Tudor Staphylococcal Nuclease plays two antagonistic roles in RNA metabolism under stress

Abstract

Adaptation to stress entails a repertoire of molecular pathways that remodel the proteome, thereby promoting selective translation of pro-survival proteins. Yet, translation of other proteins, especially those which are harmful for stress adaptation is, on the contrary, transiently suppressed through mRNA decay or storage. Proteome remodeling under stress is intimately associated with the cytoplasmic ribonucleoprotein (RNP) complexes called stress granules (SGs) and processing bodies (PBs). The molecular composition and regulation of SGs and PBs in plants remain largely unknown. Recently, we identified the Arabidopsis Tudor Staphylococcal Nuclease (TSN, Tudor-SN or SND1) as a SG- and PB-associated protein required for mRNA decapping under stress conditions. Here we show that SGs localize in close proximity to PBs within plant cells that enable the exchange of molecular components. Furthermore, we provide a meta-analysis of mRNA degradome of TSN-deficient plants suggesting that TSN might inhibit the degradation of mRNAs which are involved in stress adaptation. Our results establish TSN as a versatile mRNA regulator during stress.

Adaptation to stress depends on the availability of energy resources.¹ Stress drives cells to an energy crisis whereupon they have to reduce energy expenditure in order to survive. To this end, eukaryotic cells compartmentalize specific mRNAs and proteins in cytoplasmic ribonucleoprotein (RNP) structures known as stress granules (SGs) and processing bodies (PBs).² In these structures mRNA molecules are stored, degraded or kept silent in order to prevent energy expenditure on producing useless, surplus or even harmful proteins under stress conditions.³

Numerous components of SGs and PBs have been identified in yeast and animal models. SGs typically contain poly(A)⁺ RNA, translation initiation factors (eIFs), poly(A) binding protein (PABP) and ribosomal proteins.⁴ In contrast, PBs contain a suit of proteins involved in mRNA decay and translational repression, including subunits of decapping and exosome complexes, deadenylases, and RNA-binding proteins.⁵ Although both types of RNP complexes are present under stress conditions, they serve distinct functions. While SGs are thought to play a role in sequestering, stabilizing and storing mRNAs and translation factors,^6,7 the main function of PBs is attributed to translational repression and mRNA decay, in accordance with their composition.^8,9

Plants are sessile organisms subjected to and able to cope with a vast array of biotic and abiotic stresses throughout their lifespans. Recent studies have provided useful insight into the cell biology and biochemistry of SGs and PBs in plants. Sorenson and Bailey-Serres have found that Ubp1c, a SG-nucleating RNA-binding protein, is a component of the machinery that reprograms post-transcriptional gene expression during hypoxia.¹⁰ More recently, we characterized the processes of SG assembly and disassembly in plants under heat stress and established Tudor Staphylococcal Nuclease (TSN) as a structural and functional component of both SGs and PBs, essential for mRNA catabolism under stress.¹¹

Here we extend our previous study and show that (i) SGs localize in a close proximity to PBs in plant cells, and (ii) TSN may positively regulate stability of mRNAs involved in stress adaptation pathways.

Although SGs and PBs have distinct functions and composition, several studies performed in animal and yeast models pointed to the existence of a strong physical link between them. It was demonstrated that both complexes are likely to exchange mRNA and proteins between themselves and with polysomes.⁴ Several observations supported the idea that similar RNP shuttle is also present in plants. First, some proteins were found to localize in both SGs and PBs, such as CCCH tandem zinc finger proteins TZF1, TZF4, TZF5 and TZF6.^11,12 Second, treatment with cycloheximide, which causes trapping of mRNA in polysomes by inhibiting the translocation step during the elongation phase in protein synthesis, abolished SG and PB assembly.^11,13 By performing a co-localization study using SG- and PB-associated proteins eIF4e and DCP1, respectively, we have revealed that these RNP complexes are situated in close proximity to each other in Arabidopsis root cells under heat stress (Fig. 1, arrowheads) .

Figure 1.

Colocalization analysis of GFP-DCP1 and eIF4E proteins. 5-day-old Col Arabidopsis seedlings expressing GFP-DCP1 (green) were heat stressed at 39°C for 40 min and then immunostained with anti-elF4E (red) as described previously.²⁷ Root cells were examined by confocal laser microscopy. Control seedlings were grown at 23°C. Note a close proximity between eIF4E-SG and DCP1-PB denoted by arrowheads. Scale bars, 2 μm.

Shuttling of cytoplasmic mRNAs between polysomes, PBs and SGs has been well documented in stressed mammalian cells.¹⁴ Yet, the mechanisms and directionality of mRNA movement between PBs and SGs remain unresolved. A longstanding notion is that mRNAs stalled at a step of translation initiation are kept in SGs for storage, or directed to PBs for degradation. However, several studies have found that mRNA decay enzymes such as XRN1 can be also found in SGs, suggesting that mRNA degradation may also take place in these foci.^8,14 Likewise, we showed that TSN is localized in both SGs and PBs and that enzymatically active tandem repeat of 4 N-terminally situated SN domains confers this localization.¹¹

TSN is an evolutionarily conserved protein present in almost all eukaryotes, with the notable exception of Saccharomyces cerevisiae.¹⁵ The domain composition of TSN is also conserved, invariably comprising a tandem repeat of 4 non-canonical SN domains followed by a Tudor and a C-terminal partial SN domain.¹⁶ In animals, TSN functions in several gene expression pathways in both the nucleus and the cytoplasm, including regulation of transcription,^17-20 pre-mRNA splicing,²¹ and RNA silencing.^22,23 Arabidopsis genome has 2 partly redundant TSN genes (TSN1 and TSN2) which were shown to confer stress tolerance through the stabilization of mRNAs encoding secreted proteins ²⁴ and gibberellin 20-oxidase 3, a key enzyme in gibberellin biosynthesis.²⁵ Unlike animal TSN, in Arabidopsis TSN1 and TSN2 are exclusively cytoplasmic.^11,26 However, the role of TSN in plants seems to be extended to also encompass an opposite process, viz. mRNA decay, under stress. In our recent work, the purification of uncapped mRNA molecules, intermediates of the 5′–3′ decay pathway, from Columbia-0 (Col), Landsberg erecta (Ler) and tsn1tsn2 double knock-out plants grown under control conditions or heat stress and subsequent cDNA arrays analysis revealed that TSN is essential for mRNA decapping under stress.¹¹ While heat-stressed Col and Ler plants exhibited a pronounced increase in the accumulation of uncapped mRNA, this increase was abrogated in TSN-deficient plants (Fig. 2A). This analysis has also detected large changes in the pattern of uncapped transcripts caused by heat stress .

Figure 2.

Analysis of global mRNA decapping pattern in Arabidopsis under heat stress as affected by TSN deficiency. (A) Quantitative analysis of transcripts enriched in uncapped form in control (23°C) and heat stress (39°C for 40 min) conditions in Col, Ler and tsn1tsn2 plants. (B) GO analysis (term “Biological Process”) of transcripts enriched in uncapped form in Col, Ler and tsn1tsn2 plants under heat stress. The charts display the ratios between the percentages of mRNAs belonging to a particular GO term that show significant enrichment in uncapped form under heat stress and under control conditions. Asterisks indicate significant differences at P < 0.05, Fisher's exact test. To identify enrichment of uncapped transcripts, significance analysis was performed using TIGR MultiExperiment Viewer modelu of TM4 software.²⁸ A further description of the microarray experiment can be found elsewhere.²⁹ GOSlim annotation developed by TAIR was used to organize sets of genes into broad ontology categories.³⁰

In order to determine which categories of transcripts are enriched in the uncapped form (i.e. subject to degradation) during heat stress, we grouped them into Gene Ontology (GO) Biological Process (Fig. 2B). A comparison between the heat stress versus control condition showed that the transcripts related to categories “DNA or RNA metabolism,” “electron transport or energy” and “transcription, DNA-dependent” are less uncapped in all genetic backgrounds, indicating that these transcripts are critical for heat stress adaptation. Noteworthy, transcripts of genes encoding components of stress response and signal transduction pathways are enriched in uncapped form in the tsn1tsn2 background but not in wild-type plants (Fig. 2B). These data are in agreement with the previously reported role of TSN in stabilizing certain mRNAs encoding secreted proteins.²⁶

Here, we provide evidence that SGs and PBs are located in close proximity in plant cells, further supporting the notion that RNP components can shuttle between them. In addition, we show that transcripts related to cell signaling and stress adaptation are preferentially uncapped in tsn1tsn2 background. Further work is required to unravel the molecular mechanism that enables TSN to play 2 seemingly antagonistic roles in RNA metabolism under stress, i.e., facilitating global mRNA decapping and thus degradation, and on the other hand stabilizing particular mRNAs that are required for survival.

Funding

This work was supported by grants from Knut and Alice Wallenberg Foundation (to P.V.B.), the Swedish Research Council (to P.N.M. and P.V.B.), Pehrssons Fund (to P.V.B.), the Swedish Foundation for Strategic Research (to P.V.B.), and Olle Engkvist Foundation (to P.V.B.).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.