Genome-wide ribosome profiling reveals dynamic translational landscape in Arabidopsis seedling roots under simulated microgravity

doi:10.21203/rs.3.rs-4849677/v1

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Genome-wide ribosome profiling reveals dynamic translational landscape in Arabidopsis seedling roots under simulated microgravity

https://doi.org/10.21203/rs.3.rs-4849677/v1

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Plants can adapt to environmental change through a variety of transcriptional and translational mechanisms. Although extensive knowledge exists about plant stress responses at the transcriptional level, understanding of these responses at the translational level is limited. This study aimed to evaluate the translational dynamics of plants in a simulated microgravity environment through the utilization of a ribosome profiling assay. A comparative analysis of ribosome profiling data and RNA-seq on a global scale demonstrated a modest correlation in the alteration of gene expression between transcriptional and translational levels. However, under one third of responsive genes exhibited concurrent transcriptional and translational activities. We found that simulated microgravity simulation elicited separate transcriptional and translational responses in plants. The findings indicated a significant alteration in the translational efficiency (TE) of 1081 genes under simulated microgravity conditions. Subsequent analysis identified specific sequence characteristics, including GC content and coding sequence length, as influential factors on gene TE. The abundance of upstream open reading frames (uORFs) within gene promoters indicates a possible influence on the translation efficiency of primary open reading frames located downstream. These results suggested that in response to simulated microgravity, plants had a responsive translational mechanism that interacted with transcription in a coordinated manner.

Biological sciences/Plant sciences/Tropism

Biological sciences/Plant sciences/Plant molecular biology

Arabidopsis

simulated microgravity

ribosome profiling

translational efficiency

RNA-seq

upstream ORFs.

The gravitational field provides a constant mechanical stimulus to plants on Earth, a phenomenon that has existed since the beginning of life on the planet.¹ The evolution of plants has been greatly influenced by this constant force. They have shaped their developmental patterns and optimized their ability to obtain light, water, and mineral salts by adapting to a level of gravity at 1 × g.² Any changes to this essential parameter would significantly alter the physiological systems of plants.³ Understanding these changes is critical to improving our fundamental understanding of plant physiology and advances in the field of plant space biology.⁴ Gravity is the primary factor that distinguishes the environment of Earth from that of other celestial bodies. Plants are crucial for sustaining life on Earth as they facilitate the absorption of CO₂ and the release of O₂ and water vapor, in addition to serving as a vital food source.⁵ Consequently, their importance in bioregenerative life support systems for extended human space missions cannot be overstated.⁵

The scarcity, high cost, and time constraints of studying plant growth in microgravity are limiting factors for research. Microgravity plant biology research is made possible by space flights to International Space Station (ISS)⁶ and Chinese Space Station (CSS)⁷. However, the limited number of flights available restricts both research opportunities in low Earth orbit and access to the CSS or ISS. As a result, ground-based microgravity facilities play a critical role in the preparation of spaceflight experiments and independent studies. These ground-based facilities serve as cost-effective platforms for gravitational research and several have been established to provide simulated microgravity conditions for research purposes. Consequently, to overcome limitations, experiments can be conducted on Earth using a 3-D clinostat to simulate microgravity conditions by compensating for the unilateral influence of gravity.⁸ The creation of a simulated microgravity environment can be accomplished by subjecting a biological object to 3-D rotation within a Random Positioning Machine.^{9, 10} This study endeavored to use the 3-D clinostat technique to examine the translational effects of simulated microgravity on the growth of Arabidopsis seedling roots.

Ribosome profiling, a technique enabling the precise measurement of protein synthesis at the level of individual nucleotides through extensive sequencing of ribosome-protected mRNA fragments (RPFs), has rapidly emerged as a significant methodological advancement ^{11, 12}. This approach has been used to investigate translational dynamics across a range of organisms, including yeast, fruit flies, zebrafish, mice, and humans ^11–15. The method has also been used in Arabidopsis research for the analysis of photomorphogenesis in plants. ¹⁶ The article delved into the simulated microgravity translational response in Arabidopsis, as well as a genome-wide ribosome profiling analysis to investigate the translation processing of Arabidopsis seedling roots. The ribosome profiling data revealed a variety of mechanisms implicated in translation, demonstrating the diverse tactics used by plants to modulate gene expression in reaction to stimuli such as light, heat, hydrogen peroxide, and hypoxia.^{16, 17} These mechanisms regulated ribosome occupancy to either promote or inhibit translation. Occasionally, frameshifts or stop codon readthroughs may occur. Additionally, research has shown that open reading frames upstream (uORFs) could have a significant impact.^{16, 18}

Here, we conducted an analysis of ribosome profiling and RNA-seq data obtained from 6-day-old Arabidopsis seedlings exposed to simulated microgravity conditions. Our findings revealed a moderate association between alterations in gene expression at the transcriptional and translational levels in response to the simulated microgravity environment. Additionally, less than one-third of genes displaying differential expression were common to both levels, with a notable proportion of genes demonstrating obviously modified translational efficiencies (TEs) in response to simulated microgravity. The TE of several genes exhibited a two-fold difference and was significantly impacted by attributes in their sequences. Furthermore, a notable quantity of uORFs was identified as possessing translational capacity. These uORFs possessed the capability to impede the downstream primary ORFs translation. The study illustrated that plants possessed the capacity to react to simulated microgravity by alterations in dynamic translational that functioned independently and in coordination with transcriptional changes.

Characteristics of ribosome profiling analyses

To examine the molecular reaction to simulated microgravity, ribosome profiling and RNA-seq analyses were conducted on Arabidopsis seedling roots subjected to normal growth conditions and simulated microgravity. Three biological replicates were used for each data set, yielding a combined total of approximately 482 to 579 million reads. Ribosome profiling reads were acquired for both normal growth and simulated microgravity seedlings, as outlined in Table S1. Furthermore, RNA-seq reads were obtained for normal growth and simulated microgravity seedling roots, as detailed in Table S2.

Transfer RNA (tRNA) recognizes codons on mRNA and transfers the corresponding amino acids during translation. It plays a crucial role in determining protein synthesis and translation speed. The concentration of tRNA have a significant impact on these processes. To avoid the precipitation of tRNA along with the ribosome-nascent peptide chain complex during the precipitation process, we compared the sample data to GenBank and Rfam databases after removing ribosomal RNA using BLAST. The ribosome profiling raw reads underwent initial processing to remove rRNA sequences. Subsequently, samples were aligned to TAIR10 Arabidopsis reference genome, as described in the experimental procedures. Strong correlations for both ribosome profiling and RNA-seq data were identified by principal component analysis (PCA) between three biological replicates, as depicted in Figure S1.

Ribosome profiling in various organisms exhibits common characteristics related to the translation process. These characteristics can be considered indicative of effective ribosome profiling libraries. ¹²To assess the impact of simulated microgravity stress, we conducted a comparison of ribosome profiling data between normal growth and simulated microgravity seedling roots. Our analysis revealed that the length of RPFs was about 32 nucleotides in both samples, a length consistent with findings in mammalian systems and rice (Figs. 2a and S2a) ^{11, 16}. The observed read length was longer than the 28 nucleotides reported in yeast ^{11, 12}. The distribution of read density by ribosomes showed an advancement of three nucleotides at a time during translation, leading to the characteristic triplet periodicity ¹². Strong evidence of a three-nucleotide periodicity was identified when analyzing sequences in normal growth and simulated microgravity seedling roots (Figs. 2b and S2b). Meta-gene analysis revealed distinct peaks of read density surrounding the start and stop codons (Fig. 2c), indicating that translation initiation and termination processes may serve as the limiting factors for translation in Arabidopsis seedlings. Additionally, an elevated level of normalized read density was detected close to the start of the coding sequence (CDS), extending up to around 200 nucleotides. Tuller et al. (2010) reported that rare codons slowed-down the start of translation at the start of the CDS, so the next part of the sequence could be translated more quickly and efficiently.¹⁹

Read density distributions within the first 200 nucleotides of the CDS were found to be similar between normal growth and under simulated microgravity conditions in seedling roots (Fig. 2c). The findings with yeast under hydrogen peroxide stress differed from this. Stress led to more ribosomes at the start of translation. Additionally, small ORFs have been identified in the untranslated regions of various species, including yeast, mammals, and plants ^{20, 21}. In the seedlings under normal growth, 1.9% and 1.3% of RPFs were observed in the 3’-UTR and 5’-UTR, respectively, whereas 96.8% of RPFs were located in the gene body (Fig. 2d). Under simulated microgravity conditions, the proportion of reads mapped to the gene body increased to 97.6%, whereas in the 3’-UTR and 5’-UTR decreased to 1.4% and 1.0%, respectively (Fig. 2d). The alterations observed in the ribosome-protected footprints (RPFs) mapping to the 5’- and 3’-untranslated regions (UTRs) indicate the potential for translation modifications in these regions, consistent with previous findings in Arabidopsis.

Simulated microgravity affects gene expression both transcriptionally and translationally.

It is reported that simulated microgravity affects plant growth and development.^{10, 22, 23} By the conduct of ribosome profiling coupled with RNA-seq methodologies, we analyzed the impact of simulated microgravity on seedling roots cultivated under normal growth and simulated microgravity conditions.

Our initial study focused on delineating the alterations in gene expression induced by simulated microgravity. At the transcriptional level, we found 360 genes up-regulated and 71 down-regulated, whereas, at the translational level, 331 genes were up-regulated and 49 were down-regulated (Fig. 3a). The genes with up-regulation expression level surpassed down-regulation at both translational and transcriptional levels, suggesting a widespread enhancement in gene expression. Polysome profiling was used to validate translational promotion, as depicted in Fig. 3b. In comparison to the control group of seedlings under normal growth conditions, the polysome fraction in seedlings subjected to simulated microgravity was up-regulated from 49.5–56.1%, whereas the monosome fraction was down-regulated from 32.1–25.3%. These findings unequivocally illustrated a global up-regulation of translation in simulated microgravity-exposed seedling roots (Fig. 3b).

Subsequently, a functional analysis was conducted on the differentially expressed genes (DEGs) through Gene Ontology (GO) analysis. A significant overlap in enriched pathways was found at both the transcriptional and translational levels. Specifically, pathways related to response to stimuli, response to stress, wounding, defense response, hormone signaling pathway, and response to oxygen levels, etc. (Figure S3). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that genes were enriched at both transcriptional and translational levels in photosynthesis, hormone signaling pathway, plant-pathology interaction, nitrogen metabolism, and biosynthesis of secondary metabolites, etc (Figure S4).²⁴

It is noteworthy that a considerable proportion of genes within enriched pathways displayed disparate responses at the two levels. As illustrated in Fig. 3c, only under one third of responsive genes (31.6% up-regulated and 20.8% down-regulated) were found to be shared between the two processes (Fig. 3c), which suggested discordant changes at the two levels. To assess the collective pattern at the two distinct levels concurrently, the fold-change of gene expression measured by FPKM (fragments per kilobase of transcript per million mapped reads) was computed. The findings indicated a moderate correlation between altered expression at the transcriptional and translational levels (Fig. 4). Subsequently, genes were categorized into nine groups based on the FPKM fold change. Subsequent analysis revealed that about 40% of the responsive genes fell into the (classes C and G) concordant groups, which exhibited coordinated regulation either increasing or decreasing to a comparable degree at both levels.

Approximately 60% of responsive genes were found within the other six discordantly groups. Further analysis was conducted on genes within four discordantly regulated groups. The classes D and F genes showed regulation without significant changes in translation. In class D, transcriptionally down-regulated genes were enriched in plant hormone signal transduction and MAPK signaling pathway. Additionally, in class F, 244 up-regulated genes were observed to be enriched in nitrogen and carbon metabolism at transcriptional level. Genes within classes B and H exhibited translational regulation without discernible alterations at the expression level. Specifically, there were 167 up-regulated genes at the translational level in class B, associated with pathways related to biosynthesis of secondary metabolites and plant-pathogen interaction. Class H, on the other hand, featured 66 genes with reduced translation, showing significant enrichment for sugar metabolism and plant hormone signal transduction.

The influence of simulated microgravity stimuli resulted in notable changes in the translational efficiencies of numerous genes.

Translational efficiency (TE) is a metric that directly assesses the effectiveness of RNA utilization and is a significant factor in the process of translation ¹². Significantly, defense response and photosynthesis have been recognized as prominently enriched pathways in developing Arabidopsis seedlings under a microgravity environment, demonstrating altered at the mRNA levels ²⁵. This changed mRNA level may be related to the altered TE of these pathway genes (Fig. 5a).

We aimed to investigate the likely mechanism of plants response to simulated microgravity through alterations in TE. Despite observing a high degree of similarity in global mRNA abundance and TE among simulated microgravity seedlings, our analysis revealed 552 genes with decreased TE and 529 genes with increased TE (z-score > 2) following simulated microgravity treatment (Fig. 5a). These findings suggested that modulating gene TE may be an effective mechanism in the response of plants to simulated microgravity stimuli. To further evaluate this phenomenon, we conducted a detailed analysis of the data.

Plants demonstrate a wide range of responses to stress. To better understand changes in TE, we analyzed the distribution of genes showing decreased and increased TE across nine predetermined responsive categories. Our findings indicated that modifications in transcriptional and translational abundance influence changes in TE, as illustrated in Figure S5. Nevertheless, a majority of the genes displaying significantly altered TE were found to be regulated exclusively at either the transcriptional or translational level. For example, genes belonging to Groups F and H were solely regulated at one of these levels. Collectively, these groups account for major of genes exhibiting a notable increase in TE, as indicated by a z-score above 2. Moreover, genes within groups B, D, and E, which are subject to both transcriptional and translational regulation, may experience a substantial reduction in TE (Figure S5).

The genes translational efficiencies are influenced by their specific sequence characteristics.

Gene sequence features influenced mRNAs associated with polysomes.²⁶ The potential impact of typical characteristics (sequence length, normalized minimal free energy (NMFE), and GC content) on TE was evaluated. Our analysis revealed that genes with higher TE tended to have higher GC content, NMFE, and shorter lengths as shown in Fig. 6a-c in CDS region. Conversely, differences in these characteristics between genes with higher and lower TE in 3’-UTR were minimal. Specifically, only genes in the highest TE group exhibited shorter length and lower NMFE in the 3’-UTR compared to other groups, as depicted in Figures S6. Interestingly, for 5’-UTR, genes with higher TE exhibited a higher NMFE, shorter length, and lower GC content (Figure S6). The trends observed in the normal growth seedlings were similar to those observed in seedlings subjected to simulated microgravity (Figure S7).

Subsequently, an examination was conducted to determine whether genes exhibiting concordant and discordant expression patterns displayed differences in their sequence characteristics. The Kolmogorov-Smirnov test results demonstrated that genes within the concordant group, located in the overlapping regions of Fig. 3d, exhibited a higher GC content and lower NMFE in CDSs compared to genes within the discordant group (Fig. 6d, e).

Widespread upstream open reading frames

The prevalence of uORFs was found to decrease under simulated microgravity (Fig. 2d). This phenomenon mirrored previous findings in yeast subjected to starvation ^{12, 27}, suggesting a potential role of uORFs in this stress response. These observations highlighted the significance of elements within the 5’-UTR in modulating cellular responses to stress, particularly through the regulation of uORFs. It is widely recognized that uORFs, which are small ORFs situated in the 5’-UTR, can inhibit the translation of main ORFs by reinitiation and leaky scanning. uORFs have been reported in various species ²¹. This study facilitated the comprehensive identification of uORFs in Arabidopsis seedling roots at a genome-wide level. To identify genes containing uORFs accurately, our study focused on all Arabidopsis genes. We revealed that approximately 20% of these genes, totaling 4023 genes, were predicted to contain uORFs. The figure S8 indicates the length distribution of all predicted uORFs, statistics for the number of predicted uORFs per gene, and the length distribution of translated uORFs in the normal growth and simulated microgravity seedling roots.

The translation of main ORFs was repressed by the translated upstream ORF.

To demonstrate the distinctions between untranslated and translated uORFs, three parameters related to the mORF reinitiation (including uORF length, 5’-UTR length, and NMFE) were compared ²¹. Our results suggested that translated uORFs exhibited greater length and higher folding potential compared to untranslated uORFs (Fig. 7a). Conversely, the shorter 5'-UTR genes more likely contribute to translated uORFs (Fig. 7b). Upon further examination of the impact of 5’-UTR length, it was observed that the distance from the uORF to the transcription start site (TSS) in translated uORFs were obviously shorter and the distance from the uORF to the start codon of the mORFs in translated uORFs were obviously longer (Fig. 7c-e).

The Kozak consensus sequence, denoted as "GCCA/GCCAUGG," is a conserved motif found in the mRNA of certain eukaryotes ²⁸. ORFs containing Kozak sequences exhibit a greater likelihood of translation and increased translation efficiency. Statistical analysis should be conducted on all small open reading frames (sORFs) that contain Kozak sequence features. Motif analysis was conducted in the vicinity of the start codon for all full-length gene sequences. The sequences flanking the start codon in the genes exhibited a high degree of similarity to the Kozak sequence. Higher GC content was observed in the translated uORFs flanking sequence, as depicted in Figs. 7f.

To examine likely impact of translated uORFs on the downstream mORFs translation, we analyzed three groups TE: those predicted uORFs lacking translation, those with a single translated uORF, and those with multiple translated uORFs. Translated uORFs have been shown to significantly reduce the TE of corresponding genes, with this effect becoming more pronounced as the number of translated uORFs increases (Fig. 7g). Previous studies have indicated that uORFs can be influenced by sugar, light, and pathogenic infections ^{16, 29}. Consequently, we performed an examination of the translation efficiency (TE) of upstream open reading frames (uORFs) in seedlings cultivated under simulated microgravity conditions. Our findings indicated that the TE of uORFs exhibited comparable levels in both normal growth and simulated microgravity environments (Fig. 7h).

The combined effects of transcriptional and translational responses to simulated microgravity

Through the acquisition of ribosomal profiles and RNA-seq data, our study enabled the examination of global gene expression alterations at the translational level following exposure to simulated microgravity and investigated the interplay between translational and transcriptional responses. Our findings revealed correlation among the various changes in transcriptional and translational gene expression induced by simulated microgravity.

In the study, we observed that more than two third of the simulated microgravity response genes exhibited inconsistent changes in their transcription or translation. This observation suggested independent simulated microgravity responses occurring at the two levels. Specifically, hundreds of genes were found to be regulated solely at the translational level. This translational response, which does not necessitate the synthesis of new mRNA, is recognized for its rapid environmental adaptation capabilities ^{30, 31}. While translation is viewed traditionally as having a fine-tuning function ²⁷, our findings highlighted the significance of the independent role it plays in the simulated microgravity response. In contrast, a total of 418 class D and F genes were identified as being exclusively regulated at the transcriptional level, without observable alterations in translation. These genes played crucial roles in fundamental physiological processes. In a study involving yeast treated with sodium chloride, it was observed that mRNA levels at a specific time point exhibited a stronger correlation with protein levels at a subsequent time point, suggesting a delay in protein expression following the transcriptional activation of stress response genes ³².

Hence, we proposed that alterations in mRNA levels of class D and F genes may serve as predictive markers for subsequent changes in translation (Fig. 4). Among these genes analyzed, only an elevation in class F transcription was noted, suggesting that plants may modulate mRNA abundance to offset reduced TE and sustain essential cellular functions. An alternative interpretation is that heightened mRNA levels following stress relief could provide a reservoir for expedited translation in the future ³³.

Genes sequence characteristics can influence translation efficiency.

In addition to encoding protein functions, genome sequences also harbor a significant amount of information related to expression regulation. Our results revealed that genes exhibiting varying TEs exhibited distinct sequence characteristics, particularly within the CDS of normal growth and simulated microgravity seedling roots (Fig. 6). Furthermore, these sequence attributes were linked to divergent gene expression profiles under simulated microgravity conditions (Fig. 6b and c). The 5’-UTR uORF is a well-documented cis-regulatory element known for its translation inhibitory function ²⁹. Here, we identified that uORFs in Arabidopsis significantly reduced the TE of mORFs. Additionally, the internal ribosome entry site located in the 5′-UTR was another regulatory element that facilitated gene translation under simulated microgravity conditions through a cap-independent mechanism, as demonstrated in previous research ³⁴.

Furthermore, it was discovered that the translation of multiple Arabidopsis genes in the 5’-UTR containing the TAGGTTT motif was augmented following exposure to light¹⁶. Experimental findings also indicate that these motifs can react to light by upregulating downstream gene expression. Additionally, codon usage, poly(A) tail length, and potential secondary structures have been implicated in translational responses ¹⁹. The underlying molecular mechanisms remain to be discovered.

Various translation repression mechanisms are mediated by upstream ORFs.

Previous research has indicated that multiple uORFs in mammals had a greater inhibitory effect on protein accumulation compared to transcripts with one or no uORFs, as demonstrated by Calvo et al. (2009).³⁵ The translation repression mechanisms of uORFs in plants and mammals appeared to share similarities and can be categorized into two distinct groups: one involved the competition between uORFs and downstream mORFs for translation initiation complexes, and the other involved the interference with the restart of mORF translation. The initiation codon of uORFs competes vigorously with the initiation codon of mORFs for translation initiation complexes, and accurate recognition of the uORF initiation codon is essential for uORF-mediated inhibition ²¹. Our findings indicated that the ATG flanking sequence was highly conserved with the Kozak sequence in mORFs (Fig. 7f). In contrast, the conservation of the ATG flanking sequence and the Kozak sequence in translated uORFs was found to be low, and the sequence conservation of the untranslated uORF was the lowest (Fig. 7f).

Hence, the initiation codon of uORFs can either be disregarded or acknowledged, potentially resulting in scan leakage and translation of mORFs. Conversely, the start codon of uORFs recognition can impede the translation elongation and termination, hindering the resumption of mORFs ³⁵. The uORFs characterized by greater length and shorter proximity are deemed disadvantageous for the initiation of mORF translation, thus suggesting that inhibition may be more efficacious ²¹.

In this study, it was observed that the translated uORF exhibited a shorter distance from the CDS compared to the untranslated uORF, as depicted in Fig. 7a-e. The findings supported the view that the length and sequence background of the uORF were factors influencing its function in plants. Specifically, research conducted by Chang et al. (2000) demonstrated that the seven peptides encoded by uORFs, known as gDcADC8, found in carnations, possessed the ability to inhibit the translation of mORFs. Additionally, recent findings by Ebina et al. (2015) have revealed that frameshift mutants containing conserved peptide uORFs have been shown to inhibit mORF translation in Arabidopsis.³⁶ Furthermore, studies have indicated that peptides derived from uORFs function in a cis-acting manner to suppress the translation of mORFs. The detailed underlying molecular mechanisms remain to be discovered in plants in the future.

Plant growth conditions and treatment

Arabidopsis (Col-0) was grown in soil at 22°C under a 16-hour light/8-hour dark cycle with approximately 60% humidity. Plants grown in both normal conditions and under simulated microgravity were incubated under identical light intensity and humidity for six days. The Arabidopsis seedling root tips were then harvested and stored at − 80°C.

Recovery of ribosomal footprints

The ribosomal profiling technique was conducted following the methodology outlined in a previous study¹¹, with some adjustments. To generate ribosomal footprints (RFs), 10 µL of RNase I (NEB, Ipswich, MA, USA) and 6 µL of DNase I (NEB) were added to 400 µL of lysate, which was then incubated for 45 min at room temperature with gentle mixing on a Nutator mixer. The nuclease digestion was halted by the addition of 10 µL of SUPERase·In RNase inhibitor (Ambion, Austin, TX, USA). Following this, 100 µL of the digested RFs were introduced to the column and centrifuged at 600 × g for 2 min. Subsequently, 10 µL of 10% (wt/vol) SDS was incorporated into the elution, and RFs with a size exceeding 17 nt were isolated by using an RNA Clean and Concentrator-25 kit (Zymo Research; R1017) ³⁷. Subsequently, RNase H and DNase I enzymes were used to degrade the rRNA and any remaining DNA probes. The RFs were then subjected to additional purification using magnetic beads from Vazyme (Nangjing, China).

Library construction and sequencing

Following the acquisition of RFs, ribo-seq libraries were constructed using NEBNext® Multiple Small RNA Library Prep Set for Illumina® (catalog no. E7300S, E7300L; New England Biolabs, Ipswich, MA, USA). This involved the addition of adapters to both ends of the RFs, followed by reverse transcription and polymerase chain reaction (PCR) amplification. The 140–160 bp size PCR products were subjected to an enrichment process to generate a cDNA library, which was then sequenced using the Illumina HiSeq X10 platform by Gene Denovo Biotechnology Co. (Guangzhou, China).

Ribo-seq data analysis

Reads filtering

Reads were subjected to quality filtering by removing those with over 50% low-quality bases or over 10% N bases. Additionally, adapter sequences were trimmed, and reads falling within the 20–40 bp range were retained for further analysis. Subsequently, short reads were aligned to the ribosome RNA (rRNA) database, GenBank, and Rfam database using Bowtie2. Reads that aligned to rRNAs, tRNAs, snRNAs, snoRNAs, and miRNAs were subsequently excluded from the dataset.

Reference genome alignment

Reads were aligned to the genome using STAR with the 2-pass setting enabled, followed by the exclusion of reads mapping to noncoding RNAs. The remaining reads were then categorized into different genomic features based on their alignment position.

Three-nucleotide periodicity analysis

Three-nucleotide periodicity analysis was conducted using the riboWaltz R package.³⁸ Reads with a specific length (e.g., 30 nt) were assigned to frames 1, 2, or 3 based on their position in the 5′ ends in transcripts of protein-coding genes. Subsequently, the proportion of total reading frames in frames 1, 2, and 3 was calculated across all protein-coding genes.

Sample relationship analysis

Principal component analysis (PCA)

PCA was used to evaluate the reliability of the experimental outcomes by calculating the correlation coefficient between three replicates. The PCA was conducted with the R package g models to show how the samples are related. To identify genes that are translated differently in different samples, we used DESeq2 and edgeR. Genes with a fold-change of at least 2 and a false discovery rate (FDR) of less than 0.05 were considered significant DTGs. The DTGs were then analyzed for functions and pathways.

Gene Ontology (GO) enrichment

The GO system serves as a framework for categorizing gene functions, providing a standardized vocabulary and conceptual framework for characterizing the properties of genes and proteins across various organisms³⁹. Comprising three main components, molecular function, cellular component, and biological process, GO terms represent the fundamental units of the ontology. Initially, all DTGs were associated with corresponding GO terms within the GO database. GO terms that satisfied this criterion were considered significantly enriched in DTGs, providing insight into the primary biological functions of these genes.

Pathway enrichment analysis

Genes collaborate to fulfill various functions in biological processes. Pathway analysis is instrumental in elucidating the mechanisms by which genes operate. The KEGG serves as the primary public repository for pathways.²⁴ The significance of these findings was assessed by adjusting the p-value for FDR using a threshold of 0.05.

Gene set enrichment analysis

The software tools Gene Set Enrichment Analysis (GSEA) ⁴⁰and MSigDB were used to assess the differential expression of a set of genes within specific GO terms or pathways between two groups.

Identification of ORFs

The longest transcript from each protein-coding gene was identified and subsequently analyzed for ORFs within noncoding regions, specifically the 5'- and 3'-UTRs of established protein-coding genes. Non-canonical ORFs, ranging in size from 60 to 450 nucleotides and initiated by the start codon ATG, were extracted, encompassing both uORFs and downstream ORFs (dORFs).

Translation quantification for ORF

The reads aligned to classical noncoding RNAs were excluded from the analysis. Subsequently, the remaining RFs were used to assess the TE of all ORFs, encompassing mORF of protein-coding genes, uORFs, and dORFs. The read counts within the ORFs were tallied, and the TE was normalized using the FPKM method.

Differentially translated ORF analysis

The DESeq2 package was used to detect differentially transcribed ORFs among various sample groups. ORFs exhibiting a fold-change of ≥ 2 and an FDR of < 0.05 in a given comparison were deemed to be statistically significant.

Evaluating the coding potential of non-canonical ORFs

Two methods, including Ribosome Release Score (RRS) and ORFscore, were used to assess the coding potential of non-canonical ORFs. ORFscore was used to evaluate the coding potential of non-canonical ORFs as documented previously.⁴¹ The calculation of RRS followed the formula reported in prior studies.⁴² We additionally used the Fickett score and hexamer score from the CPAT tool ⁴³ to assess the coding potential of different ORFs.

Sequence feature analysis

The lengths and GC content of both translated and untranslated ORFs were quantified, followed by the determination of secondary structure stability using the NMFE algorithm. This stability metric was computed through ViennaRNA RNAfold and subsequently normalized.⁴⁴ P-values were derived from a student’s t-test comparing translated and untranslated ORFs. The presence of the Kozak consensus sequence, GCCA/GCCAUGG, surrounding the start codon was found to be crucial for start codon recognition and translation initiation.^{45, 46} Motif analysis around start codons of all ORFs was conducted using MEME, with significance assessed using a chi-squared test.

The influence of uORFs on mORFs

The potential impact of uORF sequence and expression on mORF translation was investigated. Scatter plots were generated to analyze the differences between uORFs and downstream mORFs in each comparison group (with FPKM ≥ 1 in at least one sample). Additionally, the influence of the Kozak sequence and the distance between uORFs and mORFs on the translation of mORFs was examined.

Annotation of small ORFs

Small ORFs (sORFs, average FPKM ≥ 1) were annotated with sORF databases (PsORF ⁴⁷ and SORFS.ORG ^{48, 49}) to identify their potential for translation. The on-canonical ORFs were also analyzed with the Pfam database to predict potential domains.

Competing Interests

The authors declare no competing interests.

Data availability

The data presented in this manuscript is accessible and can be acquired by contacting Peipei Xu.

Acknowledgments

We thank Professor WR Hu for organizing the Strategic Priority Research Program of CAS, CSU of CAS for organizing the China’s Manned Space Program. This work was supported by the National Natural Science Foundation of China (Grant No. 32171232, No. 31570859, No. 31500236, No. 31070237, No. 90917009 and No.31971172), Natural Science Foundation of Shanghai (Grant No. 22ZR1469500), CAS (No. XDA04020202-15) and the China's Manned Space Program.

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Genome-wide ribosome profiling reveals dynamic translational landscape in Arabidopsis seedling roots under simulated microgravity

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Abstract

Figures

Introduction

Results

Characteristics of ribosome profiling analyses

Widespread upstream open reading frames

Discussion

The combined effects of transcriptional and translational responses to simulated microgravity

Materials and methods

Plant growth conditions and treatment

Recovery of ribosomal footprints

Library construction and sequencing

Ribo-seq data analysis

Reads filtering

Three-nucleotide periodicity analysis

Sample relationship analysis

Principal component analysis (PCA)

Gene Ontology (GO) enrichment

Pathway enrichment analysis

Gene set enrichment analysis

Identification of ORFs

Translation quantification for ORF

Differentially translated ORF analysis

Evaluating the coding potential of non-canonical ORFs

Sequence feature analysis

The influence of uORFs on mORFs

Annotation of small ORFs

Declarations

References

Additional Declarations

Supplementary Files

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