fluorescence channels false-colored green and red, respectively [area = (97 µm)2, z depth = 21 µm]. (D–K) Maximum projection of a 16-image z stack; histograms are relative GFP intensity in 0–0.25 (dark gray bar), 0.25–0.75 (white bar), 0.75–1.25 (light gray bar), and >1.25 µm (midgray bar) diameter granules. (D and E) Reoxygenation reverses UBP1C granule formation. Five-day-old ubp1c-1 35S:UBP1C-GFP-1 cotyledon epidermal cells. Seedlings were submerged in 0.25× Murashige–Skoog under a coverslip for 60 min (D) and exposed to humidified air for 20 min (E). (F and G) Oxyrase treatment induces UBP1C granule formation. Five-day-old ubp1c-1 35S:UBP1C-GFP-1 seedling cotyledon epidermal cells after 4 min (F) and 22 min (G) of submergence in 0.25× Murashige–Skoog containing 5 units per mL−1 Oxyrase. (H–K) UBP1C granule formation in ubp1c-1 35S:UBP1C-GFP-1 hypocotyl cells is inhibited by CHX. Images of 5-d-old seedlings bathed with 0.4% dimethyl sulfoxide maintained 1 h in the light (H), in the dark (I), or in an Ar(gas)-purged chamber without (J) or with 200 ng⋅µL−1 CHX (K). (L) Cotyledon mesophyll cells of 3-d-old 35S:UBP1C-GFP-1 seedlings after 3 h under 2% O2, hybridized to Cy3-oligo(dT). (M) Hypocotyl epidermal cells of 35S:UBP1C-GFP × 35S:PAB2-mRFP first generation offspring seedlings after 60 min submergence. (N) UBP1C also localizes to the nucleus [area = (30 μm)2]. Fluorescent signals from UBP1C-GFP and DAPI were merged. Ar laser excitation at 488 (GFP, chl) and 543 nm (cy3, mRFP). Emission collection at 500–600 nm for GFP and 650–700 nm for chlorophyll, respectively (A–N). GFP/mRFP fluorescence collected in series. (Scale bars = 10 μm in D–G and 20 µm in H–M.)
Fig. 3.
Fig. 3.
UBP1C binds mRNAs with U-rich 3′-UTRs under normoxia and associates with poorly translated mRNAs during hypoxia. (A) Seedlings (ubp1c-1 35S:FLAG-UBP1C-1) grown on Murashige–Skoog containing 1% (wt/vol) sucrose in the light (L) (control), dim light for 2 h (D), 2 h hypoxia in dim light (H), or 2-h hypoxia followed by 20-min reoxygenation in dim light (R). UBP1C immunoprecipitation (IP) with anti-FLAG beads, using anti-HA IP as a negative control (M). (B) Polysomal abundance profiles of samples after treatments. (C) Immunoblot of 0.33% of the cell homogenate (input) and the post-IP supernatant (unbound) and 7.5% of the IP. Protein detection with antibodies against FLAG (α-FLAG) and ribosomal protein S6 (α-RPS6). Markers are in kilodaltons. (D) Analysis of representative RNA from total, mock IP, and UBP1C IP fractions. (E) Quantitative RT-PCR used to measure mRNAs in the UBP1C IP normalized to total abundance and compare total fold change relative to ADH1 in L. Data are mean ± SEM of three independent experiments. (F and G) Global analysis of UBP1C mRNA association. ATH1 microarrays identified 13,964 UBP1C client mRNAs. The SLR of UBP1C IP mRNA to total mRNA (T). (F) Scatter plots of transcript association with UBP1C comparing seedlings in L to those in D, H, or R. (G) Fuzzy k-means cluster analysis performed on SLR comparisons (k = 20) of UBP1C-mRNA association and ribosome-mRNA association to identify coregulated genes. Comparisons: UBP1C IP versus total RNA (IP/T) under each condition and IP/T of each treatment versus L (IP/T Δ); total mRNA abundance (T) in treatment versus L; immunopurified polysomal mRNA (FLAG-RPL18 IP) from 2 or 9 h hypoxia relative to 2 or 9 h nonstress conditions (2H/2N, 9H/9N) and 9H + 1 h air (1R/9N) and total mRNA from same samples (1). Cluster median values are represented in the heatmap. (H) Percentage nucleotide composition of 3′UTRs by cluster.
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