Intrinsically disordered plant protein PARCL colocalizes with RNA in phase-separated condensates whose formation can be regulated by mutating the PLD.

In higher plants, long-distance RNA transport via the phloem is crucial for communication between distant plant tissues to align development with stress responses and reproduction. Several recent studies suggest that specific RNAs are among the potential long-distance information transmitters. However, it is yet not well understood how these RNAs enter the phloem stream, how they are transported, and how they are released at their destination. It was proposed that phloem RNA-binding proteins facilitate RNA translocation. In the present study, we characterized two orthologs of the phloem-associated RNA chaperone-like (PARCL) protein from Arabidopsis thaliana and Brassica napus at functional and structural levels. Microscale thermophoresis showed that these phloem-abundant proteins can bind a broad spectrum of RNAs and show RNA chaperone activity in FRET-based in vitro assays. Our SAXS experiments revealed a high degree of disorder, typical for RNA-binding proteins. In agroinfiltrated tobacco plants, eYFP-PARCL proteins mainly accumulated in nuclei and nucleoli and formed cytosolic and nuclear condensates. We found that formation of these condensates was impaired by tyrosine-to-glutamate mutations in the predicted prion-like domain (PLD), while C-terminal serine-to-glutamate mutations did not affect condensation but reduced RNA binding and chaperone activity. Furthermore, our in vitro experiments confirmed phase separation of PARCL and colocalization of RNA with the condensates, while mutation as well as phosphorylation of the PLD reduced phase separation. Together, our results suggest that RNA binding and condensate formation of PARCL can be regulated independently by modification of the C-terminus and/or the PLD.

Noncell-autonomous HSC70.1 chaperone displays homeostatic feedback regulation by binding its own mRNA.

The HSC70/HSP70 family of heat shock proteins are evolutionarily conserved chaperones involved in protein folding, protein transport, and RNA binding. Arabidopsis HSC70 chaperones are thought to act as housekeeping chaperones and as such are involved in many growth-related pathways. Whether Arabidopsis HSC70 binds RNA and whether this interaction is functional has remained an open question. We provide evidence that the HSC70.1 chaperone binds its own mRNA via its C-terminal short variable region (SVR) and inhibits its own translation. The SVR encoding mRNA region is necessary for HSC70.1 transcript mobility to distant tissues and that HSC70.1 transcript and not protein mobility is required to rescue root growth and flowering time of hsc70 mutants. We propose that this negative protein-transcript feedback loop may establish an on-demand chaperone pool that allows for a rapid response to stress. In summary, our data suggest that the Arabidopsis HSC70.1 chaperone can form a complex with its own transcript to regulate its translation and that both protein and transcript can act in a noncell-autonomous manner, potentially maintaining chaperone homeostasis between tissues.

Structural and functional analysis of a plant nucleolar RNA chaperone-like protein.

Ribosome biogenesis is a key process in all eukaryotic cells that requires hundreds of ribosome biogenesis factors (RBFs), which are essential to build the mature ribosomes consisting of proteins and rRNAs. The processing of the required rRNAs has been studied extensively in yeast and mammals, but in plants much is still unknown. In this study, we focused on a RBF from A. thaliana that we named NUCLEOLAR RNA CHAPERONE-LIKE 1 (NURC1). NURC1 was localized in the nucleolus of plant cell nuclei, and other plant RBF candidates shared the same localization. SEC-SAXS experiments revealed that NURC1 has an elongated and flexible structure. In addition, SEC-MALLS experiments confirmed that NURC1 was present in its monomeric form with a molecular weight of around 28 kDa. RNA binding was assessed by performing microscale thermophoresis with the Arabidopsis internal transcribed spacer 2 (ITS2) of the polycistronic pre-rRNA precursor, which contains the 5.8S, 18S, and 25S rRNA. NURC1 showed binding activity to the ITS2 with a dissociation constant of 228 nM and exhibited RNA chaperone-like activity. Our data suggested that NURC1 may have a function in pre-rRNA processing and thus ribosome biogenesis.

Detection of RNA in Ribonucleoprotein Complexes by Blue Native Northern Blotting.

Northern blotting is a classical technique that allows the detection of specific nucleic acids using radioactive or non-radioactive probes. Normally, nucleic acids are denatured and separated by agarose or polyacrylamide gel electrophoresis and transferred and fixed to a membrane prior to detection. Here, we describe a method to analyze specific RNA in native ribonucleoprotein complexes using blue native PAGE with subsequent northern blotting, crosslinking of RNA onto a suitable membrane, and detection using non-radioactive probes.

Comparative proteomic analysis of salt-responsive proteins in canola roots by 2-DE and MALDI-TOF MS.

Salinity stress is a major abiotic stress that affects plant growth and limits crop production. Roots are the primary site of salinity perception, and salt sensitivity in roots limits the productivity of the entire plant. To better understand salt stress responses in canola, we performed a comparative proteomic analysis of roots from the salt-tolerant genotype Safi-7 and the salt-sensitive genotype Zafar. Plants were exposed to 0, 150, and 300 mM NaCl. Our physiological and morphological observations confirmed that Safi-7 was more salt-tolerant than Zafar. The root proteins were separated by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry was applied to identify proteins regulated in response to salt stress. We identified 36 and 25 protein spots whose abundance was significantly affected by salt stress in roots of plants from the tolerant and susceptible genotype, respectively. Functional classification analysis revealed that the differentially expressed proteins from the tolerant genotype could be assigned to 14 functional categories, while those from the susceptible genotype could be classified into 9 functional categories. The most significant differences concerned proteins involved in glycolysis (Glyceraldehyde-3-phosphate dehydrogenase, Fructose-bisphosphate aldolase, Phosphoglycerate kinase 3), stress (heat shock proteins), Redox regulation (Glutathione S-transferase DHAR1, L-ascorbate peroxidase), energy metabolism (ATP synthase subunit B), and transport (V-type proton ATPase subunit B1) which were increased only in the tolerant line under salt stress. Our results provide the basis for further elucidating the molecular mechanisms of salt-tolerance and will be helpful for breeding salt-tolerant canola cultivars.