Nonomura Group / Plant Cytogenetics Laboratory

Impact of protein domains on the MEL2 granule, a cytoplasmic ribonucleoprotein complex maintaining faithful meiosis progression in rice.

Manaki Mimura, Seijiro Ono, Harsha Somashekar, Ken-Ichi Nonomura

New Phytologist 2024 Jul 24. DOI:10.1111/nph.19968

Cytoplasmic ribonucleoprotein (RNP) granules are membraneless structures composed of various RNAs and proteins that play important roles in post-transcriptional regulation. While RNP granules are known to regulate the meiotic entry in some organisms, little is known about their roles in plants.

In this study, we observed the cytoplasmic granular structures of rice RNA-binding protein MEL2, which contributes to the control of meiotic entry timing, in leaf protoplasts and spore mother cells. Our results indicated that MEL2 granules colocalized with processing body and stress granule factors. The maintenance of granule properties modulated by LOTUS domain and the intrinsically disordered region (IDR) is essential for proper MEL2 function in meiosis progression (Figure). MEL2-like proteins widely found in plant kingdom conserved LOTUS domain followed by the IDR despite their diverse domain structures, suggesting the functional conservation of these domains among plant species.

Figure: Roles of MEL2 functional domains in granule formation
(a) Genomic fusion constructs of MEL2 and GFP genes, and its derivatives with  deletions (dashed lines) or mutations (asterisks) in MEL2 domains.
(b) MEL2 granule formation in spore mother cells in rice anthers transformed with the constructs in (a). The wildtype MEL2 (FL) formed granules (green) at premeiosis (0.3-0.4mm anthers), and disappeared by meiosis (0.5mm anthers). The mRING failed granule formation, and the granules of ∆LOTUS and ∆IDR were aberrantly carried over to meiocytes, all resulting in defective meiosis and pollen development.

Miyagishima Group / Symbiosis and Cell Evolution Laboratory

Development of a rapamycin-inducible protein-knockdown system in the unicellular red alga Cyanidioschyzon merolae.

Takayuki Fujiwara, Shunsuke Hirooka, Shota Yamashita, Fumi Yagisawa and Shin-ya Miyagishima

Plant Physiology (2024) kiae316 DOI:10.1093/plphys/kiae316

An inducible protein-knockdown system is highly effective for investigating the functions of proteins and mechanisms essential for the survival and growth of organisms. However, this technique is not available in photosynthetic eukaryotes. The unicellular red alga Cyanidioschyzon merolae possesses a very simple cellular and genomic architecture and is genetically tractable but lacks RNA interference machinery. In this study, we developed a protein-knockdown system in this alga. The constitutive system utilizes the destabilizing activity of the FK506-binding protein 12 (FKBP12)-rapamycin-binding (FRB) domain of human target of rapamycin kinase or its derivatives to knock down target proteins. In the inducible system, rapamycin treatment induces the heterodimerization of the human FRB domain fused to the target proteins with the human FKBP fused to S-phase kinase associated protein 1 or Cullin 1, subunits of the SCF E3 ubiquitin ligase. This results in the rapid degradation of the target proteins through the ubiquitin-proteasome pathway. With this system, we successfully degraded endogenous essential proteins such as the chloroplast division protein dynamin-related protein 5B and E2 transcription factor, a regulator of the G1/S transition, within 2 to 3 h after rapamycin administration, enabling the assessment of resulting phenotypes. This rapamycin-inducible protein-knockdown system contributes to the functional analysis of genes whose disruption leads to lethality.

Figure: A rapamycin-inducible protein knockdown system.
(A) The cellular structure of the unicellular red alga Cyanidioschyzon merolae and a differential interference contrast microscopy image.
(B) Mechanism of the rapamycin-inducible protein knockdown system. FRB tags and FKBP proteins form a heterodimer in the presence of rapamycin. The target protein fused with an FRB tag (in this figure, the fluorescent protein mVenus) and the CUL1 subunit of ubiquitin ligase fused with FKBP bind in the presence of rapamycin, resulting in the ubiquitination of FRB-mVenus. The ubiquitinated FRB-mVenus is rapidly degraded by the proteasome.
(C) Immunoblot analysis showing the decrease in mVenus-FRB protein level after the addition of rapamycin.

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Kanemaki Group / Molecular Cell Engineering Laboratory

Replication initiation sites and zones in the mammalian genome: Where are they located and how are they defined?

Xiaoxuan Zhu, Masato T. Kanemaki*
*Corresponding author

DNA Repiar (2024) 141, 103713 DOI:10.1016/j.dnarep.2024.103713

The question of how genomic DNA is replicated is one of the key themes in molecular biology, as abnormalities in DNA replication are related to genome instability, genetic disorders, cell death, and cancer. Therefore, DNA replication has been extensively studied over the years using Escherichia coli and budding yeast as model organisms. In these unicellular models, DNA replication starts from genomic regions with consensus DNA sequences. On the other hand, in mammalian cells, including humans, consensus sequences appear to be absent, and it is still not well understood where DNA replication begins and how are they defined in the genome. Hence, this review paper compiles and compares the results of large-scale high-throughput sequencing-based explorations for mapping initiation sites/zones reported so far. Furthermore, it highlights the similarities and differences in the DNA replication mechanisms between budding yeast and mammalian cells, presents a hypothetical mechanism for how replication initiation zones are defined in mammalian cells, and discusses the future directions and challenges in DNA replication research.

The first author, Dr Xiaoxuan Zhu, has been supported as an NIG post-doctoral fellow.

Figure: Differences in the mechanisms of replication initiation sites/zones selection between budding yeast and mammalian cells: In yeast, licensing factors accumulate at ARS (autonomously replicating sequence) sites containing a consensus sequence, leading to replication initiation at these ARS sites. In contrast, in mammalian cells, licensing factors are widely distributed across the genome. It is thought that the subsequent accumulation of firing factors at specific locations is crucial for forming initiation zones.