Biology subjects

Zoschke, R.

Publications and source records attributed to Zoschke, R..

9 recordsLinked to original sources

bioRxiv2025

Maturase K forms a plastidial splicing complex with a neofunctionalized branching enzyme

Chloroplast group IIA introns derive from bacterial ribozymes. Their splicing likely requires Maturase K (MatK), which has been largely inaccessible to functional analyses being itself a chloroplast intron-encoded protein. Here we show that MatK physically interacts with a conserved, essential plastid-localized homolog of starch-branching enzymes (BEs), dubbed MATURASE K INTERACTING PROTEIN1 (MKIP1). We demonstrate that MKIP1 proteins have lost BE activity and acquired an insertion enabling direct interaction with the N-terminal region of MatK. Arabidopsis MKIP1 specifically co-precipitates all known intron targets of MatK. Induced MKIP1 silencing results in pale newly emerging leaves, in which the splicing of these intron targets is strongly reduced. Our data suggest that MKIP1 functionally diverged from canonical BEs to facilitate splicing in conjunction with MatK. We propose that the N-terminus of MatK, in turn, has evolved from an RNA-binding domain into a platform for protein interaction, helping its transition towards a general splicing factor.

plant biology↗

bioRxiv2023

Guidelines for performing ribosome profiling in plants including structural analysis of rRNA fragments

Ribosome profiling (or Ribo-seq) is a technique that provides genome-wide information on the translational landscape (translatome). Across different plant studies, variable methodological setups have been described which raises questions about the general comparability of data that were generated from diverging methodologies. Furthermore, a common problem when performing Ribo-seq are abundant rRNA fragments that are wastefully incorporated into the libraries and dramatically reduce sequencing depth. To remove these rRNA contaminants, it is common to perform preliminary trials to identify these fragments because they are thought to vary depending on nuclease treatment, tissue source, and plant species. Here, we compile valuable insights gathered over years of generating Ribo-seq datasets from different species and experimental setups. We highlight which technical steps are important for maintaining cross experiment comparability and describe a highly efficient approach for rRNA removal. Furthermore, we provide evidence that many rRNA fragments are structurally preserved over diverse nuclease regimes, as well as across plant species. Using a recently published cryo-electron microscopy (cryo-EM) structure of the tobacco 80S ribosome, we show that the most abundant rRNA fragments are spatially derived from the solvent-exposed surface of the ribosome. The guidelines presented here shall aid newcomers in establishing ribosome profiling in new plant species and provide insights that will help in customizing the methodology for individual research goals.

plant biology↗

bioRxiv2023

MatK impacts Differential Chloroplast Translation of Ribosomal and Photosynthetic genes by limiting spliced tRNA-K(UUU) abundance

The protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we employed RNA immunoprecipitation followed by high-throughput sequencing (RIP-Seq) to identify MatKs preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for branch point selection, and we discovered alternative branch points in three MatK target introns, the first observed instances of alternative splicing in chloroplasts. The alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA-K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and ribosome profiling in a trnK-matK over-expressor exhibited elevated levels of the spliced tRNA-K(UUU). Our analysis revealed a significant up-shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA-K(UUU), enabling the differential expression of functional chloroplast gene groups.

plant biology↗

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↗

bioRxiv2023

A eukaryote-specific factor mediates an early step in the assembly of plant photosystem II

ABSTRACTThe initial step of oxygenic photosynthesis is the thermodynamically challenging extraction of electrons from water and the release of molecular oxygen. This light-driven process, which is the basis of life on Earth, is catalyzed by the photosystem II (PSII) within the thylakoid membrane of photosynthetic organisms. The biogenesis of PSII requires a controlled step-wise assembly process of which the early steps are considered to be highly conserved between plants and their cyanobacterial progenitors. This assembly process involves auxiliary proteins, which are likewise conserved. In the present work, we show that in plants, the early assembly step, in which the PSII reaction center (RC) is associated with the intrinsic antenna protein CP47 to form the RC47 intermediate, is facilitated by a novel eukaryote-exclusive assembly factor. This factor, we named DEAP2 for DECREASED ELECTRON TRANSPORT AT PSII, works in concert with the conserved PAM68 assembly factor. The deap2 and pam68 mutants showed similar defects in PSII accumulation and assembly of the RC47 intermediate. The combined lack of both proteins results in a loss of functional PSII and the inability of plants to grow photoautotrophically on soil. While overexpression of DEAP2 partially rescued the pam68 PSII accumulation phenotype, this effect was not reciprocal. DEAP2 accumulates at 20-fold higher levels than PAM68, together suggesting that both proteins have distinct functions. In summary, our results uncover eukaryotic adjustments to the PSII assembly process, which involve the addition of DEAP2 for the rapid progression from RC to RC47.

plant biology↗

bioRxiv2023

Utilizing high resolution ribosome profiling for the global investigation of gene expression in Chlamydomonas reinhardtii

Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Here, we established an optimized and high resolution Ribo-seq protocol for the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas). Comparing different nuclease treatments for the extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq, provided deep insight into translational dynamics and post-transcriptional control of gene expression, thoroughly covering more than 10,000 different transcripts. Our high quality Ribo-seq protocol captures the 3-nucleotide movement of elongating ribosomes along nuclear and chloroplast transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5- and 3-sections of transcripts and features of eukaryotic translation termination. These offsets reveal drastic differences between the nature of cytosolic and chloroplast translation mechanisms. Chloroplast translation is further characterized by heterogenous RPF size distribution. We found that local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly revealing an uncharacterized regulator step during PsbA/D1 synthesis. Further analyses of RPF distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light harvesting complex proteins, LHCs. Moreover, our Ribo-seq data can be utilized to survey coding sequence annotations and the expression preference of alternatively spliced transcripts in Chlamydomonas. We made these features easily accessible for the research community by attaching our Ribo-seq data to the most recent Chlamydomonas reference genome.

systems biology↗

bioRxiv2020

Neither the availability of D2 nor CP43 limits the biogenesis of PSII in tobacco

The pathway of photosystem II assembly is well understood and multiple auxiliary proteins supporting it have been identified. By contrast, little is known about rate-limiting steps controlling PSII biogenesis. In the green alga Chlamydomonas reinhardtii, biosynthesis of the chloroplast-encoded D2 reaction center subunit (PsbD) limits PSII accumulation. To determine the importance of D2 synthesis for PSII accumulation in vascular plants and elucidate the contributions of transcriptional and translational regulation, the 5-untranslated region of psbD was modified via chloroplast transformation in tobacco. A drastic reduction in psbD mRNA abundance resulted in a strong decrease of PSII content, impaired photosynthetic electron transport, and retarded growth under autotrophic conditions. Overexpression of the psbD mRNA also increased transcript abundance of psbC (the CP43 inner antenna protein), which is co-transcribed with psbD. Because translation efficiency remained unaltered, translation output of pbsD and psbC increased with mRNA abundance. However, this did not result in increased PSII accumulation. The introduction of point mutations into the Shine-Dalgarno-like sequence or start codon of psbD decreased translation efficiency without causing pronounced effects on PSII accumulation and function. These data show that neither transcription nor translation of psbD and psbC are rate-limiting for PSII biogenesis in vascular plants, and that PSII assembly and accumulation in tobacco are controlled by different mechanisms than in Chlamydomonas. One sentence summaryPSII biogenesis in tobacco is neither limited by transcript accumulation nor translation of psbD and psbC.

plant biology↗

bioRxiv2020

Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions in vivo.

The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ~30 amino acids. They are found in metazoans and plants where they localize to mitochondria or chloroplasts. In higher plants, the mTERF family comprises ~30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.

plant biology↗

bioRxiv2017

AtRsgA from Arabidopsis thaliana controls maturation of the small subunit of the chloroplast ribosome

SummaryPlastid ribosomes are very similar in structure and function to ribosomes of their bacterial ancestors. Since ribosome biogenesis is not thermodynamically favourable at biological conditions, it requires activity of many assembly factors. Here, we have characterized a homolog of bacterial rsgA in Arabidopsis thaliana and show that it can complement the bacterial homolog. Functional characterization of a strong mutant in Arabidopsis revealed that the protein is essential for plant viability, while a weak mutant produced dwarf, chlorotic plants that incorporated immature pre-16S ribosomal RNA into translating ribosomes. Physiological analysis of the mutant plants revealed smaller, but more numerous chloroplasts in the mesophyll cells, reduction of chlorophyll a and b, depletion of proplastids from the rib meristem and decreased photosynthetic electron transport rate and efficiency. Comparative RNA-sequencing and proteomic analysis of the weak mutant and wild-type plants revealed that various biotic stress-related, transcriptional regulation and post-transcriptional modification pathways were repressed in the mutant. Intriguingly, while nuclear- and chloroplast-encoded photosynthesis-related proteins were less abundant in the mutant, the corresponding transcripts were upregulated, suggesting an elaborate compensatory mechanism, potentially via differentially active retrograde signalling pathways. To conclude, this study reveals a new chloroplast ribosome assembly factor and outlines the transcriptomic and proteomic responses of the compensatory mechanism activated during decreased chloroplast function.\n\nSignificance statementAtRsgA is an assembly factor necessary for maturation of the small subunit of the chloroplast ribosome. Depletion of AtRsgA leads to dwarfed, chlorotic plants and smaller, but more numerous chloroplasts. Large-scale transcriptomic and proteomic analysis revealed that chloroplast-encoded and - targeted proteins were less abundant, while the corresponding transcripts were upregulated in the mutant. We analyse the transcriptional responses of several retrograde signalling pathways to suggest a mechanism underlying this compensatory response.

molecular biology↗