Biology subjects

Kachariya, N.

Publications and source records attributed to Kachariya, N..

3 recordsLinked to original sources

bioRxiv2023

The prion-like domain of the chloroplast RNA binding protein CP29A is required for cold-induced phase separation next to nucleoids and supports RNA splicing and translation during cold acclimation

Arabidopsis thaliana is capable of producing photosynthetic tissue with active chloroplasts at temperatures as low as 4{degrees}C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif (RRM) domains of CP29A. The resulting droplets display liquid-like properties and are found in close proximity to chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role for plant cold resistance.

plant biology↗

bioRxiv2022

Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA

RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large messenger ribonucleoprotein particle (mRNP) assemblies, these proteins often function in. Using UV-induced RNA-protein crosslinking and subsequent mass spectrometric analysis, we first identified more than 100 in vivo RNA crosslinks in 16 nuclear mRNP components in S. cerevisiae. For functional analysis, we chose Npl3, for which we determined crosslinks in its two RNA recognition motifs (RRM) and in the flexible linker region connecting the two. Using NMR and structural analyses, we show that both RRM domains and the linker uniquely contribute to RNA recognition. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains of Npl3. Notably, the npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of further mRNP components into nuclear mRNPs, establishing a function of Npl3 in nuclear mRNP assembly. Taken together, we determined the specific function of the RNA-binding activity of the nuclear mRNP component Npl3, an approach that can be applied to many RBPs in any RNA metabolic process.

biochemistry↗

bioRxiv2019

PINK1-dependent phosphorylation of Serine111 within the SF3 motif of Rab GTPases impairs effector interactions and LRRK2 mediated phosphorylation at Threonine72

Loss of function mutations in the PINK1 kinase are causal for autosomal recessive Parkinson disease (PD) whilst gain of function mutations in the LRRK2 kinase cause autosomal dominant PD. PINK1 indirectly regulates the phosphorylation of a subset of Rab GTPases at a conserved Serine111 (Ser111) residue within the SF3 motif. Using genetic code expansion technologies we have produced stoichiometric Ser111-phosphorylated Rab8A revealing impaired interactions with its cognate guanine nucleotide exchange factor (GEF) and GTPase activating protein (GAP). In a screen for Rab8A kinases we identify TAK1 and MST3 kinases that can efficiently phosphorylate the Switch II residue Threonine72 (Thr72) in a similar manner as LRRK2. Strikingly we demonstrate that Ser111 phosphorylation negatively regulates the ability of LRRK2 but not MST3 or TAK1 to phosphorylate Thr72 in vitro and demonstrate an interplay of PINK1- and LRRK2-mediated phosphorylation of Rab8A in cells. Finally, we present the crystal structure of Ser111-phosphorylated Rab8A and NMR structure of Ser111-phosphorylated Rab1B that does not demonstrate any major changes suggesting that the phosphorylated SF3 motif may disrupt effector-Switch II interactions. Overall, we demonstrate antagonistic regulation between PINK1-dependent Ser111 phosphorylation and LRRK2-mediated Thr72 phosphorylation of Rab8A suggesting that small molecule activators of PINK1 may have therapeutic potential in patients harbouring LRRK2 mutations.

biochemistry↗