Biology subjects

Muthunayake, N. S.

Publications and source records attributed to Muthunayake, N. S..

3 recordsLinked to original sources

bioRxiv2025

Rif-seq reveals Caulobacter crescentus mRNA decay is globally coordinated with transcription and translation

While transcription and translation have been shown to be coordinated with mRNA decay across various single-gene studies, their global coordination remains poorly defined. Therefore, we performed Rif-seq experiments in C. crescentus to measure genome-wide mRNA lifetimes and analyzed the impact of transcription and translation. Based upon the RNAP elongation speed, we identified that approximately 20% of mRNAs were cotranscriptionally degraded. We generated absolute quantitative estimates of mRNA copy numbers in C. crescentus, a useful systems biology resource, revealing that some gene categories have coordinated mRNA turnover. To investigate translations impact on mRNA decay, we found that translation efficiency measured by ribosome profiling correlates with mRNA lifetime. We compared the 5 P cleavage sites to ribosome occupancy and found that cleavage sites occur preferentially in regions of low ribosome occupancy. Using the translation initiation inhibitor retapamulin, which traps ribosomes at the start codon, and chloramphenicol, which arrests elongating ribosomes, shows that chloramphenicol leads to global mRNA stabilization. Surprisingly, we find that the codon adaptation index is inversely correlated with mRNA lifetime, suggesting slow translation elongation may be stabilizing mRNAs from decay. We confirmed the roles of translation initiation and elongation on mRNA lifetimes by generating synthetic YFP and mCherry reporter mRNAs. Taken together, mRNA decay is globally interconnected with transcription and translation. Highlights[bullet] Quantitative analysis yields absolute mRNA abundance and half-lives for C. crescentus [bullet]Identification of 47 new stable ncRNAs [bullet]Both transcription and translation are globally coordinated with mRNA decay [bullet]Translation initiation and elongation impact mRNA decay through ribosome occupancy

systems biology↗

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

BR-bodies provide selectively permeable condensates that stimulate mRNA decay and prevent release of decay intermediates

Biomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA-seq. We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved ncRNAs are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the build-up of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both, enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

biochemistry↗