plant ribosomal rna

S5A). S5D). Thank you for your interest in spreading the word on Plant Physiology. Ribosomal proteins (RPs) are essential structural components of ribosomes. The biosynthesis of mature rRNAs requires a complex series of post-transcriptional processing steps including nucleotide modifications, some of which take place during or immediately after transcription, while others occur in a ribosome assembly-assisted manner ( 9 , 10 ). 1, B and F). Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. The major pre-rRNA endonucleolytic cleavage sites have been determined in Arabidopsis. Mapping of the 5′ and 3′ extremities of the pre-18S rRNAs. Then, decreased pre-rRNA processing may negatively affect the processing dynamics of 45S transcript, resulting in its accumulation (Fig. Similarly, the 35S(P) fragment was further identified by primer combination 32P2 (p23/25R; Fig. The 18S rRNAs identified by primers 18P1 were validated by sequencing of 20 independent clones. S10C), indicating reduced pre-rRNA processing. Finally, target products exhibiting sharp bands at the proper molecular weight were excised for DNA sequencing and further analyzed by BLAST from the National Center for Biotechnology Information (NCBI), choosing the organism (O. sativa, japonica group; taxid:39947) and database (reference genomic sequences [refseq_genomic]) using Megablast (optimized for highly similar sequences). Dysfunction of ribosomal biogenesis (Gallagher et al., 2004; Ferreira-Cerca et al., 2005, 2007; Tafforeau et al., 2013) results in severe developmental defects in higher plants (Byrne, 2009; Fujikura et al., 2009; Horiguchi et al., 2011; Weis et al., 2015a, 2015b) and serious genetic diseases in mammals (Choesmel et al., 2007; Narla and Ebert, 2010; McCann and Baserga, 2013; Sondalle and Baserga, 2014; Bai et al., 2016). Processing of preribosomal RNA in Saccharomyces cerevisiae. These findings ultimately uncovered the rice alternative pre-rRNA processing pathways with the ITS1-first mode as the major pathway (Fig. Likewise, dysfunction of the ribosome biogenesis factor TOGR1 affected pre-rRNA processing, which resulted in severe developmental defects and hypersensitivity to heat stress in rice. 7, A and B; Supplemental Figs. Here, we identified the rRNA precursors produced during rRNA biogenesis and the critical endonucleolytic cleavage sites in the transcribed spacer regions of pre-rRNAs in rice. Mapping of the 5′ and 3′ extremities of the pre-18S rRNAs. atBRX1-1 and atBRX1-2 are involved in an alternative rRNA processing pathway in Arabidopsis thaliana. The ITS1 locus matched by the 3′ ends of these clones are indicated by black triangles as well as the number of clones. The ITS1 and ITS2 locus matched by the 5′ and 3′ ends of these DNA sequences, respectively, are indicated by black triangles as well as the number of clones. A, Structure of pre-18S rRNA intermediates identified by a set of primer combinations (in shaded box). Little is known about the RNA helicases involved in pre-60S ribosomal subunit processing and assembly in plants. In addition, the translational activity of ribosomes in the cytoplasm could be directly and dynamically fine-tuned by various environmental signals (Bailey-Serres et al., 2009; Browning and Bailey-Serres, 2015), such as dehydration stress (Kawaguchi et al., 2004), hypoxia (Branco-Price et al., 2008; Mustroph et al., 2009; Juntawong et al., 2014), heat stress (Zhang et al., 2017a), and light signals (Liu et al., 2012, 2013). A, Pre-rRNA processing intermediates detected by northern blots with specific probes, which are indicated by horizontal arrows. To visualize the intermediates of 45S pre-rRNA processing (Supplemental Fig. After transcription by RNA polymerase I and site-specific modification by small nucleolar ribonucleoproteins, the nascent 35S rRNA, the common precursor of 18S, 5.8S, and 25S rRNAs, is quickly assembled with many assembly factors and ribosomal proteins into small subunit processome/90S preribosomal particles (13 ⇓ –15). Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. Epub 2017 Feb 14. 3A), similar to the 6S intermediates in Arabidopsis (Shanmugam et al., 2017). Moreover, compared with both the mature rRNAs and other rRNA precursors, the P-A3 precursor readily detected by northern blots could be a reliable marker for rRNA biogenesis under chilling stress. 1–4; Supplemental Fig. B, Northern blots to determine pre-rRNA processing in pre-60S LSU in Nipponbare (lane 1), Zhongxian3037 (ZX3037, lane 2), and. Pre-18S rRNA intermediates are processed by endonucleolytic cleavages in the 5′ ETS and the ITS1 surrounding the 18S rRNA (Fig. C, Northern blots to detect the 45S rRNA transcript by probe 45P in Nipponbare under 4°C treatment for 0, 2, 4, and 6 h. Both blots of 45P and p42 came from the same membrane. cRT-PCR analysis was performed as previously described (Slomovic et al., 2008; Barkan, 2011; Hang et al., 2015), with slight modification (Supplemental Fig. The biogenesis of eukaryotic ribosomes is a fundamental process involving hundreds of ribosome biogenesis factors (RBFs) in three compartments of the cell, namely the nucleolus, nucleus, and cytoplasm. The ITS1 locus matched by the 3′ ends of these clones are indicated by black triangles as well as the number of clones. The locations of primer pairs (18P1 to 18P8) are shown in Figure 1A and summarized in Supplemental Tables S1 and S2. Supplemental Figure S6. 5, B–E; Supplemental Fig. The 32S pre-rRNAs were validated by sequencing of 20 independent clones (D). The number of identical clones is indicated to the right of each fragment. In the minor 5′ ETS-first pathway, the removal of the 5′ ETS in the 35S(P) transcript occurs first to generate the 32S intermediate before its split at the ITS1 cleavage site A2. The ribosomal subunits consist of a few ribosomal RNA (rRNA) species and a set of ribosomal proteins. 7D). After rDNA transcription by RNA Pol I, the 45S rRNA transcripts undergo primary cleavages at the P site in the 5′ ETS and an unknown site in the 3′ ETS to generate the 35S(P) intermediate. Pre-rRNA processing in rice roots responses to chilling stress. Northern blots to detect pre-rRNA processing in rice. 2F; Supplemental Figs. Then, cleavage at the A2 site splits the 32S rRNA into the 18S-A2 and 27SA2 intermediates, which undergo further endo- and exonucleolytic processing into mature 18S, 5.8S, and 25S rRNAs (Fig. The BoU3/NF-D complex is recruited by a conserved A123B [A(1), A(2), A(3), and B motifs] to mediate P-site cleavage in the 5′ ETS (Caparros‐Ruiz et al., 1997; Sáez-Vasquez et al., 2004a, 2004b; Samaha et al., 2010). Chilling stress inhibits rRNA biogenesis mainly at pre-rRNAs processing levels. 2014 Apr;20(4):540-50. doi: 10.1261/rna.043471.113. Besides, de novo characterization of nascent transcripts under chilling treatments using unbiased global nuclear run-on sequencing will provide us with more information at the transcriptional level (Hetzel et al., 2016). ), the Strategic Priority Research Programs (grants XDA08010202 and XDPB0403 to X.C. 2A), the intact 27SB intermediate was identified (Fig. Eukaryotic ribosome biogenesis is coupled with rRNA biogenesis, which starts in the nucleolus. Database searching was performed at NCBI. A and B, Northern blots to detect pre-rRNA processing in Nipponbare (. The definition of major and minor pathways in eukaryotes is based on the amount of marker pre-rRNA transcripts in wild type by northern-blot or pulse-chase labeling (Pendrak and Roberts, 2011; Mullineux and Lafontaine, 2012; Sloan et al., 2013; Henras et al., 2015; Weis et al., 2015a; Tomecki et al., 2017). Histone deacetylases play critical roles in many biological processes including transcriptional repression and rDNA silencing. Moreover, the abundance of P-A3 in the indica cultivar Zhongxian3037 was less than in the japonica cultivar Nipponbare (Fig. RNA. 7C). 27SA2, 27SA3, and 27SB belong to the 27S rRNA, the common precursor of 5.8S and 25S rRNAs. S4). Epub 2014 Sep 4. de la Cruz J, Gómez-Herreros F, Rodríguez-Galán O, Begley V, de la Cruz Muñoz-Centeno M, Chávez S. Curr Genet. The number of clones with additional sequences, such as polyadenylation at the 3′ end, is marked in parentheses. 2020 May 24;20(1):230. doi: 10.1186/s12870-020-02444-x. Environmental signals affect plant growth and crop yield. Ribosome biogenesis is crucial for plant growth and environmental acclimation. Among the pre-25S rRNA intermediates identified, 27SA3 exhibited uniform 5′ extremities at A3661 in “GTCAAGGAACACAG” in the ITS1 region (Fig. Supplemental Figure S7. Moreover, we found that rice and Arabidopsis have similar flanking sequences around the A2 and A3 endonucleolytic sites in the ITS1 and the P′ in the 5′ ETS, respectively (Supplemental Fig. 5E), as detected by probes p23 (Fig. Ribosomal RNA, molecule in cells that forms part of the protein-synthesizing organelle known as a ribosome and that is exported to the cytoplasm to help translate the information in messenger RNA into protein. PCR amplification with primer pairs 58P1 (58L1/58R1) and 58P2 (58L2/58R2; Fig. Supplemental Table S1. 3). ), the China Postdoctoral Science Foundation (2015M570169 and 2017T100113 to R.H.), the Key Research Program of Frontier Sciences of Chinese Academy of Sciences (grant QYZDY-SSW-SMC022 to X.C. The decreased biogenesis of P-A3 and 27SA2 under chilling treatment prompted us to investigate whether this inhibition began with the 45S transcript or at pre-rRNA processing stages. Feedback regulation of ribosome assembly. The 3′-5.8S (7S and 6S) and 5′-5.8S are pre-5.8S rRNAs. The ITS1 and ITS2 locus matched by the 5′ and 3′ ends of these DNA sequences, respectively, are indicated by black triangles and the number of clones. Ethidium bromide-stained gels show total rRNA. By contrast, we observed much less of the 32S intermediate than the 35S(P), when probed with S7A (Supplemental Fig. 1, B and E) intermediates were also amplified (Fig. 5; Supplemental Fig. Both endo- and exonucleolytic processing occur sequential and coordinately in this progress. A, Pre-rRNA processing intermediates detected…, Model of rRNA biogenesis in rice. C and D, DNA sequencing results for 32S (C) and 35S(P) precursors (D). 6). In contrast to the situation in unicellular budding yeast (Kos and Tollervey, 2010), the pulse-chase labeling approach for studying rRNA synthesis remains technically difficult in higher plants (Weis et al., 2015a). B, Pre-18S rRNA intermediates were determined in gel by cRT-PCR with primers 18P1 to 18P8. 2, B and D). Data are given as means and sd of three independent biological replicates. The number of clones containing additional sequences at the 3′ extremities are marked in parentheses (in the shaded box). Author information: (1)Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. Enter multiple addresses on separate lines or separate them with commas. DOI: https://doi.org/10.1104/pp.17.01714. 1. The S7 and p42 blots share the same loading control (D). B, Pre-25S rRNA intermediates were determined in gel by cRT-PCR with primers 25P1, 25P2, 27P1, and 27P2. Three sites (P, P′, and A1 [P2]) exist in the 5′ ETS, four (D, A2, A3, and B1) in ITS1, three (E, C2, and C1) in ITS2, and two in the 3′ ETS (B2 and B0; Sáez-Vasquez et al., 2004a, 2004b; Zakrzewska-Placzek et al., 2010; Lange et al., 2011; Weis et al., 2015a, 2015b; Sikorski et al., 2015; Tomecki et al., 2017). Pre-rRNA processing in rice shoots in response to chilling stress. Moreover, the A2 endonucleolytic site was deduced to be between A3560/C3561 in “ACCAAAACAGACCG” by comparing the 3′ ends of 18S-A2 (Fig. We used the togr1-1 mutant as the positive control (Wang et al., 2016), which exhibited an aberrant accumulation of 35S(P) and P-A3 compared with the wild type, Zhongxian3037 (Fig. The relative intensities for 25S rRNA, 45S transcripts, and P-A3 intermediates are marked in black, blue, and red, respectively. For each fragment, the number of clones obtained is indicated on the right. Sequence alignments of 5′ ETS, ITS1, ITS2, and partial 3′ ETS rDNAs between the japonica rice Nipponbare and the Arabidopsis thaliana accession Col-0. The 7S rRNA marked with “?” was detected by probe S9 (Fig. The number of clones containing additional sequences at the 3′ extremities is marked in parentheses. 1A). For seedlings in water (Supplemental Fig. three types of ribosomal RNA are present in a plant cell, mitochondrial, chloroplastic and nuclear. 5, C and D; Supplemental Fig. However, the processing sites and pathways remain largely unknown in crops, particularly in monocots such as rice (Oryza sativa), one of the most important food resources in the world. Black vertical arrows above the diagram indicate endonucleolytic cleavage sites relevant to this study. 1, A and D–F; Supplemental Fig. The steady level of 45S rRNA in vivo is the net product of rDNA transcription and subsequent pre-rRNA processing. 2A). 3, A and B) was performed to obtain both 5.8S-3′ (6S) (Fig. Then the 35S(P) transcript enters two alternative maturation pathways distinguished by the order of ITS1 cleavage and 5′ ETS removal. For circular RT-PCR assays (Figs. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. TruSeq Stranded Total RNA with Ribo-Zero Plant | For plant transcriptome studies Capture a comprehensive view of the plant transcriptome with this RNA-Seq library prep workflow. For short DNA probes, oligonucleotides labeled with [γ-32P]ATP (Perkin-Elmer; BLU002A001MC) by T4 polynucleotide kinase (New England Biolabs; M0201) were used to detect precursor RNAs. A and B, Structure of 3′-5.8S identified by 58P1 (58L1/58R1; A) and 5′-5.8S by 58P2 (58L2/58R2; B), respectively. Also, constitutive expression of TOGR1 enhanced the tolerance of rice to heat stress (Wang et al., 2016). In higher plants, the U3 small nucleolar ribonucleoprotein (U3 snoRNP) was first purified from cauliflower inflorescences as the Nuclear Factor D complex (Sáez-Vasquez et al., 2004a, 2004b) and from Brassica oleracea as BoU3 (B. oleracea U3) complex (Samaha et al., 2010). In the major ITS1-first pathway, the 35SP transcript is split at ITS1 endonucleolytic site A3 into P-A3 and 27SA3 precursors. We next used the sequences at the 5′ and 3′ extremities of the identified processing intermediates to define the processing sites. The small subunit contains 18S ribosomal RNAs (rRNAs) and more than 30 ribosomal proteins, while the large subunit contains the 25S/28S, 5.8S, and 5S rRNAs and more than 40 ribosomal proteins (Yusupova and Yusupov, 2014). 5A) were designed to detect the pre-18S rRNAs in the pre-40S SSU (Fig. Uncoupled processing of 5′ ETS removal and ITS1 cleavage during the processing of early transcripts resulted in alternative rRNA biogenesis pathways (Hang et al., 2014; Weis et al., 2015a, 2015b; Tomecki et al., 2017). S6B, S7A, and S7B). 4D). A, Structure of early pre-rRNA intermediates identified (in shaded box) by two pairs of primers: 32P1 and 32P2. The locations of the P and A3 endonucleolytic sites were consistent with a previous report (Wang et al., 2016). 2014;42(17):11180-91. doi: 10.1093/nar/gku787. However, in contrast to the model dicot species Arabidopsis, rRNA maturation in monocot crops remains unexplored. Similarly, the P′ site of P′-A3 was at G1634/A1635 of TCGGAAGACGACAG in the 5′ ETS (Fig. Epub 2019 Jun 25. The number of copies of the ribosomal RNA genes varies considerably between the different plant species studied. This observation indicated that (1) the complete trimming of the 3′ ETS region occurred from 35S(P) to 32S in rice, in 3′→5′ exonucleolytic processing (Lange and Gagliardi, 2010) by presently unknown enzymes. Remove rRNA from plant leaf, seed, and root tissue. COVID-19 is an emerging, rapidly evolving situation. To this end, the DNA oligonucleotide 18c (Fig. Briefly, 10 μg of total RNA extracted from Nipponbare panicles was self-ligated into circular RNA by T4 RNA ligase 1 (New England Biolabs; M0204S; Supplemental Fig. We further found that two pre-rRNA processing pathways, distinguished by the order of 5′ ETS removal and ITS1 cleavage, coexist in vivo. Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. S9 and S10). Citrus exocortis viroid causes ribosomal stress in tomato plants. Hybridization was performed overnight at 45°C (for short probes) or 65°C (for long probes) as previously described (Hang et al., 2014). S7C). rRNA biogenesis at the level of pre-rRNA processing is an ideal and reliable molecular diagnostic reflecting ribosome biogenesis and ribosome assembly status in vivo (Mullineux and Lafontaine, 2012; Tomecki et al., 2017). Published May 2018. Then, 0.15 to ∼0.20 g of shoots and roots were harvested in the same way every 2 h for two or three intervals. Although 18S-A2 could be detected by S7, its low abundance in wild-type rice makes it harder to distinguish from 18S-A3 by northern-blot assay. Three biological replicates were performed and a representative result is shown here. Rice rDNAs mainly occur as a cluster on chromosome 9 in Nipponbare, the well-annotated japonica rice genome (Goff et al., 2002; Kawahara et al., 2013; Sakai et al., 2013). Two rice (Oryza sativa) subtypes were used in this work: Nipponbare belongs to the japonica subspecies (O. sativa ssp. no. 6045). The ITS1 and ITS2 locus matched by the 5′ and 3′ ends of these DNA sequences, respectively, are indicated by black triangles and the number of clones. The ITS1-first mode in rice resembles that in Arabidopsis (Hang et al., 2014; Weis et al., 2015a, 2015b) and mammalian systems, rather than the unicellular budding yeast, in which the 5′ ETS-first mode is the dominant pathway (Mullineux and Lafontaine, 2012; Henras et al., 2015). For this purpose, the closest tomato homologue to the A. thaliana transcription factor was identified ( Supplementary Figure S3 ) and specific oligonucleotides were designed ( Supplementary Table S1 ). Epub 2014 Feb 18. Although 18S-A2 could be detected by S7, its low abundance in wild-type rice makes it harder to distinguish from 18S-A3 by northern-blot assay. S6B and S7B), which further confirmed the A3 site in rice to be between G3660/A3661 detected by P-A3, P′-A3, and 18S-A3 (Fig. Supplemental Figure S2. Mapping of the 5′ and 3′ extremities of the 35S(P) and 32S transcripts. The corresponding genes for the 18S, 5.8S and 25S rRNA, encoded by the nuclear genome, are composed in transcription units which are located as rDNA (ribosomal DNA) repeats in the NOR (nucleolus … Mapping of the 5′ and 3′ extremities of the pre-25S rRNAs. 4C). Moreover, exposing rice to chilling stress resulted in the inhibition of rRNA biogenesis mainly at the pre-rRNA processing level, suggesting that these energy-intensive processes may be reduced to increase acclimation and survival at lower temperatures. The numbers below each lane represent the intensity ratio of each signal relative to the 0 h sample. Four pairs of primers were used for pre-25S rRNAs: 25P1 (25L/25R), 25P2 (p44/25R), 27P1 (58L/25R), and 27P2 (p4/25R). A and B, Northern blots to detect pre-rRNA processing in Nipponbare (japonica) rice under 4°C treatment for 0, 2, 4, and 6 h, with probes S7 (A) and p42 (B). 6). The asterisk detected by probe S7 represents the mature 16S rRNAs. 5A), recognized pre-5.8S rRNAs and 27S rRNAs in the pre-60S LSU (Fig. Furthermore, northern-blot assays showed that the major ITS1-first and the minor 5′ ETS-first processing pathways coexist in vivo to ensure rRNA maturation in rice. Mapping of the 5′ and 3′ extremities of the 35S(P) and 32S transcripts. A, Structure…, Mapping of the 5′ and 3′ extremities of the pre-5.8S rRNAs. S10A), after 2 h in the dark, 0.15 to ∼0.20 g samples of shoots and roots were harvested separately as 0-h controls. Cytogenetic features of rRNA genes across land plants: analysis of the Plant rDNA database. S8–S11), seedlings were grown in soil or water in growth chambers (12-h-light/12-h-dark cycle with light intensity of 200 μmol quanta m−2 s−1 and 80% humidity, unless otherwise specified) at 28°C for 10 d after germination. Abstract. The number of identical clones are indicated to the left (A) and right (B) of each fragment, respectively. Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively.  |  In order to confirm the observed ribosomal stress caused by CEVd in tomato plants, the induction of the ribosomal stress response mediator NAC082 was studied in infected tomato plants. The 3′-5.8S (7S and 6S) and 5′-5.8S are pre-5.8S rRNAs. S8). We do not capture any email address. 6). The P′-A3 intermediates identified by primers 18P2 and 18P8 were validated by sequencing of 21 independent clones (E). Given that several RP genes are up‐regulated in the apum23 mutant, and that the rpl4 mutant, which lacks ribosomal protein L4 (RPL4) is resistant to streptomycin (Rosado et al., 2010), it is likely that the apum23 plant has altered ribosomal functions or has an aberrant population of ribosomes that are unable to bind streptomycin. This variation in pre-rRNA processing between these two rice subspecies may come from genome variation during evolution (Huang et al., 2012), a possibility that will require further examination in the future. Alternatively, the identification of 32S rRNA, the intact 18S-ITS1-5.8S-ITS2-25S intermediate ranging from site A1 to B2, defines the minor 5′ ETS-first pathway, which coexists in Arabidopsis and involves ITS1 cleavage after complete removal of the 5′-ETS (Hang et al., 2014; Weis et al., 2014, 2015b). 58P1 and 58P2 ( 58L2/58R2 ; Fig of P′-A3 was at G1634/A1635 of TCGGAAGACGACAG in same! 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