del Río Fernández, E. Gutiérrez Díaz

Taking into account that HRR.1 and HRR.2 could be targeted to SGs and/or PBs and the assembling of both structures depends on non-translated transcripts flux, the requirement of HRR proteins for assembling of such cytoplasmic RNA granules was investigated. For this, the assembly and putative dynamic exchange of components between these cytoplasmic mRNP-containing complexes was disrupted by chemical treatment. The application of cycloheximide (CHX) would inhibit the translation by blocking the mRNA release from polysomal complexes. Conversely, the application of another

133 translation inhibitor, puromycin (PUR), would have an inverse effect of CHX, by destabilising of polysomes and releasing the transcripts that were being translated. While CHX application promotes the ribosomes stalling on transcripts and inhibits the formation of stress granules, PUR application promotes the SG assembling. These results have been explained by the requirement of the inhibition of translation initiation, for SG formation (Anderson and Kedersha 2002; Weber et al. 2008).

When tobacco BY2 cells transformed with pHRR::GFP6-HRR.1 and pHRR::GFP6-HRR.2 transgenes were treated with CHX, under standard conditions (23ºC), the expression of HRR.1 and HRR.2 fusions slightly increased (Figure 3.40, C and D), comparing with non-treated BY2 cells (Figure 3.40, A and B). As previously observed (Figures 3.38 and 3.39), under HS treatment (38ºC, 60 min), a high number of fluorescent cytoplasmic aggregates was detected on pHRR::GFP6-HRR.1 and pHRR::GFP6-HRR.2 transgenic BY2 cells (Figure 3.40, E and F). However, when both transformant BY2 cells were treated with CHX and subsequently heat-stressed, a marked reduction in size and number of cytoplasmic granules was observed (Figure 3.40, G and H). The same transformant BY2 cells when treated with PUR and then heat-stressed, they displayed a re- assemblying of cytoplasmic granules (Figure 3.40, I and J)

In standard conditions, the exposition of transformant BY2 cells to CHX promoted a small accumulation of both tagged HRR proteins. In part, these results corroborate with bioinformatics data (BAR browser), which predict that HRR is margely up-regulated under CHX treatment (10 µM, by three hours). Besides cycloheximide treatment, HS condition also influences the HRR activity. Such as previously shown, a large amount of cytoplasmic granules was observed in both transformant BY2 cells (Figure 3.38). However, the spatial distribution of both HRR fusion proteins was different. GFP-HRR.1 fusion-containing granules were mostly found close to nuclear periphery and in cytoplasm, while GFP-HRR.2 fusion was more randomly dispersed in the cytoplasm. These results suggest that HRR proteins possess specific subcellular dynamics. The presence of HRR.1 fusion protein close to the nuclear membrane indicates that this protein could be recruited for mRNA nuclear export or translation initiation process. In mammalian models, the translation initiation is characterised as ‘pionner round’, consisting in the ribosomal scanning (searching by PTCs and EJC displacing) and remodelating of mRNP (Ishigaki et al. 2001; Chang et al. 2007).

The HRR.2 fusion, seems to be located in SGs, or PBs or in both, during HS treatment. The dynamic of SGs and PBs assembling is mostly dependent from mRNP homeostasis, not only in standard conditions as during stressful conditions. Hence, the exposition of transformant BY2 cells (GFP6-HRR.1 and GFP6-HRR.2) to translation inhibitors cycloheximide (CHX) and puromycin (PUR) allows to infer if HRR proteins are involved in formation of such RNA granules. The CHX treatment before HS imposition allows the evaluation of dynamic influx of stalled mRNPs from SGs to PBs,

135 through the increase/decrease of PBs number and size. The formation of SGs and PBs has been described to be inhibited by application of CHX, in stresses cells (Weber et al. 2008). In addition, CHX-treated HeLa cells presented SGs dissociated into their constituents that were dissolved in the cytoplasm (Nadezhdina et al. 2010). Occurring the SG dissolution under CHX treatment, the mRNP flux between SGs and PBs is interrupted and RNA granules disappear. The decreased number and size of cytoplasmic aggregates after CHX treatment and subsequent HS imposition indicates that HRR.1 and HRR.2 could be involved in SG and PB assembling. As a result, the composition of PBs would change, reducing their size and number. Conversely, the treatment with puromycin promoted the cytoplasmic aggregates assembling, after the HS treatment. This translation inhibitor is an aminoacyl tRNA analogue that destabilizes polysomes by promoting premature termination (Kedersha et al. 2000). Altogether, HRR proteins are suggested to be involved in dynamic flux of mRNPs between SG and/or PBs cytoplasmic RNA granules.

As a conclusion, the results suggest that the products of HRR alternative splicing, HRR.1 and HRR.2 proteins, could follow different subcellular pathways, during the thermotolerance responses upon HS conditions. Once translated, their targeting and intracellular accumulation appears to be somewhat different. In early responses to HS, HRR.1 could promote the SG assembly, participating in recruitment of stalled and housekeeping mRNPs, possibly through the protein-protein interactions with other RNA-binding proteins. The untranslated mRNPs are then screened for (1) storage, (2) reintegration into translation program/process or (3) moved to PBs, where are expected to be degraded. All these tasks can only be afforded by a dynamic exchange of components between SGs and PBs, in which HRR.1 is likely to be involved. Even though HRR.2 could be early integrated into SGs, most of its corresponding GFP6 signal was observed in cytoplasmic granules similar to PBs, which were smaller than SGs. HRR.2 could play a regulatory function during mRNA decay and transcriptional regulation, upon HS conditions. The modified HRR.2 binding motif (in RRM domain) could be sufficient for altering the RNA and protein interaction activities. A specific set of transcripts could then be drived for degradation, including their transcripts. In PBs, the transcripts can be degraded through 5’-3’ degradation pathway (NMD), or HRR.2 transcripts could be also degraded by exosome (3’-5’ degradation), generating small RNAs. Ultimately, these molecules might be recruited to the nucleus, where could influence the transcriptional activity.

(Left page) Figure 3.40 Intracellular dynamics of HRR.1 and HRR.2 fusions under HS conditions and chemical treatment (CHX and PUR). Transformant BY2 cells harbouring the pHRR::GFP-HRR.1 and pHRR::GFP6-HRR.2 transgenes were observed using a fluorescence microscope. Cells were observed under standard conditions (A and B,

respectively), after being treated with cycloheximide (CHX, 100 µg.ml-1_{; C and D, respectively) or upon HS treatment}

(38ºC, 60 min; E and F, respectively). The transformant BY2 cells harbouring pHRR::GFP6-HRR.1 (G, I) and

pHRR::GFP6-HRR.2 transgenes (H, J) were treated with CHX (100 µg.ml-1_{) (G and H) or treated with puromycin (PUR,}

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