Piyada Juntawong

Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis

Significance Plant survival in a highly varied environment requires flexibility in gene regulation. To capture dynamics of mRNA translation at the genome scale, we precisely mapped individual ribosomes to mRNAs of whole seedlings under control and low-oxygen conditions. The results demonstrate nearly 100-fold variation in the efficiency of translation of individual mRNAs under both conditions and provide unique insights into posttranscriptional and translational regulation modulated by low-energy stress in Arabidopsis thaliana. Translational regulation contributes to plasticity in metabolism and growth that enables plants to survive in a dynamic environment. Here, we used the precise mapping of ribosome footprints (RFs) on mRNAs to investigate translational regulation under control and sublethal hypoxia stress conditions in seedlings of Arabidopsis thaliana. Ribosomes were obtained by differential centrifugation or immunopurification and were digested with RNase I to generate footprint fragments that were deep-sequenced. Comparison of RF number and position on genic regions with fragmented total and polysomal mRNA illuminated numerous aspects of posttranscriptional and translational control under both growth conditions. When seedlings were oxygen-deprived, the frequency of ribosomes at the start codon was reduced, consistent with a global decline in initiation of translation. Hypoxia–up-regulated gene transcripts increased in polysome complexes during the stress, but the number of ribosomes per transcript relative to normoxic conditions was not enhanced. On the other hand, many mRNAs with limited change in steady-state abundance had significantly fewer ribosomes but with an overall similar distribution under hypoxia, consistent with restriction of initiation rather than elongation of translation. RF profiling also exposed the inhibitory effect of upstream ORFs on the translation of downstream protein-coding regions under normoxia, which was further modulated by hypoxia. The data document translation of alternatively spliced mRNAs and expose ribosome association with some noncoding RNAs. Altogether, we present an experimental approach that illuminates prevalent and nuanced regulation of protein synthesis under optimal and energy-limiting conditions.

Getting the message across: cytoplasmic ribonucleoprotein complexes

mRNA-ribonucleoprotein (mRNP) complexes mediate post-transcriptional control mechanisms in the cell nucleus and cytoplasm. Transcriptional control is paramount to gene expression but is followed by regulated nuclear pre-mRNA maturation and quality control processes that culminate in the export of a functional transcript to the cytoplasm. Once in the cytosol, mRNPs determine the activity of individual mRNAs through regulation of localization, translation, sequestration and turnover. Here, we review how quantitative assessment of mRNAs in distinct cytoplasmic mRNPs, such as polyribosomes (polysomes), has provided new perspectives on post-transcriptional regulation from the global to gene-specific level. In addition, we explore recent genetic and biochemical studies of cytoplasmic mRNPs that have begun to expose RNA-binding proteins in an integrated network that fine-tunes gene expression.

Dynamic light regulation of translation status in Arabidopsis thaliana

Light, a dynamic environmental parameter, is an essential regulator of plant growth and development. Light-regulated transcriptional networks are well documented, whereas light-regulated post-transcriptional regulation has received limited attention. In this study, dynamics in translation of cytosolic mRNAs were evaluated at the genome-level in Arabidopsis thaliana seedlings grown under a typical light/dark diurnal regime, shifted to darkness at midday, and then re-illuminated. One-hour of unanticipated darkness reduced levels of polysomes by 17% in a manner consistent with inhibition of initiation of translation. This down-regulation of translation was reversed within 10 min of re-illumination. Quantitative comparison of the total cellular population of transcripts (the transcriptome) to those associated with one or more 80S ribosome (the translatome) identified over 1600 mRNAs that were differentially translated in response to light availability. Unanticipated darkness limited both transcription and translation of mRNAs encoding components of the photosynthetic machinery. Many mRNAs encoding proteins associated with the energy demanding process of protein synthesis were stable but sequestered in the dark, in a rapidly reversible manner. A meta-analysis determined these same transcripts were similarly and coordinately regulated in response to changes in oxygen availability. The dark and hypoxia translationally repressed mRNAs lack highly supported candidate RNA-regulatory elements but are characterized by G + C-rich 5′-untranslated regions. We propose that modulation of translation of a subset of cellular mRNAs functions as an energy conservation mechanism.

Isolation of plant polysomal mRNA by differential centrifugation and ribosome immunopurification methods.

Polyribosomes (polysomes) form as multiple ribosomes engage in translation on a single mRNA. This process is regulated for individual mRNAs by both development and the environment. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts by high-speed differential centrifugation. These ribonucleoprotein complexes can be further purified by centrifugation through sucrose density gradients. By fractionation of the gradient the amount of an individual mRNA in a sub-population of polysomes can be quantitatively determined. Here, we describe methods for the isolation and quantification of polysome complexes from plant tissues. The mRNA obtained can be further analyzed by methods that evaluate polysomal mRNA abundance at the individual transcript or global level. A modification of the conventional polysome isolation procedure is described for transgenic Arabidopsis thaliana that express an epitope-tagged version of ribosomal protein L18 (RPL18) that facilitates capture of ribosomes from crude cell extracts by a one-step immunoprecipitation method.

Posttranscriptional Control of Photosynthetic mRNA Decay under Stress Conditions Requires 3′ and 5′ Untranslated Regions and Correlates with Differential Polysome Association in Rice

Abiotic stress, including drought, salinity, and temperature extremes, regulates gene expression at the transcriptional and posttranscriptional levels. Expression profiling of total messenger RNAs (mRNAs) from rice (Oryza sativa) leaves grown under stress conditions revealed that the transcript levels of photosynthetic genes are reduced more rapidly than others, a phenomenon referred to as stress-induced mRNA decay (SMD). By comparing RNA polymerase II engagement with the steady-state mRNA level, we show here that SMD is a posttranscriptional event. The SMD of photosynthetic genes was further verified by measuring the half-lives of the small subunit of Rubisco (RbcS1) and Chlorophyll a/b-Binding Protein1 (Cab1) mRNAs during stress conditions in the presence of the transcription inhibitor cordycepin. To discern any correlation between SMD and the process of translation, changes in total and polysome-associated mRNA levels after stress were measured. Total and polysome-associated mRNA levels of two photosynthetic (RbcS1 and Cab1) and two stress-inducible (Dehydration Stress-Inducible Protein1 and Salt-Induced Protein) genes were found to be markedly similar. This demonstrated the importance of polysome association for transcript stability under stress conditions. Microarray experiments performed on total and polysomal mRNAs indicate that approximately half of all mRNAs that undergo SMD remain polysome associated during stress treatments. To delineate the functional determinant(s) of mRNAs responsible for SMD, the RbcS1 and Cab1 transcripts were dissected into several components. The expressions of different combinations of the mRNA components were analyzed under stress conditions, revealing that both 3′ and 5′ untranslated regions are necessary for SMD. Our results, therefore, suggest that the posttranscriptional control of photosynthetic mRNA decay under stress conditions requires both 3′ and 5′ untranslated regions and correlates with differential polysome association.

Profiling of Translatomes of in Vivo–Grown Pollen Tubes Reveals Genes with Roles in Micropylar Guidance during Pollination in Arabidopsis

This work described the use of a method for the specific isolation of ribosome-associated mRNAs of in vivo–grown pollen tubes to identify over 500 genes with transcripts highly enriched or specific to in vivo–grown pollen tubes. T-DNA insertion alleles of a number of these genes conferred defects in late stages of the pollination process by haploid gametes. Transcriptome profiling has been used to identify genes expressed in pollen tubes elongating in vitro; however, little is known of the transcriptome of in vivo–grown pollen tubes due to the difficulty of collecting pollen that is elongating within the solid maternal gynoecium. Using a pollen-specific promoter (ProLAT52) to generate epitope-tagged polysomal-RNA complexes that could be affinity purified, we obtained mRNAs undergoing translation (the translatome) of in vivo–grown pollen tubes from self-pollinated gynoecia of Arabidopsis thaliana. Translatomes of pollen grains as well as in vivo– and in vitro–cultured pollen tubes were assayed by microarray analyses, revealing over 500 transcripts specifically enriched in in vivo–elongating pollen tubes. Functional analyses of several in vivo mutants (iv) of these pollination-enhanced transcripts revealed partial pollination/fertilization and seed formation defects in siliques (iv2, iv4, and iv6). Cytological observation confirmed the involvement of these genes in specialized processes including micropylar guidance (IV6 and IV4), pollen tube burst (IV2), and repulsion of multiple pollen tubes in embryo sac (IV2). In summary, the selective immunopurification of transcripts engaged with polysomes in pollen tubes within self-fertilized florets has identified a cohort of pollination-enriched transcripts that facilitated the identification of genes important in in vivo pollen tube biology.

Translating Ribosome Affinity Purification (TRAP) followed by RNA sequencing technology (TRAP-SEQ) for quantitative assessment of plant translatomes.

Translating Ribosome Affinity Purification (TRAP) is a technology to isolate the population of mRNAs associated with at least one 80S ribosome, referred as the translatome. TRAP is based on the expression of an epitope-tagged version of a ribosomal protein and the affinity purification of ribosomes and associated mRNAs using antibodies conjugated to agarose beads. Quantitative assessment of the translatome is achieved by direct RNA sequencing (RNA-SEQ), which provides accurate quantitation of ribosome-associated mRNAs and reveals alternatively spliced isoforms. Here we present a detailed procedure for TRAP, as well as a guide for preparation of RNA-SEQ libraries (TRAP-SEQ) and a primary data analysis. This methodology enables the study of translational dynamic by assessing rapid changes in translatomes, at organ or cell-type level, during development or in response to endogenous or exogenous stimuli.

Characteristics and Significance of Intergenic Polyadenylated RNA Transcription in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) genome is the most well-annotated plant genome. However, transcriptome sequencing in Arabidopsis continues to suggest the presence of polyadenylated (polyA) transcripts originating from presumed intergenic regions. It is not clear whether these transcripts represent novel noncoding or protein-coding genes. To understand the nature of intergenic polyA transcription, we first assessed its abundance using multiple messenger RNA sequencing data sets. We found 6,545 intergenic transcribed fragments (ITFs) occupying 3.6% of Arabidopsis intergenic space. In contrast to transcribed fragments that map to protein-coding and RNA genes, most ITFs are significantly shorter, are expressed at significantly lower levels, and tend to be more data set specific. A surprisingly large number of ITFs (32.1%) may be protein coding based on evidence of translation. However, our results indicate that these “translated” ITFs tend to be close to and are likely associated with known genes. To investigate if ITFs are under selection and are functional, we assessed ITF conservation through cross-species as well as within-species comparisons. Our analysis reveals that 237 ITFs, including 49 with translation evidence, are under strong selective constraint and relatively distant from annotated features. These ITFs are likely parts of novel genes. However, the selective pressure imposed on most ITFs is similar to that of randomly selected, untranscribed intergenic sequences. Our findings indicate that despite the prevalence of ITFs, apart from the possibility of genomic contamination, many may be background or noisy transcripts derived from “junk” DNA, whose production may be inherent to the process of transcription and which, on rare occasions, may act as catalysts for the creation of novel genes.

Ribosome Profiling: A Tool for Quantitative Evaluation of Dynamics in mRNA Translation

Translational regulation is important for plant growth, metabolism, and acclimation to environmental challenges. Ribosome profiling involves the nuclease digestion of mRNAs associated with ribosomes and mapping of the generated ribosome-protected footprints to transcripts. This is useful for investigation of translational regulation. Here we present a detailed method to generate, purify, and high-throughput-sequence ribosome footprints from Arabidopsis thaliana using two different isolation methods, namely, conventional differential centrifugation and the translating ribosome affinity purification (TRAP) technology. These methodologies provide researchers with an opportunity to quantitatively assess with high-resolution the translational activity of individual mRNAs by determination of the position and number of ribosomes in the corresponding mRNA. The results can provide insights into the translation of upstream open reading frames, alternatively spliced transcripts, short open reading frames, and other aspects of translation.