Translation of mRNA into functional proteins is a fundamental process underlying many aspects of plant growth and development. Yet, the role of translational regulation in plants across diverse tissue types, including seeds, is not well known due to the lack of methods targeting these processes. Studying the seed translatome could unveil seed-specific regulatory mechanisms, offering valuable insights for breeding efforts to enhance seed traits. Polysome profiling is a widely used technique for studying mRNAs being translated. However, the traditional method is time-consuming and has a low polysome recovery rate; therefore, it requires substantial starting material. This is particularly challenging for species or mutants with limited seed quantities. Additionally, seed polysome fractions often yield low quality RNA due to the abundance of various compounds that interfere with conventional RNA extraction protocols. Here we present a robust polysome extraction method incorporating a size-exclusion step for polysome concentration streamlined with a rapid RNA extraction method optimized for seeds. This protocol works across multiple plant species and offers increased speed and robustness, requiring less than half the amount of seed tissue and time compared to conventional methods while ensuring high polysome recovery and yield of high-quality RNA for downstream experiments. These features make this protocol an ideal tool for studying seed translation efficiency and hold broad applicability across various plant species and tissues. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Robust polysome extraction for seeds Basic Protocol 2: Rapid fraction total RNA extraction.

The coordinated action of transcriptional and post-transcriptional machineries shapes gene expression programs at steady state and determines their concerted response to perturbations. We have developed Nanodynamo, an experimental and computational workflow for quantifying the kinetic rates of nuclear and cytoplasmic steps of the RNA life cycle. Nanodynamo is based on mathematical modelling following sequencing of native RNA from cellular fractions and polysomes. We have applied this workflow to triple-negative breast cancer cells, revealing widespread post-transcriptional RNA processing that is mutually exclusive with its co-transcriptional counterpart. We used Nanodynamo to unravel the coupling between transcription, processing, export, decay and translation machineries. We have identified a number of coupling interactions within and between the nucleus and cytoplasm that largely contribute to coordinating how cells respond to perturbations that affect gene expression programs. Nanodynamo will be instrumental in unravelling the determinants and regulatory processes involved in the coordination of gene expression responses.

GEMIN5 is a multifunctional protein involved in various aspects of RNA biology, including biogenesis of snRNPs and translation control. Reduced levels of GEMIN5 confer a differential translation to selective groups of mRNAs, and biallelic variants reducing protein stability or inducing structural conformational changes are associated with neurological disorders. Here, we show that upregulation of GEMIN5 can be detrimental as it modifies the steady state of mRNAs and enhances alternative splicing (AS) events of genes involved in a broad range of cellular processes. RNA-Seq identification of the mRNAs associated with polysomes in cells with high levels of GEMIN5 revealed that a significant fraction of the differential AS events undergo translation. The association of mRNAs with polysomes was dependent on the type of AS event, being more frequent in the case of exon skipping. However, there were no major differences in the percentage of genes showing open-reading frame disruption. Importantly, differential AS events in mRNAs engaged in polysomes, eventually rendering non-functional proteins, encode factors controlling cell growth. The broad range of mRNAs comprising AS events engaged in polysomes upon GEMIN5 upregulation supports the notion that this multifunctional protein has evolved as a gene expression balancer, consistent with its dual role as a member of the SMN complex and as a modulator of protein synthesis, ultimately impinging on cell homoeostasis.

The relationship between transcription and protein expression is complex. We identified polysome-associated RNA transcripts in the somata and central terminals of mouse sensory neurons in control, painful (plus nerve growth factor), and pain-free conditions (Nav1.7-null mice). The majority (98%) of translated transcripts are shared between male and female mice in both the somata and terminals. Some transcripts are highly enriched in the somata or terminals. Changes in the translatome in painful and pain-free conditions include novel and known regulators of pain pathways. Antisense knockdown of selected somatic and terminal polysome-associated transcripts that correlate with pain states diminished pain behavior. Terminal-enriched transcripts included those encoding synaptic proteins (e.g., synaptotagmin), non-coding RNAs, transcription factors (e.g., Znf431), proteins associated with transsynaptic trafficking (HoxC9), GABA-generating enzymes (Gad1 and Gad2), and neuropeptides (Penk). Thus, central terminal translation may well be a significant regulatory locus for peripheral input from sensory neurons.

The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.

Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs.

mRNA therapeutics are revolutionizing the pharmaceutical industry, but methods to optimize the primary sequence for increased expression are still lacking. Here, we design 5\'UTRs for efficient mRNA translation using deep learning. We perform polysome profiling of fully or partially randomized 5\'UTR libraries in three cell types and find that UTR performance is highly correlated across cell types. We train models on our datasets and use them to guide the design of high-performing 5\'UTRs using gradient descent and generative neural networks. We experimentally test designed 5\'UTRs with mRNA encoding megaTALTM gene editing enzymes for two different gene targets and in two different cell lines. We find that the designed 5\'UTRs support strong gene editing activity. Editing efficiency is correlated between cell types and gene targets, although the best performing UTR was specific to one cargo and cell type. Our results highlight the potential of model-based sequence design for mRNA therapeutics.

In recent years, numerous evidence has been accumulated about the extent of A-to-I editing in human RNAs and the key role ADAR1 plays in the cellular editing machinery. It has been shown that A-to-I editing occurrence and frequency are tissue-specific and essential for some tissue development, such as the liver. To study the effect of ADAR1 function in hepatocytes, we have created Huh7.5 ADAR1 KO cell lines. Upon IFN treatment, the Huh7.5 ADAR1 KO cells show rapid arrest of growth and translation, from which they do not recover. We analyzed translatome changes by using a method based on sequencing of separate polysome profile RNA fractions. We found significant changes in the transcriptome and translatome of the Huh7.5 ADAR1 KO cells. The most prominent changes include negatively affected transcription by RNA polymerase III and the deregulation of snoRNA and Y RNA levels. Furthermore, we observed that ADAR1 KO polysomes are enriched in mRNAs coding for proteins pivotal in a wide range of biological processes such as RNA localization and RNA processing, whereas the unbound fraction is enriched mainly in mRNAs coding for ribosomal proteins and translational factors. This indicates that ADAR1 plays a more relevant role in small RNA metabolism and ribosome biogenesis.

Translational control is important in all life, but it remains a challenge to accurately quantify. When ribosomes translate messenger (m)RNA into proteins, they attach to the mRNA in series, forming poly(ribo)somes, and can co-localize. Here, we computationally model new types of co-localized ribosomal complexes on mRNA and identify them using enhanced translation complex profile sequencing (eTCP-seq) based on rapid in vivo crosslinking. We detect long disome footprints outside regions of non-random elongation stalls and show these are linked to translation initiation and protein biosynthesis rates. We subject footprints of disomes and other translation complexes to artificial intelligence (AI) analysis and construct a new, accurate and self-normalized measure of translation, termed stochastic translation efficiency (STE). We then apply STE to investigate rapid changes to mRNA translation in yeast undergoing glucose depletion. Importantly, we show that, well beyond tagging elongation stalls, footprints of co-localized ribosomes provide rich insight into translational mechanisms, polysome dynamics and topology. STE AI ranks cellular mRNAs by absolute translation rates under given conditions, can assist in identifying its control elements and will facilitate the development of next-generation synthetic biology designs and mRNA-based therapeutics.

Given their highly polarized morphology and functional singularity, neurons require precise spatial and temporal control of protein synthesis. Alterations in protein translation have been implicated in the development and progression of a wide range of neurological and neurodegenerative disorders, including Huntington\'s disease (HD). In this study we examined the architecture of polysomes in their native brain context in striatal tissue from the zQ175 knock-in mouse model of HD. We performed 3D electron tomography of high-pressure frozen and freeze-substituted striatal tissue from HD models and corresponding controls at different ages. Electron tomography results revealed progressive remodelling towards a more compacted polysomal architecture in the mouse model, an effect that coincided with the emergence and progression of HD related symptoms. The aberrant polysomal architecture is compatible with ribosome stalling phenomena. In fact, we also detected in the zQ175 model an increase in the striatal expression of the stalling relief factor EIF5A2 and an increase in the accumulation of eIF5A1, eIF5A2 and hypusinated eIF5A1, the active form of eIF5A1. Polysomal sedimentation gradients showed differences in the relative accumulation of 40S ribosomal subunits and in polysomal distribution in striatal samples of the zQ175 model. These findings indicate that changes in the architecture of the protein synthesis machinery may underlie translational alterations associated with HD, opening new avenues for understanding the progression of the disease.