Information

6.9: Ribozymes - RNA Enzymes - Biology

6.9:  Ribozymes - RNA Enzymes - Biology


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Ribozymes

Any molecule that displays any of the catalytic motifs seen in the earlier chapters (general acid/base catalysis, electrostatic catalysis, nucleophilic catalysis, intramolecular catalysis, transition state stabilization) can be a catalyst. These can fold to form unique 3D structures which can have active sites with appropriate functional groups or nonprotein "cofactors" (metal ions, vitamin derivatives) that participate in catalysis. It is now known that RNA, which can form complicated secondary and tertiary structures as seen in the 3D image of the ribozyme from Tetrahymena thermophila, can as well.

Figure: ribozyme from Tetrahymena thermophila,

RNA molecules that act as enzymes are called ribozymes. This property of some RNA's was discovered by Sidney Altman and Thomas Czech, who were awarded the Nobel Prize in Chemistry in 1989. In contrast to protein enzymes which are true catalysts in that they are used over again, this is an example of a single use ribozyme. Other ribozymes are true catalysts and can carry out RNA slicing by transesterification (splicesome) and peptidyl transfer (in ribosomes). The mechanisms of catalysis of the hepatitis delta virus ribozyme include general acid/base catalysis.

Figure: mechanisms of catalysis

The hairpin ribozyme from satellite RNAs of plant viruses is 50 nucleotides long, and can cleave itself internally, or , in a truncated form, can cleave other RNA strands in a transesterification reaction. The structure consists of two domains, stem A required for binding (self or other RNA molecules) and stem B, required for catalysis. Self-cleavage in the hairpin ribozyme occurs in stem A between an A and G bases (which are splayed apart) when the 2' OH on the A attacks the phosphorous in the phosphodiester bond connecting A and G to form an pentavalent intermediate.

Figure: Self-cleavage in the hairpin ribozyme

Rupert & Ferr�-D'Amar� (2001) solved the crystal structure of a hairpin ribozyme with a non-cleavable substrate analog containing a in which a 2'-OCH3 was substituted for the nucleophilic 2'-OH group. See the applet below.

A recent study by Rupert et al. (2002) shows that A38 in Stem B appears to be able to interact with the products (the cleaved A now in the form of a cyclic phosphodiester with itself) and the departing G, and also with a transition state pentavalent analog of the sessile A-G bond in which the phosphodiester linking A and G in the substrate is replaced with a pentavalent vanadate bridge between A and G. However, A 38 does not appear to react with the sessile A -G groups in the normal substrate, indicating that the main mechanism used by this ribozyme is transition state binding. Since RNA molecules have fewer groups available for acid/base and electrostatic catalysis (compared to protein enzymes), ribozymes, presumably the earliest type of biological catalyst, probably make more use of transition state binding as their predominant mode of catalytic activity.

Figure: Active Site of Hairpin Ribozyme: Transition State Binding

Recently, the crystal structure of a purple bacterium group I self-splicing intron (which catalyze the removal of itself) interacting with both exons in a state prior to their ligation was determined (Adams, P. et al.). The structure shows both exons in close proximity. Nucleophilic attack of the 3'OH of the 5' exon on a distorted phosphate at the intron-3'-exon junction. Two metal ions reside on either side of the labile phosphodiester bond at the intron-3'exon junction, and are held in place by 6 phosphates.

A novel use of ribozymes was recently reported by Winkler et. al. They discovered that the 3' end of the mRNA of the gene glmS (from Gram-positive bacteria) which encodes an amidotransferase (catalyzing the formation of glucosamine-6-phosphate from glutamine and fructose-6-phosphate) is a ribozyme. A glucosamine-6-phosphate binding site in the ribozyme (3' end of the mRNA) binds this sugar, inducing autocleavage of the ribozyme. This inhibits, by an uncertain mechanism, the formation of the amidotransferase from the remaining part of the mRNA. This mechanism of regulation of gene expression through ribozyme activity might prove to be common.

In order for RNA molecules to have acted as both catalysts and carriers of genetic information in the original RNA world, RNA must have the potential to self replicate. Shechner et al. looked at the core structure of a class I ligase ribozyme able to polymerize RNA. This ligase was synthesized and enhanced through The tripod structure allows more RNA solvent interaction then often found in ribozymes. Several common structural motifs were also found to be present including the GNRA-triloop, uridine-turn and the frameshifting pseudoknot motifs. GNRA-triloop and uridine-turn motifs are short thermodynamically stable segments that cap the end of the helical regions. Varieties of these are found in many RNA structures although they don’t seem to be necessary for activity. The frameshifting pseudoknot motif is almost identical in structure to small viral ribosomal frameshifting pseudoknots and is adjacent to a new motif named “A-minor triad”. The A-minor triad is responsible for the coordination with Mg2+, although only when inserted in-between certain sequences. Triphosphoguanosine (called G1) at the 5’ terminus of the ribozyme acts as the electrophile for the RNA ligation reaction. Other segments of the ribozyme, such as the J1/2, have highly specific contacts which orient G1 and allow the RNA to fold more efficiently. Two residues, cytosine 12 (C12) and uracil 48 (U48) bind to Mg2+. Other sections of the RNA also promote specificity to assist in the replication process. A model for catalysis and the transition state of the ribozyme polymerase is similar to that for protein RNA polymerases, as shown in the figure below

Figure: Comparison of Transition State Models of Ribozyme and Protein RNA Polymerases (after Schechner et al)

A divalent Mg2+ in the active site of the ribozyme enhances the nuclophilicity of the 3-OH on the primer, which attached the the terminal phosphate of the G(1)TP substrate to form a pentavalent intermediate. The Mg cation is stabilized by oxygens on P 29 and 30 of the ribozyme. The Mg ion also stabilizes the developing charge in the transition state and in the charge in the intermediate. Stabilization of analogous divalent cations in the protein polymerase occurs through Asp side changes in the protein.

  • Hairpin Ribozyme Add 1HP6.pdb

Jmol: Updated Self-splicing Group I intron with both exons Jmol14 (Java) | JSMol (HTML5)

Jmol: Updated L1 Ligase Ribozyme Jmol14 (Java) | JSMol (HTML5) Not done; Fix

  • 3D Structure of Ribozyme with Active Site - From the Howard Hughes Medical Institute.

The Biggest Ribozyme - The Ribosome

Protein synthesis from a mRNA template occurs on a ribosome, a nanomachine composed of proteins and ribosomal RNAs (rRNA). The ribosome is composed of two very large structural units. The smaller unit (termed 30S and 40S in bacteria and eukaryotes, respectively) coordinates the correct base pairing of the triplet codon on the mRNA with another small adapter RNA, transfer or tRNA, that brings a covalently connected amino acid to the site. Peptide bond formation occurs when another tRNA-amino acid molecule binds to an adjacent codon on mRNA. The tRNA has a cloverleaf tertiary structure with some intrastranded H-bonded secondary structure. The last three nucleotides at the 3' end of the tRNA are CpCpA. The amino acid is esterified to the terminal 3'OH of the terminal A by a protein enzyme, aminoacyl-tRNA synthetase.

Covalent amide bond formation between the second amino acid to the first, forming a dipeptide, occurs at the peptidyl transferase center, located on the larger ribosomal subunit (50S and 60S in bacteria and eukaryotes, respectively). The ribosome ratchets down the mRNA so the dipeptide-tRNA is now at the the P or Peptide site, awaiting a new tRNA-amino acid at the A or Amino site. The figure below shows a schematic of the ribosome with bound mRNA on the 30S subunit and tRNAs covalently attached to amino acid (or the growing peptide) at the A and P site, respectively.

Figure: Prokaryotic Ribosme - P and A sites

A likely mechanism (derived from crystal structures with bound substrates and transition state analogs) for the formation of the amide bond between a growing peptide on the P-site tRNA and the amino acid on the A-site tRNA is shown below. Catalysis does not involve any of the ribosomal proteins (not shown) since none is close enough to the peptidyl transferase center to provide amino acids that could participate in general acid/base catalysis, for example. Hence the rRNA must acts as the enzyme (i.e. it is a ribozyme). Initially it was thought that a proximal adenosine with a perturbed pKa could, at physiological pH, be protonated/deprotonated and hence act as a general acid/base in the reaction. However, none was found. The most likely mechanism to stabilize the oxyanion transition state at the electrophilic carbon attack site is precisely located water, which is positioned at the oxyanion hole by H-bonds to uracil 2584 on the rRNA. The cleavage mechanism involves the concerted proton shuffle shown below. In this mechanism, the substrate (Peptide-tRNA) assists its own cleavage in that the 2'OH is in position to initiate the protein shuttle mechanism. (A similar mechanism might occur to facilitate hydrolysis of the fully elongated protein from the P-site tRNA.) Of course all of this requires perfect positioning of the substrates and isn't that what enzymes do best? The main mechanisms for catalysis of peptide bond formation by the ribosome (as a ribozyme) are intramolecular catalysis and transition state stabilization by the appropriately positioned water molecule.

Figure: Mechanism Peptide Bond Formation by the Ribosome

The crystal structure of the eukaryotic ribosome has recently been published (Ben-Shem et al). It is significantly larger (40%) with mass of around 3x106 Daltons. The 40S subunit has one rRNA chain (18) and 33 associated proteins, while the larger 60S subunit has 3 rRNA chains (25S, 5.8S and 5S) and 46 associated proteins. The larger size of the eukaryotic ribosome facilitates more interactions with cellular proteins and greater regulation of cellular events. The Jmol structure of a bacterial 70S ribosome showing mRNA and tRNA interactions is shown below.


New catalytic structures from an existing ribozyme

Although protein enzymes with new catalytic activities can arise from existing scaffolds, less is known about the origin of ribozymes with new activities. Furthermore, mechanisms by which new macromolecular folds arise are not well characterized for either protein or RNA. Here we investigate how readily ribozymes with new catalytic activities and folds can arise from an existing ribozyme scaffold. Using in vitro selection, we isolated 23 distinct kinase ribozymes from a pool of sequence variants of an aminoacylase parent ribozyme. Analysis of these new kinases showed that ribozymes with new folds and biochemical activities can be found within a short mutational distance of a given ribozyme. However, the probability of finding such ribozymes increases considerably as the mutational distance from the parental ribozyme increases, indicating a need to escape the fold of the parent.


The Ribosome Challenge to the RNA World

An RNA World that predated the modern world of polypeptide and polynucleotide is one of the most widely accepted models in origin of life research. In this model, the translation system shepherded the RNA World into the extant biology of DNA, RNA, and protein. Here, we examine the RNA World Hypothesis in the context of increasingly detailed information available about the origins, evolution, functions, and mechanisms of the translation system. We conclude that the translation system presents critical challenges to RNA World Hypotheses. Firstly, a timeline of the RNA World is problematic when the ribosome is incorporated. The mechanism of peptidyl transfer of the ribosome appears distinct from evolved enzymes, signaling origins in a chemical rather than biological milieu. Secondly, we have no evidence that the basic biochemical toolset of life is subject to substantive change by Darwinian evolution, as required for the transition from the RNA world to extant biology. Thirdly, we do not see specific evidence for biological takeover of ribozyme function by protein enzymes. Finally, we can find no basis for preservation of the ribosome as ribozyme or the universality of translation, if it were the case that other information transducing ribozymes, such as ribozyme polymerases, were replaced by protein analogs and erased from the phylogenetic record. We suggest that an updated model of the RNA World should address the current state of knowledge of the translation system.

This is a preview of subscription content, access via your institution.


Results

Confirmation of infection with RSV and RBSDV in rice seedlings

To confirm the infection of rice plants with the two plant viruses, disease development was observed. SBPH-infested rice plants harbouring RBSDV began to exhibit plant growth abnormalities, such as dwarfism and leaf darkening. At 20 days-post-transplantation (dpt), these plants showed more serious developmental abnormalities. At 60 dpt, SBPH-infested rice plants infected with RSV showed leaf yellowing, stripe, chlorosis, and slower plant growth (Fig. 1A). Leaves were collected from these symptomatic rice plants for RT-PCR and western blotting analyses to detect the target viruses (Fig. 1B, C). The specific pairs of primers corresponding to RSV and RBSDV are shown in Additional file 2: Table S1. RT-PCR showed a specific band with the expected size appeared in RSV- and RBSDV-infected rice plants compared with mock-treated plants, respectively. These results and those of western blots using RSV NS3 and RBSDV p10 antisera (Fig. 1B, C) confirmed the independent infection of the plants by the viruses following inoculation.

Flow chart of the investigation of m 6 A methylation during infection of rice by RBSDV or RSV. A Symptoms of RBSDV- and RSV-infected rice plants in plastic buckets at 60 days post infection (dpt). The left plant is a mock-treated plant, the middle two plants are infected with RBSDV, and the plant on the right is infected with RSV. B RT-PCR and western blot (WB) detection of RSV using a specific pair of primers corresponding to RdRp and anti-NS3 specific antiserum. Total proteins were stained with Coomassie brilliant blue (CBB), which was treated as the loading control. C RT-PCR and WB were performed to detect RBSDV in rice using a specific pair of primers corresponding to CP. Anti-p10 specific antiserum was carried out for the WB. D Experimental flow chart of m 6 A-IP-seq and RNA-seq using RBSDV- and RSV-infected plants. NGS, next-generation sequencing. MeRIP, methylated RNA immunoprecipitation

Transcriptome-wide mapping of m 6 A in rice

To obtain a transcriptome m 6 A methylation modification map in rice, the mock-, RSV-, and RBSDV-infected rice samples were used for further analyses. The m 6 A analysis procedures were described, and the samples that were used for input (non-IP control), m 6 A-based RNA Immunoprecipitation (m 6 A-IP), and RNA-seq were clearly indicated (Fig. 1D). A series of IP, input, and mRNA libraries were constructed and sequenced, respectively (Additional file 2: Table S2). Samples of these series libraries came from mock-, RSV-, and RBSDV-infected rice leaves at 60 dpt. Each treatment was performed with two biological replicates. Raw sequencing data were further processed for adaptor and low-quality base removal. The obtained clear reads were aligned to the rice reference genome (Oryza sativa. IRGSP-1.0). Read distribution analysis showed that all the m 6 A-IP samples were highly enriched around the stop codon and within 3′-untranslated regions (3′-UTRs), which are in line with the previous reports in HIV-infected T cells and maize and suggested the m 6 A-IP sequencing data are reliable and with a high authenticity [6, 15] (Additional file 1: Fig. S1). The m 6 A-IP-seq analyses detected more than 26,000 m 6 A peaks in each individual treatment and biological replicate (Fig. 4A, Additional file 1: Fig. S2). For each treatment (one individual virus infection), high-confidence peaks were identified (Additional file 2: Table S3). Briefly, the regions that overlapped in at least one of the two replicates were designated high-confidence m 6 A peak regions. Confident peaks from different experimental conditions were further integrated into a unique m 6 A peak map. Consequently, a total of 26,390, 27,038, and 26,675 unique m 6 A peaks with high confidence (p < 0.05, fold change > 1.5) for mock-, RBSDV-, and RSV-infected samples were detected, respectively (Fig. 4A, Additional file 1: Fig. S2). After comparison with mock treatment, the RBSDV- and RSV-infected samples displayed 8011 and 6603 different regulated peaks, accounting for an average of approximately 1 m 6 A peak within transcription units from each gene. Among these differential m 6 A peaks, there are 3897 and 2900 new peaks appeared upon RBSDV and RSV infection, and 4113, and 3702 common peaks in RBSDV- and RSV-infected sample compared with mock-treated rice, and 1503 differential m 6 A peaks were both appeared in RBSDV- and RSV-infected sample (Additional file 2: Table S4). These results suggested that 48.7% and 43.9% differential m 6 A peaks newly appeared upon RBSDV- and RSV infection of rice, respectively, which also indicated that m 6 A methylation was tightly associated with viruses’ infection of the plant. At the genomic level, these unique m 6 A-methylated peaks for the three treatments were unevenly distributed across each rice chromosome. The common peak density was also mapped. The gene density according to the previously reported data is presented in Fig. 2, and the m 6 A peak distribution density was highly consistent with the corresponding gene density on the same chromosome position in the mock sample.

Circos plots of the m 6 A methylome in rice plants infected with RBSDV or RSV. The six rings from the outside to the inside show the genomic positions (1st), gene density (2nd), peak density of mock-treated rice plants (3rd), peak density of RSV-treated rice plants (4th), peak density of RBSDV-treated rice plants (5th), and the common peak density of RBSDV- and RSV-infected rice plants (6th)

Widespread m 6 A methylation of RSV and RBSDV genomic RNA

The m 6 A-IP experiment was performed twice. The peak calling method used was stringent (false discovery rate < 0.01). The two replications of the next generation sequencing (NGS) data revealed high correlations with the bound RNAs (0.990), which indicated the high replicability of the sequencing results. The clear reads obtained by NGS were also aligned to the reference RBSDV and RSV genomic RNAs, and the m 6 A peaks spanning the full sequences of different segments of viruses were mapped (Fig. 3, Additional file 2: Table S5). In particular, clusters of m 6 A peaks were clearly observed in the 5′ terminal of RBSDV genomic S1, S2, S3, S4, S5, S6, S9, and S10, and some discrete peaks appeared in S4, and S7 (Fig. 3A, red arrows). In RSV-infected sample, the main m 6 A peak clusters were located on the genomic RNA2, RNA3, and RNA4, and compared to the input, several clearly m 6 A peaks were in the RNA1 to RNA4, two m 6 A peaks located to the 3′ terminal of RNA1 (Fig. 3B, red arrows). We also exhibited the fine m 6 A peaks that distributed to the each viral genomic RNAs (Additional file 2: Table S5), and the viral-specific m 6 A peaks that distributed on RBSDV S5, S6, and S9 (Fig. 3C) and RSV RNA1, RNA2, and RNA3 (Fig. 3D) were selected and exhibited. Our results suggested that the m 6 A modification often occurred in the 5′-terminal of the genomic RNAs of RBSDV, while they were random distributed on the RSV genome. These maybe resulted from the characteristic of the two completely different plant viruses (RSV is a single-strand RNA virus, while RBSDV is a double-stranded RNA virus). Taken together, in addition to the mRNA of the host plant, viral mRNAs could also be N 6 -methyladenosine methylated under interactions between virus and rice, and the m 6 A distribution pattern on viral genomic RNA was specific and novel.

Circos plots of the m 6 A methylome in RBSDV and RSV genomic RNAs. A Distribution of m 6 A methylated reads on the ten RBSDV genomic RNAs. Six rings from outside to inside show genomic positions (1st), reads distribution of RBSDV_1_Input (2nd), reads distribution of RBSDV_1_IP (3rd), reads distribution of RBSDV_2_Input (4th), reads distribution of RBSDV_2_IP (5th), and the GC content of the genomic RNA (6th). B Distribution of m 6 A methylated reads on the four RSV genomic RNAs. Six outer rings were indicated similarly to RBSDV above. C m 6 A methylation peaks in the full-length RBSDV segment 5 (upper panel), 6 (middle panel), and 9 (bottom panel). The detail peaks regions and viral gene annotation are shown in the Additional file 2: Table S6. Top numbers show the full length of the analysed RNA segments, and bp is the short name of base-pair. Blue colour marked line shows the m 6 A peak region on viral genome, and number 1 and 2 mean the two replicate of the m 6 A-IP-sequencing. D Distribution of m 6 A peaks on the RSV genomic RNA1 (upper panel), RNA2 (middle panel), and RNA4 (bottom panel). The detail peak regions and viral gene annotation are shown in the Additional file 2: Table S6. Top numbers show the full-length of the analysed genomic RNAs, and the nt means nucleotide. Other marks are similar with Fig. 3C

Activation of rice m 6 A RNA methylation levels upon virus infection

To explore the changes of m 6 A RNA methylation levels in virus-infected rice, the sequencing data were analysed. Collectively, there were 15,977, 16,854, and 16,267 m 6 A methylated genes that corresponding to mock-, RBSDV-, and RSV-infected rice, respectively (Additional file 2: Table S6). The findings clearly indicated that rice m 6 A RNA methylation was enriched under RSV and RBSDV infection. In terms of differential m 6 A peak number, the enriched peaks were also increased in rice infected with viruses (Fig. 4A, Additional file 2: Table S6). Meanwhile, the confidence of the peak significances was determined by calculating the correlations of the peak numbers and the p value (Fig. 4B). The horizontal ordinate dimension (-log10[q-value]) of most of the m 6 A peaks ranged from 4 to 10. The findings indicated that most of the peaks were highly confident and credible. The differential m 6 A peaks were selected and compared to those obtained for mock-treated rice plants. There were 8,010 and 6,602 unique and different m 6 A peaks for RBSDV- and RSV-infected rice plants compared to the mock-treated sample, respectively (Fig. 4C). For the digital exhibition of the m 6 A signal intensity, a violin plot was used to show the fold enrichment distribution (Fig. 4D). The median number of the peak enrichment-fold was approximately 40 and 76 (Additional file 2: Table S7). To explore the confidence of the different peaks deposited in RBSDV- and RSV-infected samples, the significance distribution of different m 6 A peaks was analysed by correlation with the p- value of each peak and peak number (Fig. 4E). The main differential regulated m 6 A peaks were deposited in 3 on the horizontal axis. The finding indicated that the different m 6 A peaks of rice mRNA under viral infection was confident and reliable. Taken together, the m 6 A modification levels of rice mRNAs were enriched under infection by plant viruses.

Activation of rice m 6 A methylation by virus infection. A Histograms show the number of unique peak in mock-treated and RBSDV- and RSV-infected rice plants. The Y-axis represents the peak number, and the X-axis represents the treatments. B Significant distribution analysis of the different peaks in mock-treated and RBSDV- and RSV-infected rice samples. The Y-axis represents the peak number, and the X-axis represents the negative value of the logarithm of the p- value base-10. C Histogram showing the different regulated numbers of m 6 A methylated genes in RBSDV- and RSV-infected rice compared with mock-treated rice samples. D Comparisons of the fold change of the different regulated peaks in RBSDV- and RSV-infected rice plants. The Y-axis represents the logarithmic of peak folded-enrichment base-2. E Significant distribution analysis of the different peaks in RBSDV- and RSV-infected rice samples. The Y-axis represents the numbers of different regulated peaks, and the X-axis represents the negative value of the logarithmic value of the p- value base-10

Association of m 6 A with genes that were not actively expressed in virus-infected rice plants

RNA deep sequencing (input samples) of the 60 dpt rice plants of mock-, RBSDV-, and RSV-infected samples with two biological replicates were performed, and each replicate was used to examine the correlation between m 6 A modification and gene expression in rice. The euclidian distance (ED) of the two replicates’ reads was small, and the square colour of the two was close (Fig. 5A). The findings suggested that the experiments were reproducible between the two replicates. Based on the confident sequencing results, the m 6 A peaks of each treatment were annotated to the genes. Rice genes that could be annotated by the m 6 A peaks were called m 6 A gene, and that couldn’t be annotated were called non-m 6 A gene in the next analyses. According to the Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) (Additional file 2: Table S6), these genes were divided into three groups (FPKM < 1, 1 < FPKM < 5, and FPKM > 5), and the ratio of gene number of each category were calculated and showed by the heatmap (Fig. 5B). Either in the mock-treated sample or the viruses’ infected rice, most of the analysed genes (m 6 A or non-m 6 A genes) were mainly distributed in the low expressed category (FRKM < 1) (Fig. 5B). Compared to the mock-treated sample, the ratio of m 6 A methylated genes were increased in low expressed category of RSV- and RBSDV-infected samples (Fig. 5B). When these genes (m 6 A and non-m 6 A) were divided into highly expressed genes (FRKM ≥ 1) and genes that were not highly expressed (FRKM < 1), the expression of genes that was high or low were showed in mock-, RBSDV-, and RSV-infected samples (Fig. 5C), and we found that most genes were non-methylated and distributed in low expression category in mock-, RBSDV-, and RSV-infected rice samples (Fig. 5C). The m 6 A methylated genes were slightly enriched in the low expression category upon viruses’ infection of rice compared to the mock-treated sample (Fig. 5C). Either in mock-treated or in RSV-infected and RBSDV-infected samples, the m 6 A-methylated genes were faintly expressed at a higher level than those of non-m 6 A methylated genes (Fig. 5D), and the highly expressed genes displayed a lower m 6 A occupancy (Fig. 5B). Hence, the m 6 A methylation mainly occurred in low expressed genes either in mock-treated or in RSV- and RBSDV-infected rice samples and the m 6 A modified gene number were slightly enriched in the low expressed category upon viruses’ infection.

Analyses of the relationship between m 6 A methylation with expression levels of the target gene in rice under plant virus infection. A Euclidian distance (ED) coefficients among gene expression profiles generated by RNA-seq analysis of the two biological replicates of the three treatments. RNA-seq was performed simultaneously with m 6 A-IP-seq with total RNA extracted at 60 dpt. A lower value means a closer ED of the two compared objects, and with higher reproducibility of the two replicates. B The percentage of rice m 6 A methylated and un-methylated genes at a defined FPKM levels (< 1, 1–5, and > 5). Different colour densities indicate different percentages of the corresponding gene. C Comparisons of number of non-m 6 A methylated genes and number of m 6 A methylated genes in their gene bodies with high (FPKM > 1) and low (FPKM > 1) expression levels in the three treatments. Relationships between gene expression and number were tested using the chi-square test. “*” indicate p- value < 0.05, and “**” means p- value < 0.01. D Box plot comparing FPKM expression levels between non-m 6 A methylated genes and m 6 A methylated genes in the three treatments. A two-tailed unpaired Student’s t -test was performed to calculate the p- values of these three treatments

Different regulated m 6 A-methylated genes in virus-infected rice plants

To investigate which pathway-related genes were m 6 A methylated, the obtained different m 6 A peaks were mapped to the rice reference genome. The gene was divided into 5′-UTR, 3′-UTR, first exon region, and exon excluding the 5′-UTR and 3′-UTR in the protein-coding region. The sequenced clear m 6 A data were aligned to these functional elements. We found that 38%, 42%, and 47% of the m 6 A modification sites were located on the 3′-UTR of the genes in RBSDV-infected, RSV-infected, and common RBSDV and RSV peaks, respectively (Fig. 6A). Furthermore, 23%, 23%, and 24% of the m 6 A modification sites were deposited on the 5′-UTR regions of the aforementioned genes (Fig. 6A). These results indicated that most of the m 6 A sites were located in the non-coding region of genes. Gene ontology (GO) analyses were performed to provide insight into the biological functions of different m 6 A modifications in rice. The methylated genes were involved in multiple molecular functions, particularly in binding and catalytic activity (Fig. 6B). Thus, m 6 A modifications may act as epigenetic markers that mediate molecular interactions between rice and plant viruses. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the different m 6 A modification-related genes were performed. The five enriched pathways were carbohydrate metabolism, translation, protein folding/sorting/degradation, amino acid metabolism, and signal transduction (Fig. 6C). these results suggested that m 6 A modification was widely involved in and closely related to the intracellular carbohydrate metabolism, amino acid metabolism, and protein translation/folding/sorting/degradation in RSV- and RBSDV- infected rice sample.

GO and KEGG analyses of different m 6 A methylated genes in RBSDV- and RSV-infected rice plants. A Percentages of the different gene bodies (5′-UTR, 3′-UTR, 1st exon, and other exons) in different m 6 A methylated genes in rice infected with RBSDV alone, RSV alone, or with both. B GO analysis of the different m 6 A methylated genes in rice infected with RBSDV alone, RSV alone, or with both compared with mock-treated rice plant. C KEGG analysis of the different m 6 A methylated genes in rice infected with RBSDV alone, RSV alone, or with both

Analysis of consensus motifs related to m 6 A modifications

To investigate whether there are conserved motifs in clear sequencing reads of mock-, RBSDV-, and RSV-infected rice samples, the MEME suite software was used for de novo scanning of the enriched motifs. In total, 172,801, 152,136, and 168,666 raw m 6 A peaks appeared in the sequencing data corresponding to the mock-, RBSDV-, and RSV-infected rice samples (Additional file 2: Table S8). When the parameter was set as the fold enrichment (FC) > 1.5 (p < 0.05), 26,389, 27,038, and 26,675 significantly enriched m 6 A peaks were identified in mock-, RBSDV-, and RSV-infected rice samples, respectively. These significantly enriched peaks were performed to do de novo searching of the common consensus by software. The “CGBCGKC” (B = C, G, or T K = G or T), “CGRCGVC” (R = A or G V = A, G or C), and “CGHCGDCG” (H = A, C or T D = A, G or T) motifs were enriched in mock-, RBSDV-, and RSV-infected rice m 6 A methylated reads, respectively, using the DREME suite (Fig. 7A, C, and E). MEME suite scanning revealed the enrichment of the longer conserved motifs “CSYCBCCGCCSYCGCCGCSSY”, “GCGGCGGCGRCGGCG”, and “YCGCCGCCGBCGCCG” in m 6 A methylated reads of mock-, RBSDV-, and RSV-infected rice (Fig. 7B, D, and F). Besides, the enriched top five consensus in mock-, RBSDV-, and RSV-infected rice were selected and exhibited (Additional file 2: Table S9), and results showed the dynamic of the ranking with viruses’ infection. The top four widely studied motifs in other species were selected and analyses using the DREME and MEME suites in the mock-, RBSDV-, and RSV-infected rice samples (Additional file 2: Table S10), and the results showed that the most common motifs still were “RRACH” (68%), and “URUAY” (28%). Taken together, these results implied that the common recognition mechanism of the m 6 A methylated sites during the plant-virus interaction, and viruses’ infection caused the dynamics of the top five consensus ranking.

Identification of predominant consensus motifs containing m 6 A methylation sites in mock-treated and RBSDV- and RSV-infected rice plant using DREME and MEME suites. A Sequences logo representations of the consensus motifs containing m 6 A sites in mock-treated rice samples. B The most enriched consensus motif in RBSDV-infected samples. C The most enriched consensus motif in RSV-infected samples

Rice m 6 A methylation modifications display sensitive and dynamic responses to infection of rice plants by viruses

Abiotic and biotic stress, such as heat, cold, salt, and drought, and a variety of fungal, bacterial, viral, and nematode plant pathogens often affect the growth and development of rice plants, which poses a significant threat to the yield and restricts the global distribution of rice plants. In addition to the visual phenotype changes to these stresses, there are sophisticated and fine gene expression and regulation networks behind. Hence, we wondered whether mRNA m 6 A modification was involved in the interactions between rice and plant viruses. LC-MS/MS was used to track the m 6 A methylation dynamics of rice mRNA in plants infected with RBSDV or RSV. The difference in the potential epigenetic basis of the virus infection on rice plants was also investigated. Compared to the mock treatment, the N 6 -methyladenosine level was significantly increased in RBSDV- and RSV-infected rice compared to the mock-treated sample just at the one-day-post-infection (dpi), and the FC of the m 6 A content was further increased with the infection time extended from 2 days to 16 days (Fig. 8A). These results demonstrated that the m 6 A methylation was sensitive and dynamic upon viruses’ infection. The FC of the m 6 A content in the RBSDV-infected samples was approximately 5 times higher than that in the mock-treated sample at 16 days, and 3 times higher in the RSV-infected sample (Fig. 8A). Total RNAs were extracted, then the mRNAs were isolated. Nucleic acid-based dot-blot analyses were performed. The blotting results showed that with the increase in mRNA loading, the colour reaction of virus-infected rice plants became stronger compared with the mock-treated samples (Fig. 8B). These findings indicated that the infection of plants with viruses markedly increased the N 6 -methyladenosine level of plant mRNAs, and the N 6 -methyladenosine level was tightly correlated with virus infection of rice plants.

Rice m 6 A methylation levels are positively associated with the expression of key genes involved in antiviral RNA silencing pathways and plant hormone signals. A Comparisons of m 6 A methylation levels of the respect mock-treated and RBSDV- and RSV-infected rice plants at 0, 1, 2, 4, 8, and 16 dpi by LC-MS/MS. Error bars indicate mean ± SD, with three biological replicates. B Dot-blot analysis of m 6 A levels in extracted total RNA from samples at 16 dpi using the specific anti-m 6 A antibodies. The left side of the membrane depicts the amount of loaded mRNA from mock-treated and RBSDV- and RSV-infected rice plants, respectively. C qRT-PCR analysis of the relative expression of OsAGO18 in mock-treated and RBSDV- and RSV-infected rice plants at 0, 1, 2, 4, 8, and 16 dpi. D Relative expression of the OsSLRL1 in the three treatments at 0, 1, 2, 4, 8, and 16 dpi. E Analysis of m 6 A methylation levels on different fragments of OsAGO18 by m 6 A-IP-qPCR. The upper panel indicates the gene structures of OsAGO18 labelled with fragments amplified in the m 6 A-IP-qPCR assay. The results of positions 1 and 12 were chosen for figure exhibition. F m 6 A-IP-qPCR assay of the m 6 A methylation levels of different fragments on OsSLRL1. Similarly, the upper panel represents the gene structures labelled with fragments amplified in the m 6 A-IP-qPCR analyses. Results of positions 2, 3, and 4 were selected for the display in the figure. Error bars denote mean ± SD, n = 3 biological replicates in all qRT-PCR assays

Correlation of m 6 A levels with expression of key genes involved in plant antiviral RNA silencing pathway and hormone metabolism

The m 6 A methylation level of rice total mRNAs was strongly correlated with virus infection of the plants (Fig. 8B). This prompted us to explore the potential molecular mechanisms. We focused on the virus infection of rice and explored whether m 6 A modifications were involved in the regulation of the key genes’ expression that related to virus infection. Based on the m 6 A-IP sequencing results (Additional file 2: Table S11), the m 6 A methylated candidate genes, ARGONAUTE 18 (OsAGO18), and SLENDER RICE LIKE 1 (OsSLRL1), were selected. OsAGO18 was reported to participate in anti-virus RNA silencing pathways in rice by binding small interfering RNAs (siRNAs) [39]. The expression of OsAGO18 can be activated by jasmonate (JA) signalling [40]. OsSLRL1 is a DELLA family protein that regulates plant hormone metabolism and mediates the growth and development of plants [41,42,43]. qRT-PCR was performed and the relative expression levels of the selected candidate genes were determined in plants infected by the virus from 0 to 16 days. OsAGO18 was upregulated by 14-fold and 4-fold in RBSDV- and RSV-infected rice plants, respectively, compared with mock-treated plants, while the OsSLRL1 was respectively downregulated by 12-fold and 25-fold in RBSDV- and RSV-infected rice plants, respectively (Fig. 8C, D). Based on these results, we concluded that the antiviral RNA silencing pathway was activated, and the synthesis and degradation of plant hormones controlling the growth, development, and basal resistance to pathogens were regulated in virus-infected rice plants.

To validate whether the m 6 A modifications were correlated with the expression of key genes, m 6 A methylation-based RNA immunoprecipitation and qRT-PCR technology (m 6 A-IP-qPCR) was performed. The m 6 A levels in position 12 of OsAGO18, not position 1 or other regions, was increased by approximately 13-fold and 11-fold in RBSDV- and RSV-infected samples, respectively, compared with mock-infected rice plants (Fig. 8E). The m 6 A levels in positions 2 and 4 of OsSLRL1 were increased by 16-fold and 12-fold in RBSDV-infected rice plants, respectively, but not in position 3 (approximately 6-fold and not responsive to virus infection) (Fig. 8F). For RSV-infected samples, m 6 A levels in positions 2 and 4 of OsSLRL1 were not significantly changed (approximately 6- and 4-fold, respectively) under virus infection, whereas the m 6 A level was markedly up-regulated at position 3 (Fig. 8F). The collective findings indicated that changes in the relative expression of OsAGO18 and OsSLRL1 tightly correlated with the changes in their m 6 A modification level and that the m 6 A methylation of different gene regions may have different effects on its expression. These findings could contribute to a deeper understanding of the molecular basis of interactions between rice and viruses.

Involvement of rice m 6 A modification in the regulation of the expression of the main m 6 A modification machinery components in virus-infected plants

RNA m 6 A modifications occur in many eukaryotes, and it was done by the m 6 A methylation machinery. The WRITER, READER, ERASER, and methyl synthetases that produce the DONOR (methyl) are the four main components of the m 6 A methylation machinery. To investigate whether the gene expression of the main components of m 6 A methylation machinery can be affected by m 6 A modifications, qRT-PCR was performed. The relative gene expression levels of five WRITER genes (OsMAT1, OsMAT2, OsMAT3, OsMAT4, and OsFIP), five ERASER genes (OsALKBH10B, OsALKBH9B-1, Os05g0401500, OsALKBH9B-2, and Os03g0238800), 12 READER genes (OsYTH1–12), and five S-adenosyl-I-methionine synthetases (OsSAM1, OsSAM1L, OsSAM2, OsSAM2L, and OsSAM3) were determined in mock and virus-infected rice samples. The relationship between the relative expression level of target genes and m 6 A modification sites was counted by combination analyses (Additional file 2: Table S12), and no any fixed regular pattern has been found.

Compared with mock-treated samples, the OsMAT3 and OsMAT4 WRITER genes were significantly increased in RSV-infected samples, whereas they did not change in RBSDV-infected samples (Fig. 9A). For ERASER genes, the expression of OsALKBH10 was significantly suppressed in both the RBSDV- and RSV-infected samples, both Os05g0401500 and OsALKBH9B-2 were up-regulated in RSV-infected samples, whereas the expression of OsALKBH9B-2 was increased in RRSDV-infected samples (Fig. 9B). For READER genes, the expression levels of OsYTH1, OsYTH3, OsYTH5, and OsYTH7 were up-regulated in RSV-infected samples, whereas OsYTH8 was significantly decreased. Only the expression level of OsYTH6 was increased in RBSDV-infected samples (Fig. 9C). For the methyl donor producer, the OsSAM1 and OsSAM2 expression levels were significantly down-regulated in both RBSDV- and RSV-infected samples, whereas the expression of OsSAM2L and OsSAM3 in RSV-infected samples were up-regulated, and the expression of OsSAM3 was also markedly increased in RBSDV-infected samples (Fig. 9D). Based on the relative expression results, we integrated the m 6 A-IP sequencing data with the m 6 A methylation machinery in rice (Additional file 2: Table S12). Modification of m 6 A occurs in genes of m 6 A methylation machinery under plant viruses’ infection. For the donor producer, OsSAM2 was m 6 A methylated in both RBSDV- and RSV-infected samples. For the WRITER genes, OsMTA3 and OsMTA4 were m 6 A methylated in RBSDV- and RSV-infected samples, respectively. For ERASER genes, the m 6 A modification occurred on OsALKBH10B and OsALKBH9B in RSV-infected samples, whereas no ERASER genes were m 6 A methylated in RBSDV-infected samples. For READER genes, OsYTH01, OsYTH10, OsYTH11, and OsYTH12 in RBSDV-infected samples, and OsYTH05 and OsYTH08 in RSV-infected samples were m 6 A methylated (Fig. 9E). In summary, the m 6 A modifications occurred in the genes that encoding the plant m 6 A methylation machinery, and probably regulated the dynamics of the target gene expression, which may act as a main post-translational gene expression regulation strategy under plant virus infection.

Integrated analyses of the main components of m 6 A methylation machinery in rice with m 6 A methylation modifications and gene expression in plants infected with viruses. A Relative expression levels of five “WRITER” components in rice plants by qRT-PCR analyses. B qRT-PCR analysis of the relative expression of five “ERASER” components in rice plants. C Relative expression levels of twelve “READER” component genes were determined by qRT-PCR. D Relative expression levels of five methyl “DONER” synthesis genes were analysed by qRT-PCR. E Rice m 6 A methylation pathways and related m 6 A methylated genes under plant virus infections. Blue coloured letters indicate the m 6 A methylated genes of certain treatments. For instance, RBSDV: OsMTA3, means the OsMTA3 gene was methylated in RBSDV-infected sample. All qRT-PCR assays were performed with three biological replicates, and the error bars denote the mean ± SD

Involvement of rice m 6 A modification in regulating the expression of the main antiviral RNA silencing components in virus-infected rice plants

To investigate whether m 6 A methylation modification was involved in the regulation of the main host antiviral RNA silencing pathways genes and the reported resistance genes corresponding to these two viruses. Genes, which included nine DCLs, five RNA-dependent RNA polymerases (RDR), 17 ARGONAUTE genes (OsAGOs), seven RBSDV resistance genes (OsGDI homologous), and two RSV resistance genes (OsSOT1 and OsStvb-i), were selected for relative expression analyses. Integrated analyses of the antiviral genes with m 6 A modification sites on the gene body and relative expression level were determined in virus-infected rice plants.

qRT-PCR revealed that both the OsDCL2b-1 and OsDCL2b-2 were significantly up-regulated in RBSDV- and RSV-infected samples than in mock-treated samples (Fig. 10A). In addition, the expression of OsDCL1b was greatly suppressed in RBSDV-infected samples, whereas it was increased in RSV-infected samples (Fig. 10A). Compared to the mock-treated samples, the expression of OsRDR1 and OsRDR3 in both RBSDV- and RSV-infected samples was markedly increased, and OsRDR2 was upregulated only in RSV-infected samples (Fig. 10B). In RBSDV-infected samples, only the expression of OsAGO18 was up-regulated significantly (Fig. 8C), whereas in the RSV-infected samples, OsAGO5c, OsAGO12, OsAGO13, OsAGO14, and OsAGO18 were markedly increased (Figs. 8C and 10C). Concerning resistance genes, seven ZmRabGDI (Rab GTPase dissociation inhibiter) homologous genes in rice, which were defined as RBSDV resistance genes on maize [44], were screened out. The RSV resistance genes OsSOT1 (Sulfotransferase 1) [45] and OsStvb-i (Stripe disease resistance i) [46], were also chosen. A total of nine genes were selected for further gene expression analyses. OsGDI-1-1, OsGDI-1-2, OsGDI-2, and OsSOT1 were significantly decreased both in RBSDV- and RSV-infected rice plants compared to that in the mock-treated sample (Fig. 10D). In contrast, OsGDIα was markedly upregulated in RBSDV- and RSV-infected rice plants (Fig. 10D). The collective findings indicated that OsDCL2b-1, OsDCL2b-2, OsRDR1, OsRDR3, OsAGO12, OsAGO13, and OsAGO18 were the main antiviral genes in the innate immune RNA silencing pathways, and that OsGDIα, OsGDI-1-1, OsGDI-1-2, OsGDI-2, and OsSOT1 may act as resistance genes for both RBSDV and RSV.

Integrated analyses of main antiviral RNA silencing pathway-related genes with m 6 A methylation modifications and gene expression levels in rice infected with viruses. A Relative expression levels of nine OsDCL genes in mock-treated and RBSDV- and RSV-infected samples. B Relative expression levels of five OsRDR genes in mock-treated and RBSDV- and RSV-infected samples using qRT-PCR analyses. C Relative expression levels of 17 OsAGO genes were determined with respect to mock-treated and RBSDV- and RSV-infected samples using qRT-PCR analyses. D Relative expression levels of nine resistance genes, including seven OsGDI genes, one OsSOT1 gene, and one OsStvb-i were determined in mock-treated and RBSDV- and RSV-infected samples using qRT-PCR analyses. E Rice antiviral RNA silencing pathways and the related m 6 A methylated genes in virus-infected plants. Blue coloured letters depict the m 6 A methylated genes of a certain treatment, as detailed in Fig. 9E. All qRT-PCR assays were performed with three biological replicates. The error bars denote the mean ± SD

To analyse the potential roles of m 6 A methylation in the expression of 57 selected genes in RNA silencing pathways and viral resistance genes, we systematically analysed their m 6 A modification status according to the results of m 6 A-IP-seq (Additional file 2: Table S13). For the main RNA silencing pathway genes, OsAGO1c, OsAGO2, OsAGO18, and OsRDR1 were m 6 A methylated in RBSDV-infected rice. In RSV-infected samples, OsDCL1a, OsDCL3b, OsDCL4, OsAGO2, OsAGO12, OsAGO5c, OsAGO17, OsAGO18, OsRDR1, and OsRDR3 were significantly methylated (Fig. 10E). In contrast, no viral resistance genes were m 6 A methylated (Fig. 10E). These results indicated the possible involvement of m 6 A methylation in the main antiviral RNA silencing pathways. The methylation may act as a fine regulator to mediate the spatial and temporal expression of target genes in arm race of plant-virus interaction.

Integrated analyses of relative expression profile and m 6 A modification of seven phytohormone metabolism-related genes

Viruses have various strategies to reprogram the host’s cellular status to one that is prone to viral replication and spread. In plants, the specific environment created by viruses usually refers to phytohormone regulations of nearly all aspects of plant physiology, including development, growth, defence, and reproduction [47]. These phytohormones, which include jasmine acid (JA), salicylic acid (SA), abscisic acid (ABA), auxin, cytokinin (CTK), ethylene (ET), and brassinosteroids (BR), have significant roles in plant development and physiological regulations and are also involved in defence against pathogens [48, 49]. Previous findings suggest that phytohormones are strongly associated with virus infection and symptom development. We investigated whether these phytohormones are regulated by m 6 A modification and whether the expression profiles of their metabolic genes are altered by virus infection of rice plants. qRT-PCR was performed to determine the relative expression of the seven main phytohormone metabolism-related genes (Additional file 1: Fig. S3–S9). Further, integrated analyses of their relative expression and m 6 A modification sites were performed (Additional file 1: Fig. S10, Additional file 2: Table S14).

For the JA pathway, the biosynthesis genes OsLOX8 and OsLOX9 were markedly up-regulated in both RSV- and RBSDV-infected plants. OsJMT1-1, OsAOS2, and OsLOX2 were markedly decreased, whereas OsAOS1 and OsLOX5 were not changed (Additional file 1: Fig. S3A). In addition, OsJMT1, OsLOX1, and OsHPL3 were decreased in RSV-infected samples, whereas OsJMT1 was increased in RBSDV-infected plants (Additional file 1: Fig. S3A). The expression profiles of 21 JA responsive genes were further investigated. OsPR1a, OsWRKY28, OsPR2, and OsPR5-4 were significantly increased in both RSV- and RBSDV-infected plants, OsPR5-3, OsMYB2, OsMYB55/56-1, OsWRKY10, OsRbohA, OsRbohB, and OsRbohC were markedly decreased, and OsPR1b, OsPR5-2, OsbZIP52, and OsRbohE were not changed. OsPR5-1 and OsPR1 were increased in RBSDV- and RSV-infected rice, respectively (Additional file 1: Fig. S3B and S3C). These results indicated that the activated JA pathways mainly depend on the markedly increased expression of biosynthesis-related OsLOX8 and OsLOX9, and the up-regulated OsPR1a, OsWRKY28, OsPR2, and OsPR5-4 responsive genes.

For the SA pathway, we investigated the biosynthesis-related genes OsICS1, OsPAL, OsPAL1, OsAIM1, OsCM, and OsEDS1) and the response genes (OsPR1-101, OsWRKY45-1, OsWRKY45-2, OsSGT1, OsPR1b, OsPR1a, OsPR1-12, OsPR1-21, OsPR1-22, OsPR1-51, and OsPR1-121). OsICS1, OsPAL, and OsPAL1 were significantly suppressed in both the RBSDV- and RSV-infected plants, OsCM and OsEDS1 were not altered, and only OsAIM was slightly up-regulated in RSV-infected plants (Additional file 1: Fig. S4A). These results indicated that SA biosynthesis was suppressed upon virus infection. OsWRKY45-1, OsWRKY45-2, and OsRR1-51 were significantly increased in both the RBSDV- and RSV-infected plants, whereas the OsSGT1, OsPR1-21, OsPR1-22, and OsPR1-121 were dramatically decreased. OsPR1-101 and OsPR1a were markedly up-regulated in RSV-infected plants but significantly down-regulated in RBSDV-infected plants (Additional file 1: Fig. S4B and S4C). These results suggested that the SA pathway was suppressed by virus infection through down-regulation of the main biosynthesis genes.

For the ABA pathway, qRT-PCR revealed that OsNCED3 was significantly up-regulated in RBSDV-infected samples, whereas in RSV-infected plants, OsNCED1-1 was up-regulated. OsABA was suppressed in both RBSDV- and RSV-infected plants. Other biosynthesis-related genes, including OsABA1, OsbZIP72, and OsbZIP23, were unchanged in RBSDV- and RSV-infected plants (Additional file 1: Fig. S5A). ABA deactivation genes OsABA8OX1, OsABA8OX2, and OsABA8OX3 were significantly up-regulated in RBSDV-infected plants. In RSV-infected plants, OsABA8OX2 and OsABA8OX3 were slightly decreased (Additional file 1: Fig. S5B). The collective results indicated that the ABA pathway was activated by RBSDV infection, but was suppressed in RSV-infected plants.

For the Auxin pathway, the relative expression levels of auxin-related metabolic genes were affected differently by virus infection. Of the analysed biosynthesis genes, OsYUCCA1, OsYUCCA5, OsYUCCA6, OsYUCCA9, OsAO-2, OsAO3-L, and OsAOO3-2 were significantly down-regulated upon RBSDV and RSV infection, whereas OsYUCCA8, OsAO-1, OsAAO, and OsAAO3-1 were up-regulated (Additional file 1: Fig. S6A). The OsGH3.8 and OsGH3.2 auxin transformation genes were markedly increased in both RBSDV- and RSV-infected plants (Additional file 1: Fig. S6B). The OsPIN1A, OsPIN1B, OsPIN2, OsPILS7a, and OsPILS7b auxin transportation genes were markedly down-regulated in RBSDV- and RSV-infected plants (Additional file 1: Fig. S6C), while only OsPIN3A was increased (Additional file 1: Fig. S6D). The OsIAA3 and OsIAA7 signalling genes were markedly decreased in both RBSDV- and RSV-infected plants (Additional file 1: Fig. S6E). The other signalling gene (OsIAA20) was significantly up-regulated in RBSDV-infected samples, whereas it was severely suppressed in RSV-infected plants (Additional file 1: Fig. S6E). These results showed that the auxin pathway was suppressed in both RBSDV- and RSV-infected plants.

For the CTK pathway, genes responsible for CTK biosynthesis, including OsIPT3 and OsIPT7, were dramatically decreased in both RBSDV- and RSV-infected plants (Additional file 1: Fig. S7A). In contrast, the CTK transformation genes or oxidase genes (OsCKX4 and OscZOGT1) were significantly up-regulated in both RBSDV- and RSV-infected plants (Additional file 1: Fig. S7B). The OsPR8, OsPR10a-1, OsPR1a-1, OsPR1b, and OsPR10a-2 CTK responsive genes were markedly increased in both RBSDV- and RSV-infected plants (Additional file 1: Fig. S7C & S7D). Most of the analysed genes (OsPR2-1, OsPR2-2, OsPR3, OsPR4, OsPR5-1, and OsPR6) were suppressed (Additional file 1: Fig. S7D). Other responsive genes, which included OsPR1a-2, were dramatically upregulated in RBSDV-infected samples, but not in RSV-infected samples. The expression profile of OsPR9 was completely opposite (Additional file 1: Fig. S7D). These results showed that the CTK pathway in rice was suppressed by viral infection.

For the ET pathway, the OsACS2 biosynthesis gene was significantly up-regulated in both RBSDV- and RSV-infected plants, as was OsACS1 in RBSDV-infected plants (Additional file 1: Fig. S8). Most of the oxidase genes (OsACO7, OsACO1, and OsACO2) were dramatically decreased in both the RBSDV- and RSV-infected plants (Additional file 1: Fig. S8). The collective results indicated that the ET pathway was activated in rice upon virus infection.

For the BR pathway, 24 genes, including 14 biosynthesis-related genes and 10 signalling genes, were analysed. Most of the biosynthesis genes (OsD11, OsDS11-L, OsCPD1, OsGSK2, OsRAV1, OsRAV2, OsRAVL1, and OsBZR1) were markedly suppressed in both RBSDV- and RSV-infected plants, whereas OsDWARF4 was significantly up-regulated (Additional file 1: Fig. S9A). The four signalling genes (OsBRI1-1, OsBRI1-2, OsBAK1-4, and OsBAK1-10) were dramatically decreased in RBSDV- and RSV-infected plants. The other four genes (OsI-BAK1, OsBAK1-2, OsBAK1-3, and OsBAK1-8) were severely downregulated, and two genes (OsBAK1-6 and OsBAK1-9) did not change (Additional file 1: Fig. S9B). The expression profiles of these genes were synchronously changed in the RBSDV- and RSV-infected plants. These results suggested that the BR pathway was suppressed in rice plants upon virus infection.

Seven phytohormone metabolism-related genes, including 154 genes, were analysed by qRT-PCR (Additional file 1: Fig. S10A). Of these genes, m 6 A modification was detected in 37 and 19 genes in RBSDV- and RSV-infected samples, respectively. m 6 A methylation was detected in nine genes in RBSDV- and RSV-infected plants (Additional file 1: Fig. S10B, Additional file 2: Table S14). m 6 A-IP-sequencing data mapped these m 6 A methylation sites to different regions of target genes. Most of the peaks were located in the coding sequence (CDS) region (Additional file 1: Fig. S10C). Furthermore, we analysed the relationship between m 6 A methylation sites on gene bodies and the relative gene expression levels. In most cases, the target gene was downregulated if the m 6 A site was located in the 5′-UTR, CDS, and CDS/3′-UTR, with the location in the 3′-UTR often being associated with up-regulation of the target gene (Additional file 1: Fig. S10D). Hence, a viral infection could regulate the expression of target genes, and the regulation mode was varied in different manners for the same genes under the two different viral infection conditions.

Widely integrated analyses of the relationship between m 6 A methylation regions and expression levels using RNA-seq and m 6 A-IP-seq

To investigate the relationship between the location sites of m 6 A methylation and the relative expression profiles, we analysed the common regulated peaks that appeared in both the RBSDV- and RSV-infected plants. We classified the reads that extended 5′-UTR to the start codon (5′-UTR/CDS), reads that extended the stop codon to the 3′-UTR (CDS/3′-UTR), and other reads that did not go beyond two obviously distinct regions to the corresponding 5′-UTR, CDS, or 3′-UTR (Additional file 2: Table S15).

In total, 3,734 and 3,336 peaks were analysed in RBSDV- and RSV-infected plants, respectively (Additional file 1: Fig. S10E and S10F). In RBSDV-infected plants, the relative expression level of 44.45% m 6 A modified genes was not changed, 29.73% were downregulated, and 25.82% were up-regulated. Most m 6 A sites were located in the CDS and 3′-UTR regions (Additional file 1: Fig. S10E). The number of genes that were upregulated, downregulated, and unchanged were increased gradually when the m 6 A methylation occurred in the 5′-UTR/CDS and CDS/3′-UTR region (Additional file 1: Fig. S10E). In RSV-infected plants, the majority of m 6 A modified genes (69.04%) were downregulated. Most m 6 A sites (49.34%) were located in the CDS region and 3′-UTR. CDS and 3′-UTR location of the m 6 A modified sites often indicated that the target genes were downregulated (Additional file 1: Fig. S10F), and the m 6 A methylation was mostly occurred in CDS and 3′-UTR region in both RSV- and RBSDV-infected samples. Taken together, these results indicated that the regulatory roles of post-transcriptional m 6 A modification differed upon RBSDV or RSV infection of rice plants and that certain m 6 A sites on specific genes may have specific functions. The common regular pattern between expression level and m 6 A methylation region, which are suitable for all m 6 A methylated genes, have not been found so far. This may be due to the fact that different viruses cause plant disease through hijacking different host’s signal pathways, and different genes own different sequences characteristics.


An RNA World?

  • DNA encodes the genetic information of proteins but
  • DNA replication and transcription requires proteins.

But if RNA can serve both as a

  • repository of information (in its sequence of nucleotides) and as a
  • catalyst,
  • involve RNA acting on RNA (not protein)
  • and (except for Ribonuclease P) are self-limited.

Yes, the ribosome turns out to be a ribozyme.

The Ribosome is a Ribozyme

Ribosomes are huge aggregates containing 3 (4 in eukaryotes) rRNA molecules and scores of protein molecules.

The three-dimensional structure of the large (50S) subunit of a bacterial ribosome was published in August 2000. It clearly shows that formation of the peptide bond that links each amino acid to the growing polypeptide chain is catalyzed by the 23S RNA molecule in the large subunit. The 31 proteins in the subunit probably provide the scaffolding needed to maintain the three-dimensional structure of the RNA.

Link to discussion of ribosome structure and function.

RNA polymerization by RNA

In today's world, RNA polymerases &mdash made of protein &mdash make the RNA molecules (using the antisense strand of DNA as a template [View]). Could RNA alone have done it?

It can be done in the laboratory. Wochner, A. et al. report in Science, 332:209, 8 April 2011, their creation of a synthetic RNA molecule that when presented with single-stranded RNA templates, polymerizes ribonucleotide triphosphates into strands of RNA complementary to the template. Their synthetic RNA polymerase was able to faithfully incorporate up to 95 nucleotides into complementary strands of RNA. One product was a functional endonuclease ribozyme. (By the autumn of 2013, they were able to copy a template of 206 nucleotides.)


More on RNA Catalysis

With the discovery nearly thirty years ago that RNA can catalyze reactions with proficiencies that approach those of protein enzymes, the central dogma of biology with RNA as a simple carrier molecule between DNA and proteins was overturned. Today RNA is recognized as an active catalyst in biology, in self-splicing of group I and group II introns, in various small ribozymes, and also as the catalytic center of the ribosome and spliceosome. These findings, and the fundamental ability of RNA to act both as an efficient information carrier and functional macromolecule led to proposal of an RNA World early in evolution.

We explore RNA catalysis to learn about the catalytic potential of RNA and to decipher what is fundamental to all biological catalysts through comparison with protein enzyme catalysis.

These studies also define the unique properties of RNA and proteins lead to catalytic and behavioral distinctions.

The fundamental properties and behaviors of RNA molecules that we uncover teach us about how the potential function of RNA early in evolution and about the function of RNA molecules in modern-day biology. This knowledge may also be applied as RNA is co-opted for medical, technological and industrial applications.

Energy from binding interactions can be used to facilitate reactions of bond substrates, a fundamental precept of enzymology posited by Jencks for protein enzymes and demonstrated in our studies of RNA enzymes.

We currently focus on the group I ribozyme, the most well-studied catalytic RNA in both structure and function. We harness previous studies, including multiple crystal structures, a robust phylogeny model, and a defined kinetic and thermodynamic framework for the Tetrahymena group I ribozyme, to delve more deeply into questions about catalytic RNA and, in particular, how an RNA scaffold can be used to sculpt an active site and how RNA achieves specific and strong molecular recognition.

We are also very interested in RNA conformational changes, as these transitions are key elements in nearly all RNA-mediated events.

Nearly all RNA-mediated events incolve conformational changes. We can learn about these transitions from studying catalysis and folding in model systems.

To answer these and additional questions, we use techniques including site-directed mutagenesis and site-specific chemical modifications to alter both the ribozyme itself and its substrates. The replacement of single functional groups within a complex RNA structure with multiple related functionalities is straightforward, whereas the corresponding replacements in proteins remain challenging. Function is probed via pre-steady state kinetics, and structure is probed using a battery of chemical footprinting approaches. Recent advances allow us to incorporate probes of local dynamics and single molecule fluorescence assays of functional conformational transitions as well as carry out high-throughput structure-function studies.

Finally, our collaboration with the Greenleaf lab allows us to now simultaneously probe thousands of mutants, allowing for the first time the depth and breadth of information needed to understand how an RNA scaffold established a functional active site.


Watch the video: A trajectory of Azoarcus ribozyme RNA folding. (October 2022).