Bacterial Ribonucleoprotein bodies (BR-bodies) play an essential role in organizing RNA degradation via phase separation in the cytoplasm of bacteria. BR-bodies mediate multi-step mRNA decay through the concerted activity of the endoribonuclease RNase E coupled with the 3′-5′ exoribonuclease Polynucleotide Phosphorylase (PNPase). In vivo, studies indicated that the loss of PNPase recruitment into BR-bodies led to a significant build-up of RNA decay intermediates in Caulobacter crescentus. However, it remained unclear whether this is due to a lack of colocalized PNPase and RNase E within BR-bodies or whether PNPase’s activity is stimulated within the BR-body. We reconstituted RNase E’s C-terminal domain with PNPase towards a minimal BR-body in vitro to distinguish these possibilities. We found that PNPase’s catalytic activity is accelerated when colocalized within the RNase E biomolecular condensates, partly due to scaffolding and mass action effects. In contrast, disruption of the RNase E-PNPase protein–protein interaction led to a loss of PNPase recruitment into the RNase E condensates and a loss of ribonuclease rate enhancement. We also found that RNase E’s unique biomolecular condensate environment tuned PNPase’s substrate specificity for poly(A) over poly(U). Intriguingly, a critical PNPase reactant, phosphate, reduces RNase E phase separation both in vitro and in vivo. This regulatory feedback ensures that under limited phosphate resources, PNPase activity is enhanced by recruitment into RNase E’s biomolecular condensates.
35The multicellular embryo, and ultimately the entire organism, is a derivative of the fertilized 36 egg cell. Unlike in animals, transcription factor networks orchestrating faithful egg 37 development are still largely unknown in plants. We have identified that egg cell 38 differentiation in Arabidopsis require interplay between evolutionarily conserved onco-protein 39 homologs RETINOBLASTOMA-RELATED (RBR) and redundant MYB proteins 40 MYB64/MYB119. RBR physically interacts with the MYBs; and with plant-specific 41 transcription factors belonging to the RWP-RK-domain (RKD) family and LEAFY 42 COTYLEDON1 (LEC1), which participate in development of egg cells and inherent stress 43 response. RBR binds to most of these egg cell-expressed loci at the DNA level, partially 44 overlapping with sites of histone methylation H3K27me3. Since deregulation of RKDs 45 phenocopies mutants of RBR and the MYBs in terms of cell proliferation in the egg cell 46 spatial domain, all the corresponding proteins are likely required to restrict parthenogenetic 47 cell divisions of the egg cells. Cross-talk among these transcription factors, and direct 48 regulation by RBR, govern egg cell development and expression of egg-to-zygotic polarity 49 factors of the WUSCHEL RELATED HOMEOBOX family. Together, a network of RBR-50 centric transcription factors underlies egg cell development and stress response, possibly, in 51 combination with several other predicted nodes. 52 53 54 55 Key words 56 egg cell | transcription factor | RETINOBLASTOMA RELATED | MYB | RKD | stress | 57 parthenogenesis 58 59 4 Author summary 60 61The RETINOBLASTOMA protein is one of the core components of the Eukaryotic 62 cell cycle, and corresponding evolutionary homologs have been implicated not only 63 to repress cell division but also to control differentiation and development. How 64 RETINOBLASTOMA RELATED (RBR) associate with other higher order regulators 65 to control faithful egg cell development in sexual plants is pivotal for manipulation of 66 successful reproduction in general, and engineering of parthenogenesis when 67 asexual or apomictic seed progeny are desirable over sexual plants. Using a suite of 68 molecular methods, we show that a RBR-associated transcription factor network 69 operates to specify egg cells in Arabidopsis. Complex cross-regulation within these 70 transcription factors seems to be necessary for successful maternal egg cell to 71 zygotic transition and reproductive stress response. Detailed genetic analysis 72 implicate that RBR and its interactive partners belonging to MYB and RWP-RK 73 transcription factor families are possibly required to prevent parthenogenesis of the 74 sexual egg cells. Novel RBR networks and stress nodes explained in this study 75 might help to improve our understanding of sexual and asexual reproduction. 76 77 78 79 80 Proper differentiation of the egg cells is pivotal for sexual reproduction as well as 81 parthenogenesis. In flowering plants, the egg cells are terminally differentiated within 82 the miniature ...
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