Delayed Protein Changes During Seed Germination

Over the past decade, ample transcriptome data have been generated at different stages during seed germination; however, far less is known about protein synthesis during this important physiological process. Generally, the correlation between transcript levels and protein abundance is low, which strongly limits the use of transcriptome data to accurately estimate protein expression. Polysomal profiling has emerged as a tool to identify mRNAs that are actively translated. The association of the mRNA to the polysome, also referred to as translatome, provides a proxy for mRNA translation. In this study, the correlation between the changes in total mRNA, polysome-associated mRNA, and protein levels across seed germination was investigated. The direct correlation between polysomal mRNA and protein abundance at a single time-point during seed germination is low. However, once the polysomal mRNA of a time-point is compared to the proteome of the next time-point, the correlation is much higher. 35% of the investigated proteome has delayed changes at the protein level. Genes have been classified based on their delayed protein changes, and specific motifs in these genes have been identified. Moreover, mRNA and protein stability and mRNA length have been found as important predictors for changes in protein abundance. In conclusion, polysome association and/or dissociation predicts future changes in protein abundance in germinating seeds.

mRNA- and Factor-Driven Dynamic Variability Controls eIF4F-Cap Recognition for Translation Initiation.

mRNA 5′ cap recognition by eIF4F is a key step in eukaryotic translational control. While different mRNAs respond differently to eIF4F–directed regulation, the molecular basis for this variability remains unclear. We developed single-molecule fluorescence assays to directly observe eIF4F–mRNA interactions. We uncovered a complex interplay of mRNA features with factor activities that differentiates cap recognition between mRNAs. eIF4E–cap association rates are anticorrelated with mRNA length. eIF4A leverages ATP binding to differentially accelerate eIF4E–mRNA association; the extent of this acceleration correlates with translation efficiency in vivo. eIF4G lengthens eIF4E–cap binding to persist on the initiation timescale. The full eIF4F complex discriminates between mRNAs in an ATP-dependent manner. After eIF4F–mRNA binding, eIF4E is ejected from the cap by eIF4A ATP hydrolysis. Our results suggest features throughout mRNA coordinate in controlling cap recognition at the 5ʹ end, and suggest a model for how eIF4F–mRNA dynamics establish mRNA sensitivity to translational control processes.

Changes in mRNA Degradation Efficiencies under Varying Conditions Are Regulated by Multiple Determinants in Arabidopsis thaliana

Multiple mechanisms are involved in gene expression, with mRNA degradation being critical for the control of mRNA accumulation. In plants, although some trans-acting factors and motif sequences have been identified in deadenylation-dependent mRNA degradation, endonucleolytic cleavage-dependent mRNA degradation has not been studied in detail. Previously, we developed truncated RNA-end sequencing (TREseq) in Arabidopsis thaliana and detected G-rich sequence motifs around 5′ degradation intermediates. However, it remained to be elucidated whether degradation efficiencies of 5′ degradation intermediates in A. thaliana vary among growth conditions and developmental stages. To address this issue, we conducted TREseq of cultured cells under heat stress and at three developmental stages (seedlings, expanding leaves and expanded leaves) and compared 5′ degradation intermediates data among the samples. Although some 5′ degradation intermediates had almost identical degradation efficiencies, others differed among conditions. We focused on the genes and sites whose degradation efficiencies differed. Changes in degradation efficiencies at the gene and site levels revealed an effect on mRNA accumulation in all comparisons. These changes in degradation efficiencies involved multiple determinants, including mRNA length and translation efficiency. These results suggest that several determinants govern the efficiency of mRNA degradation in plants, helping the organism to adapt to varying conditions by controlling mRNA accumulation.

ArfB can displace mRNA to rescue stalled ribosomes

Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.

Transcriptome-Wide Analysis of Interplay between mRNA Stability, Translation and Small RNAs in Response to Neuronal Membrane Depolarization

Experience-dependent changes to neural circuitry are shaped by spatially-restricted activity-dependent mRNA translation. Although the complexity of mRNA translation in neuronal cells is widely appreciated, translational profiles associated with neuronal excitation remain largely uncharacterized, and the associated regulatory mechanisms are poorly understood. Here, we employed ribosome profiling, mRNA sequencing and small RNA sequencing to profile transcriptome-wide changes in mRNA translation after whole cell depolarization of differentiated neuroblast cultures, and investigate the contribution of sequence-specific regulatory mechanisms. Immediately after depolarization, a functional partition between transcriptional and translational responses was uncovered, in which many mRNAs were subjected to significant changes in abundance or ribosomal occupancy, but not both. After an extended (2 h) post-stimulus rest phase, however, these changes became synchronized, suggesting that there are different layers of post-transcriptional regulation which are temporally separated but become coordinated over time. Globally, changes in mRNA abundance and translation were found to be associated with a number of intrinsic mRNA features, including mRNA length, GC% and secondary structures; however, the effect of these factors differed between both post-depolarization time-points. Furthermore, small RNA sequencing revealed that miRNAs and tRNA-derived small RNA fragments were subjected to peak changes in expression immediately after stimulation, during which these molecules were predominantly associated with fluctuations in mRNA abundance, consistent with known regulatory mechanisms. These data suggest that excitation-associated neuronal translation is subjected to extensive temporal coordination, with substantial contributions from a number of sequence-dependent regulatory mechanisms.

Identification of CDH11 as an ASD risk gene by matched-gene co-expression analysis and mouse behavioral studies

In the study of autism spectrum disorder (ASD) by gene co-expression analysis (GCA), we found that four gene features, including gene size, mRNA length, mRNA abundance, and guanine-cytosine content, profoundly affect gene co-expression profiles. To circumvent the potential interference of these confounding factors on GCA, we developed the “matched-gene co-expression analysis” (MGCA) to investigate gene co-expression relationships. This method demonstrated the convergent expression profile of high confidence ASD risk genes and effectively revealed convergent molecular pathways of ASD risk genes. Application of MGCA to two ASD candidate genes CDH11 and CDH9 showed association of CDH11, but not CDH9, with ASD. Mouse behavioral studies showed that Cdh11-null mice, but not Cdh9-null mice, have multiple autistic-like behavioral alterations. This study confirmed that CDH11 is an important ASD risk gene and demonstrated the importance of considering matched gene features in the analysis of gene co-expression.

Hallmarks of slow translation initiation revealed in mitochondrially localizing mRNA sequences

ABSTRACTThe mRNA of some, but not all, nuclear encoded mitochondrial proteins localize to the periphery of mitochondria. Previous studies have shown that both the nascent polypeptide chain and an mRNA binding protein play a role in this phenomenon, and have noted a positive correlation between mRNA length and mitochondrial localization. Here, we report the first investigation into the relationship between mRNA translation initiation rate and mRNA mitochondrial localization. Our results indicate that translation initiation promoting factors such as Kozak sequences are associated with cytosolic localization, while inhibiting factors such as 5′ UTR secondary structure correlate with mitochondrial localization. Moreover, the frequencies of nucleotides in various positions of the 5′ UTR show higher correlation with localization than the 3′ UTR. These results indicate that mitochondrial localization is associated with slow translation initiation. Interestingly this may help explain why short mRNAs, which are thought to initiate translation rapidly, seldom localize to mitochondria. We propose a model in which translating mRNA has reduced mobility and tends not to reach mitochondria. Finally, we explore this model with a simulation of mRNA diffusion using previously estimated translation initiation probabilities, confirming that our model can produce localization values similar to those measured in experimental studies.

A decrease in transcription capacity limits growth rate upon translation inhibition

In bacterial cells, inhibition of ribosomes by sublethal concentrations of antibiotics leads to a decrease in growth rate despite an increase in ribosome content. The limitation of ribosomal activity results in an increase in the level of expression from ribosomal promoters; this can deplete the pool of RNA polymerase (RNAP) that is available for the expression of non-ribosomal genes. However, the magnitude of this effect remains to be quantified. Here, we use the change in the activity of constitutive promoters with different affinities for RNAP to quantify the change in the concentration of free RNAP. The data are consistent with a significant decrease in the amount of RNAP available for transcription of both ribosomal and non ribosomal genes. Results obtained with different reporter genes reveal an mRNA length dependence on the amount of full-length translated protein, consistent with the decrease in ribosome processivity affecting more strongly the translation of longer genes. The genes coding for the β and β’ subunits of RNAP are amongst the longest genes in theE. coligenome, while the genes coding for ribosomal proteins are among the shortest genes. This can explain the observed decrease in transcription capacity that favors the expression of genes whose promoters have a high affinity for RNAP, such as ribosomal promoters.ImportanceExposure of bacteria to sublethal concentrations of antibiotics can lead to bacterial adaptation and survival at higher doses of inhibitors, which in turn can lead to the emergence of antibiotic resistance. The presence of sublethal concentrations of antibiotics targeting translation results in an increase in the amount of ribosomes per cell and a decrease in the cells’ growth rate. In this work, we have found that inhibition of ribosome activity can result in a decrease in the amount of free RNA polymerase available for transcription, thus limiting the protein expression rate via a different pathway than what was expected. This result can be explained by our observation that long genes, such as those coding for RNA polymerase subunits, have a higher probability of premature translation termination in the presence of ribosome inhibitors, while expression of short ribosomal genes is affected less, consistent with their increased concentration.

Imaging of single mRNA translation repression reveals diverse interactions with mRNP granules

During cellular stress mRNAs exit translation and accumulate in stress granules and P-bodies, although the dynamics of these interactions remain unclear. We imaged in real-time single mRNAs, their translational output, and mRNA-granule interactions during stress. We observed single mRNAs interacting with stress granules and P-bodies, with mRNAs moving bidirectionality between them. While translating mRNAs only interact with RNP granules dynamically, non-translating mRNAs can form stable associations that rigidly immobilize the mRNA within the granule. Imaging thousands of individual mRNA-granule interactions showed the probability of stable association increases with both mRNA length and granule size. Therefore, the recruitment of mRNAs to RNP granules involves both highly dynamic and stable interactions, influenced by several parameters, demonstrating a new layer of complexity in mRNA regulation during stress.One Sentence SummarymRNAs interact with stress granules and P-bodies in stable and dynamic manners influenced by ribosome association, mRNA length, and granule size.

The formation of intramolecular secondary structure brings mRNA ends in close proximity

ABSTRACTA number of protein factors regulate protein synthesis by bridging mRNA ends or untranslated regions (UTRs). Using experimental and computational approaches, we show that mRNAs from various organisms, including humans, have an intrinsic propensity to fold into structures in which the 5’ end and 3’ end are ≤ 7 nm apart irrespective of mRNA length. Computational estimates performed for ∼22,000 human transcripts indicate that the inherent proximity of the ends is a universal property of most, if not all, mRNA sequences. Only specific RNA sequences, which have low sequence complexity and are devoid of guanosines, are unstructured and exhibit end-to-end distances expected for the random coil conformation of RNA. Our results suggest that the intrinsic proximity of mRNA ends may facilitate binding of translation factors that bridge mRNA 5’ and 3’ UTRs. Furthermore, our studies provide the basis for measuring, computing and manipulating end-to-end distances and secondary structure in mRNAs in research and biotechnology.