Project description:Translational regulation at the stage of initiation impacts the number of ribosomes translating each mRNA molecule. For example, multiple ribosomes can engage on a single mRNA forming a polysome, resulting in highly efficient protein synthesis. However, the translational activity of single 80S ribosomes on mRNA (monosomes) is less well understood, even though these 80S monosomes represent the dominant ribosomal complexes in many tissues. Here, we used cryo-EM to determine the translational activity of 80S monosomes across different tissues in Drosophila melanogaster. We discovered that while head and embryo 80S monosomes are highly translationally active, testis and ovary 80S monosomes are translationally inactive. RNA-Seq analysis of head monosome- and polysome-translated mRNAs, revealed that head 80S monosomes preferentially translate mRNAs with TOP motifs, short 5’-UTRs, short ORFs and are enriched for uORFs. Overall, these findings highlight that regulation of translation initiation, and therefore the number of ribosomes bound per mRNA, varies substantially across tissues.
Project description:Transfer RNA (tRNA) repertoires vary greatly across genomes, shaped by genetic drift and selection. A peculiar pattern across prokaryotes is the near-complete absence of tRNAs with unmodified adenine at the 34th (wobble) position (i.e., tRNAANN). Each of these tRNAs are just a single mutation away from several other tRNAs. Hence, their persistent absence suggests fundamental but hitherto unclear constraints. We engineered 36 Escherichia coli strains expressing each theoretically possible tRNAANN to determine their functionality and fitness effects. Notably, there was no evidence of broad toxicity due to these tRNAs. All five tRNAANN tested underwent post-transcriptional maturation and all seven tested compensated for the deletion of their respective native tRNABNN (carrying G, C or U at the 34th position), demonstrating that tRNAANN are translationally active. Furthermore, tRNAANN from four-fold degenerate (4D) codon boxes were unmodified and were generally neutral or beneficial, whereas tRNAANN from two-fold degenerate (2D) boxes underwent A34-to-I34 modification and were more likely to impair fitness. We suggest superwobbling by tRNAANN — decoding an entire four-codon set — as one mechanism underlying these differential fitness effects. Maximal degeneracy in 4D boxes buffers or exploits tRNAANN superwobbling via synonymous decoding, whereas constrained degeneracy in 2D boxes renders it deleterious, likely through amino acid misincorporation. Thus, these differential fitness effects, sharpens the paradox of neutral or beneficial yet absent 4D tRNAANN, while beginning to empirically unravel underlying causes for the absence of 2D tRNAANN.
Project description:mRNAs associated with microtubules during interphase, metaphase and the midbody stage of cytokinesis were sequenced. Selective midbody-localized RNAs were identified and their translational characteristics were studied.
Project description:Initiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. The absence or failure of DNA replication can result in bacterial cell growth arrest or death. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. For this purpose, DNA replication was blocked using a CRISPRi approach specifically targeting DnaA boxes 6 and 7, which are essential for replication initiation. We characterized the phenotype of these cells and analyzed the overall changes in the proteome using quantitative mass spectrometry. Cells with arrested replication were elongating and not dividing but showed no evidence of DNA damage response (DDR). Moreover, these cells did not cease translation over time. This study sets the ground for future research on non-replicating but translationally active B. subtilis, which might be valuable for biotechnological applications.
Project description:MicroRNAs (miRNAs) are master regulators that act in response to development signals or environmental stimuli by mediating the cleavage of their target mRNAs or repressing their translation; however, the mechanisms by which miRNA dynamics are precisely regulated under salt stress are yet to be fully elucidated. SOS2 modulates the reconstruction of the salt-responsive transcriptome by post-translationally modifying SE.
Project description:The CCA-adding enzyme adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. Here, we report deep sequencing analysis of the 3' ends of tRNA-Ser-CGA and tRNA-Ser-UGA from S. cerevisiae strains and show that hypomodified mature tRNAs are subjected to CCACCA (or poly(A) addition) as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addtion is a universal mechanism for controlling tRNA levels and preventing errors in translation. 121 samples analyzed in total, representing time courses of 10 different yeast strains; Biological replicates for each time point are included
Project description:Retrons are prokaryotic reverse transcriptase systems that produce multicopy single-stranded DNA (msDNA), yet the principles by which they mediate antiviral defense remain largely unresolved. Here, we dissect the mechanism of Eco2, a minimal retron composed of a single reverse transcriptase–nuclease fusion protein. Cryogenic electron microscopy and hydrogen-deuterium exchange mass spectrometry reveal the structures and dynamics of a trimeric nucleoprotein complex assembled within a branched msDNA scaffold, which cages the TOPRIM nuclease. We show that the phage-encoded endonucleases DenB initiates msDNA degradation, thereby unblocking the nuclease active site. Activated Eco2 cuts tRNAs, resulting in translational shutdown for antiphage defense. We further identify ribosomal protein S1 as a putative RNA chaperone that associates with the msDNA precursor. These findings provide insights into the molecular mechanisms of minimal retrons and establish a structural basis for the engineering of Eco2.
Project description:The regulation of translation is crucial for cells to rapidly adapt to changing conditions. Although the transcriptional changes under inflammatory conditions are intensely studied, not much is known about translational changes. Therefore, this study aimed at identifying translationally deregulated targets in inflammatory settings.