Heat Shock Protein HSP101 Affects the Release of Ribosomal Protein mRNAs for Recovery after Heat Shock

Abstract

Heat shock (HS) is known to have a profound impact on gene expression at different levels, such as inhibition of protein synthesis, in which HS blocks translation initiation and induces the sequestration of mRNAs into stress granules (SGs) or P-bodies for storage and/or decay. SGs prevent the degradation of the stored mRNAs, which can be reengaged into translation in the recovery period. However, little is known on the mRNAs stored during the stress, how these mRNAs are released from SGs afterward, and what the functional importance is of this process. In this work, we report that Arabidopsis HEAT SHOCK PROTEIN101 (HSP101) knockout mutant (hsp101) presented a defect in translation recovery and SG dissociation after HS Using RNA sequencing and RNA immunoprecipitation approaches, we show that mRNAs encoding ribosomal proteins (RPs) were preferentially stored during HS and that these mRNAs were released and translated in an HSP101-dependent manner during recovery. By ¹⁵N incorporation and polysome profile analyses, we observed that these released mRNAs contributed to the production of new ribosomes to enhance translation. We propose that, after HS, HSP101 is required for the efficient release of RP mRNAs from SGs resulting in a rapid restoration of the translation machinery by producing new RPs.

Figure 1.

hsp101 knockout mutant is affected in polysome recovery. A to F, Polysome profiles on 7-d-old seedlings in the wild type (A–C) or hsp101 (D–F). A and D, Control 22°C. B and E, HS for 1 h at 37°C. C and F, Two hours of recovery at 22°C after HS. G, Percentage of polysomes for each condition. n = 3 biological repeats, mean ± sd. A t test was performed between wild type and hsp101 for control (P > 0.1), at 1 h 37°C (P > 0.1), and at 2 h recovery (P < 0.05).

Figure 2.

hsp101 knockout mutant is affected in SG dissociation after HS. A to L, Monitoring of SG formation using pPABP2-RFP-PABP2 in wild type (A–F) or the hsp101 background (G–L). A, D, G, and J, Control at 22°C. B, E, H, and K, HS for 1 h at 37°C. C, F, I, and L, Two hours recovery at 22°C after HS. Pictures are representative of at least three independent analyses. White lines correspond to 10 μm. M, SG quantification in wild type and hsp101 after 1 h at 37°C and after 2 h recovery. n = 5 biological repeats, mean ± sd. A t test was performed between wild type and hsp101 at 1 h 37°C (P > 0.1), at 2 h recovery (P < 0.001), and for hsp101 between 1 h 37°C and 2 h recovery (P < 0.01). Quantification was performed on the same volume of stacks for each condition.

Figure 3.

Translation efficiency of mRNAs released from translation during HS is affected in the hsp101 mutant during recovery phase. A, Workflow used to identify transcripts stored in SG and released from translation during recovery. Only mRNAs with an FC between 0.5 and 2 on total RNA population and FC below 0.5 on polysomal RNA population in wild type (wt) and hsp101 were kept. B and C, TE of the 3309 mRNAs during HS versus recovery period in wt (B) and hsp101 (C). D, The log₂ value of TE of the 3309 mRNAs during recovery period in wt versus hsp101. Red dots correspond to mRNAs affected in translation recovery in hsp101 (2103). The black line corresponds to a linear regression of the 3309 dots.

Figure 4.

Translation efficiency of cytoplasmic RP mRNAs in wild type and hsp101 under normal condition, after 1 h at 37°C, and after 2 h recovery. Heat map of cytoplasmic RP mRNA translation efficiency in log₂ value. The heat map was performed on the 219 cytoplasmic RP mRNAs identified in RNA-seq database. One hundred seventy-nine of them are grouped in the same cluster. The numbers of transcripts and the mean value of TE of this cluster are marked. Red values correspond to a high translation efficiency and green values to a low translation efficiency. wt, Wild type.

Figure 5.

Determination of RP mRNA translation efficiency by qPCR. A and B, Translation efficiency of the At5g02960 and At3g44010 genes determined by qPCR. Translation efficiency was determined as the ratio of mRNA quantity between polysomal and total RNA values. Values are normalized to ACTIN7 level. n = 3 biological repeats, mean ± sd.

Figure 6.

RP mRNAs are associated with UBP1 during HS and released after stress. A, Western-blot analysis of UBP1a-GFP immunoprecipitation efficiency using GFP antibody. B to E, PCR amplification (30 cycles) of the At3g44010 (RPS29B), At5g02960 (RPS23B), At1g52300 (RPL37B), and At5g09810 (ACTIN7) genes after RNA immunoprecipitation with the 35S-UBP1a-GFP line. Wild-type line was used as a negative control. 1, Input UBP1a; 2, input wild type; 3, eluate UBP1a; 4, eluate wild type.

Figure 7.

New ribosomes can be produced during recovery independently of 40S and 60S reassembly. A, Polysome profiles analysis in wild type after HS 1 h at 37°C, and after 30 min, 1 h, and 2 h recovery at 22°C. Polysome profile analysis was performed three times on three biological repeats. The area of 40/60S and 80S/polysomes were determined for each condition and normalized according to the respective area for HS condition. Data are presented below each profile. n = 3 biological repeats, mean ± sd. B, Polysome recovery after HS on liquid medium supplemented with actinomycin D. C, ¹⁵N incorporation in polysomal proteins after 2 h recovery with or without actinomycin D treatment; n = 3 biological repeats, mean ± sd. Puromycin treatment was used as a negative control. Values were normalized by ¹⁵N natural abundance.