Biology subjects

Nandana, V.

Publications and source records attributed to Nandana, V..

6 recordsLinked to original sources

bioRxiv2025

Molecular mechanisms of recruitment, function and regulation of UPF1 in histone mRNA decay

Animal replication-dependent histone mRNAs end in a conserved stem loop (SL) instead of the canonical poly(A) tail present in all other eukaryotic mRNAs. Degradation of the histone SL at the end of the S-phase is initiated by the stem-loop binding protein SLBP and its interplay with the RNA helicase UPF1 and the exoribonuclease 3hExo. We report direct interactions between SLBP and UPF1 and show that the unstructured SLBP N-terminus wraps around the UPF1 helicase core, contacting it at multiple sites. Although binding of SLBP to UPF1 impedes unwinding activity, it is critical for efficient histone mRNA decay in cells, as unwinding of the SL facilitates degradation by 3hExo. Here we show that the UPF1-activator, UPF2, binds 3hExo, and that UPF2-mediated activation of UPF1 overrides the inhibitory effect of SLBP. Our results highlight the intricate network of UPF1-centric protein-protein and protein/RNA interactions that fine-tunes its unwinding activity and orchestrates timely and efficient degradation of histone mRNA.

biochemistry↗

bioRxiv2025

Bacterial IF2's N-terminal IDR drives cold-induced phase separation and promotes fitness during cold stress

Translation initiation factor 2 (IF2) plays an essential role in bacterial cells by delivering the fMet-tRNAfMet to the ribosome pre-initiation complex. IF2 is known to have an N-terminal disordered region which is present across bacterial species, yet its function is not fully understood. Deletion of the IDR in E. coli showed no phenotypes at normal growth temperature (37{degrees}C); however, this IDR was found to be required for growth at cold temperatures (15{degrees}C). Since large IDRs can drive phase separation of various RNA binding proteins into biomolecular condensates, we investigated whether E. coli IF2 could phase separate. We discovered that IF2s N-terminal IDR drives phase separation in E. coli and C. crescentus, suggesting that IF2 condensation is a conserved property. Finally, using E. coli, we found that the IDR strongly drives phase separation in the cold, suggesting IF2 condensates promote fitness during cold stress. HighlightsO_LIIF2s IDR promotes phase separation with RNA. C_LIO_LICold temperature promotes IF2 condensation with RNA. C_LIO_LIIF2s IDR promotes fitness during cold shock. C_LI

microbiology↗

bioRxiv2024

Stress Changes the Bacterial Biomolecular Condensate Material State and Shifts Function from mRNA Decay to Storage

Bacterial ribonucleoprotein bodies (BR-bodies) are dynamic biomolecular condensates that play a pivotal role in RNA metabolism. We investigated how BR-bodies significantly influence mRNA fate by transitioning between liquid- and solid-like states in response to stress. With a combination of single-molecule and bulk fluorescence microscopy, biochemical assays, and quantitative analyses, we determine that BR-bodies promote efficient mRNA decay in a liquid-like condensate during exponential growth. On the other hand, BR-bodies are repurposed from sites of mRNA decay to reservoirs for mRNA storage under stress; a functional change that is enabled by their transition to a more rigid state, marked by reduced internal dynamics, increased molecular density, and prolonged residence time of ribonuclease E. Furthermore, we manipulated ATP levels and translation rates, and we conclude that the accumulation of ribosome-depleted mRNA is a key factor driving BR-body rigification, and that condensate maturation further contributes to this process. Upon nutrient replenishment, stationary-phase BR-bodies disassemble, releasing stored mRNAs for rapid translation, demonstrating that BR-body function is governed by a reversible mechanism for resource management. These findings reveal adaptive strategies by which bacteria regulate RNA metabolism through condensate-mediated control of mRNA decay and storage.

biophysics↗

bioRxiv2023

Caulobacter crescentus RNase E condensation contributes to autoregulation and fitness

RNase E is the most common RNA decay nuclease in bacteria, setting the global mRNA decay rate and scaffolding formation of the RNA degradosome complex and BR-bodies. To properly set the global mRNA decay rate, RNase E from Escherichia coli and neighboring {gamma}-proteobacteria were found to autoregulate RNase E levels via the decay of its mRNAs 5 UTR. While the 5 UTR is absent from other groups of bacteria in the Rfam database, we identified that the -proteobacterium Caulobacter crescentus RNase E contains a similar 5 UTR structure that promotes RNase E autoregulation. In both bacteria, the C-terminal IDR of RNase E is required for proper autoregulation to occur, and this IDR is also necessary and sufficient for RNase E to phase-separate, generating BR-bodies. Using in vitro purified RNase E, we find that the IDRs ability to promote phase-separation correlates with enhanced 5 UTR cleavage, suggesting that phase-separation of RNase E with the 5 UTR enhances autoregulation. Finally, using growth competition experiments we find that a strain capable of autoregulation rapidly outcompetes a strain with a 5 UTR mutation that cannot autoregulate, suggesting autoregulation promotes optimal cellular fitness.

biochemistry↗

bioRxiv2023

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions

Bacterial RNP bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We found 111 BR-body enriched proteins, including several RNA binding proteins, many of which are also recruited directly to in vitro reconstituted RNase E droplets, showing BR-bodies are more complex than previously assumed. While most BR-body enriched proteins that were tested cannot phase separate, we identified five that undergo RNA-dependent phase separation in vitro, showing other RNP condensates interface with BR-bodies. RNA degradosome protein clients are recruited more strongly to RNase E droplets than droplets of other RNP condensates, implying that client specificity is largely achieved through direct protein-protein interactions. We observe that some RNP condensates assemble with preferred directionally, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body associated proteins have the capacity to dissolve the condensate. Finally, we find that RNA dramatically stimulates the rate of RNase E phase separation in vitro, explaining the dissolution of BR-bodies after cellular mRNA depletion observed previously. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing. HighlightsO_LIBR-body proteomics identified 111 proteins enriched in BR-bodies. C_LIO_LIBR-bodies associate with an interconnected network of RNP condensates. C_LIO_LIBR-body condensation is modulated by its interaction network. C_LIO_LIRNA is required for rapid BR-body condensation. C_LI Graphical AbstractSummary of the BR-body protein interactome. Lines between two protein circles represent a direct interaction.

microbiology↗

bioRxiv2019

A RETINOBLASTOMA-RELATED transcription factor network governs egg cell differentiation and stress response in Arabidopsis

The multicellular embryo, and ultimately the entire organism, is a derivative of the fertilized egg cell. Unlike in animals, transcription factor networks orchestrating faithful egg development are still largely unknown in plants. We have identified that egg cell differentiation in Arabidopsis require interplay between evolutionarily conserved onco-protein homologs RETINOBLASTOMA-RELATED (RBR) and redundant MYB proteins MYB64/MYB119. RBR physically interacts with the MYBs; and with plant-specific transcription factors belonging to the RWP-RK-domain (RKD) family and LEAFY COTYLEDON1 (LEC1), which participate in development of egg cells and inherent stress response. RBR binds to most of these egg cell-expressed loci at the DNA level, partially overlapping with sites of histone methylation H3K27me3. Since deregulation of RKDs phenocopies mutants of RBR and the MYBs in terms of cell proliferation in the egg cell spatial domain, all the corresponding proteins are likely required to restrict parthenogenetic cell divisions of the egg cells. Cross-talk among these transcription factors, and direct regulation by RBR, govern egg cell development and expression of egg-to-zygotic polarity factors of the WUSCHEL RELATED HOMEOBOX family. Together, a network of RBR-centric transcription factors underlies egg cell development and stress response, possibly, in combination with several other predicted nodes.\n\nAuthor summaryThe RETINOBLASTOMA protein is one of the core components of the Eukaryotic cell cycle, and corresponding evolutionary homologs have been implicated not only to repress cell division but also to control differentiation and development. How RETINOBLASTOMA RELATED (RBR) associate with other higher order regulators to control faithful egg cell development in sexual plants is pivotal for manipulation of successful reproduction in general, and engineering of parthenogenesis when asexual or apomictic seed progeny are desirable over sexual plants. Using a suite of molecular methods, we show that a RBR-associated transcription factor network operates to specify egg cells in Arabidopsis. Complex cross-regulation within these transcription factors seems to be necessary for successful maternal egg cell to zygotic transition and reproductive stress response. Detailed genetic analysis implicate that RBR and its interactive partners belonging to MYB and RWP-RK transcription factor families are possibly required to prevent parthenogenesis of the sexual egg cells. Novel RBR networks and stress nodes explained in this study might help to improve our understanding of sexual and asexual reproduction.

genetics↗