Model of rRNA biogenesis in rice. The 18S-A2 intermediates identified by primers 18P1 and 18P8 were validated by sequencing of 33 independent clones (C). The resulting precursor-rRNA (pre-rRNA) transcript undergoes systematic processing, where multiple endonucleolytic and exonucleolytic cleavages remove the external and internal transcribed spacers (ETS and ITS). 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). The 3′-5.8S (7S and 6S) and 5′-5.8S are pre-5.8S rRNAs. Mapping of the 5′ and 3′ extremities of the pre-18S rRNAs. Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. 27SA2, 27SA3, and 27SB belong to the 27S rRNA, the common precursor of 5.8S and 25S rRNAs. In general, budding yeast pre-rRNA has two major endonucleolytic sites in the 5′ ETS (A0 and A1), five in ITS1 (D, A2, A3, B1L, and B1S), three in ITS2 (E, C2, and C1), and two in the 3′ ETS (B2 and B0; Mullineux and Lafontaine, 2012; Woolford and Baserga, 2013; Henras et al., 2015; Tomecki et al., 2017). 5B). The northern-blot assays were performed as described (Hang et al., 2014), with slight modification. Sequence alignments of 5′ ETS, ITS1, ITS2, and partial 3′ ETS rDNAs between the japonica rice Nipponbare and the Arabidopsis thaliana accession Col-0. 4C). Although Pumilio proteins have been characterized in many eukaryotes, their role in plants is unknown. Supplemental Figure S3. 5A). Using cryo-electron microscopy, the assembly of the 90S/SSU processome (Kornprobst et al., 2016; Zhang et al., 2016; Johnson et al., 2017; Sun et al., 2017) and pre-60S LSU (Gamalinda et al., 2014; Greber, 2016; Greber et al., 2016; Wu et al., 2016; Ma et al., 2017) were further resolved. ), the Young Scientist Foundation of State Key Laboratory of Plant Genomics (2015D0129-03 to R.H.), and the State Key Laboratory of Plant Genomics. 5A), recognized pre-5.8S rRNAs and 27S rRNAs in the pre-60S LSU (Fig. S6A and S7B). Similarly, the 27SA3 and 27SA2 sites were detected by primer combination 27P2 (Fig. The P-A3, P′-A3, and 18S-A3 intermediates further confirmed the A3 site to be between G3660/A3661 in “GTCAAGGAACACAG” in the ITS1 region (Supplemental Figs. 1–4; Supplemental Fig. 5E), as detected by probes p23 (Fig. Pre-rRNA processing in rice shoots in response to chilling stress. Chilling stress inhibits pre-rRNA processing, shown by the time-course reduction of P-A3 and 27SA2 in both ITS1-first and 5′ ETS-first processing pathways, respectively (A and B). These intermediates are degraded sequentially by the nuclear exosome complex (LaCava et al., 2005; Houseley et al., 2006; Doma and Parker, 2007; Lange et al., 2009; Losh and van Hoof, 2015; Thoms et al., 2015). Error bars represent sd. RiboMinus™ Plant Kit for RNA-Seq is the complete solution for transcriptome isolation and enrichment of the true whole transcriptome through selective depletion of ribosomal RNA in plant species. 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). 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focus on coordinated action of endo- and exoribonucleases, Ribosomes as sensors of heat and cold shock in, The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts, Nucleolar DEAD-box RNA helicase TOGR1 regulates thermotolerant growth as a pre-rRNA chaperone in rice, The economics of ribosome biosynthesis in yeast, Temperature sensitive mutations affecting ribosome synthesis in, Plant-specific features of ribosome biogenesis, The 60S associated ribosome biogenesis factor LSG1-2 is required for 40S maturation in, atBRX1-1 and atBRX1-2 are involved in an alternative rRNA processing pathway in, Diverse roles of assembly factors revealed by structures of late nuclear pre-60S ribosomes, High-resolution structure of the eukaryotic 80S ribosome, Arabidopsis thaliana XRN2 is required for primary cleavage in the pre-ribosomal RNA, Mutations in eIF5B confer thermosensitive and pleiotropic phenotypes via translation defects in, Stepwise and dynamic assembly of the earliest precursors of small ribosomal subunits in yeast, Rice and cold stress: methods for its evaluation and summary of cold tolerance-related quantitative trait loci, Natural variation in CTB4a enhances rice adaptation to cold habitats, Photoperiod- and thermo-sensitive genic male sterility in rice are caused by a point mutation in a novel noncoding RNA that produces a small RNA, RNase Z(S1) processes UbL40 mRNAs and controls thermosensitive genic male sterility in rice, Arabidopsis small nucleolar RNA monitors the efficient pre-rRNA processing during ribosome biogenesis, BRASSINOSTEROID-SIGNALING KINASE5 Associates with Immune Receptors and Is Required for Immune Responses, Developmental Programming of Thermonastic Leaf Movement, by The American Society of Plant Biologists, National Natural Science Foundation of China, Ribosomal RNA Biogenesis and Its Response to Chilling Stress in Oryza sativa. In the major ITS1-first pathway, the 35SP transcript is split at ITS1 endonucleolytic site A3 into P-A3 and 27SA3 precursors. Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. Image J was used to quantify band intensity (Schneider et al., 2012). 1A; Supplemental Fig. Primary transcripts generated by RNA Polymerase I are first processed at P in the 5′ ETS and at an unknown site in the 3′ ETS to generate 35S(P), which undergoes further pre-rRNA processing by alternative pathways distinguished by the order of ITS1 splitting and 5′ ETS removal, to generate mature 18S, 5.8S, and 25S rRNAs. 3A) and 5′-5.8S (Fig. Then, multiple endonucleolytic and exonucleolytic processing steps sequentially and coordinately remove the ETS and ITS regions to release mature 18S, 5.8S, and 25S/28S rRNAs. 1, A and D–F; Supplemental Fig. Sequence identities were determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) in NCBI Global Alignment tool. Eight pairs of primers were used: 18P1 (18L/18R1), 18P2 (18L/18R3), 18P3 (p23/18R3), 18P4 (p24/18R3), 18P5 (S5/18R3), 18P6 (p24/18R2), 18P7 (p23/18R2), and 18P8 (18L/18R2). A, Structure of pre-18S rRNA intermediates identified by a set of primer combinations (in shaded box). 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. Weis BL, Palm D, Missbach S, Bohnsack MT, Schleiff E. RNA. See this image and copyright information in PMC. Mapping of the 5′ and 3′ extremities of the pre-18S rRNAs. 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. three types of ribosomal RNA are present in a plant cell, mitochondrial, chloroplastic and nuclear. We propose that currently unknown transacting factors or higher-order rRNA structures (Phipps et al., 2011; Kornprobst et al., 2016; Zhang et al., 2016; Johnson et al., 2017; Sun et al., 2017) may contribute to site selection in both species in vivo. In WT vegetative cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. The P′-A3 intermediates identified by primers 18P2 and 18P8 were validated by sequencing of 21 independent clones (E). 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. Mapping of the 5′ and 3′ extremities of the pre-25S rRNAs. Recent work showed that the DEAD-box RNA helicase TOGR1 (Thermo-tolerant Growth Required 1), the rice homolog of Rrp3 (rRNA processing protein 3) in S. cerevisiae (O’Day et al., 1996) and DDX47 in Homo sapiens (Sekiguchi et al., 2006), is required for rice thermo-tolerant growth, acting as a key chaperone for rRNA homeostasis by fine-tuning pre-rRNA processing (Wang et al., 2016). To test for a potential relationship between chilling stress and ribosome biogenesis at the level of pre-rRNA processing in rice, we performed northern-blot assays with rice shoots after a time-course chilling treatment (Fig. RNA samples from two biological replicates were loaded and detected in parallel. Supplemental Figure S9. However, their function in pre-rRNA processing remains poorly understood. Then, endonucleolytic cleavage at the A2 site in ITS1 splits the 90S processome/SSU into pre-40S and pre-60S particles, which further undergo a series of endo- and exonucleolytic processing events and finally mature into the 40S and 60S subunits, respectively (Venema and Tollervey, 1999; Woolford and Baserga, 2013; Fernández-Pevida et al., 2015; Henras et al., 2015). 2F), but the 3′ extremities of the 18S-A2 fragments we identified were much more heterogeneous (Fig. 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. 5D; Supplemental Fig. The 18S-A3 intermediates identified by primers 18P2 and 18P8 were validated by sequencing of 58 independent clones (D). Eight pairs of primers were used: 18P1 (18L/18R1), 18P2 (18L/18R3), 18P3 (p23/18R3), 18P4 (p24/18R3), 18P5 (S5/18R3), 18P6 (p24/18R2), 18P7 (p23/18R2), and 18P8 (18L/18R2). S5). Different rRNA precursors are marked. Published May 2018. Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. D, Simplified model that the inhibition of rRNA biogenesis in rice by chilling stress predominantly occurs at posttranscriptional level. S6A; Supplemental Table S2). The P-A3 intermediate and its direct precursor 35S(P) were both readily detected by the 5′ ETS probe p23 (Fig. The results suggested that active polyadenylation-dependent RNA processing systems, such as those mediated by the TRAMP (Jia et al., 2011; Lange et al., 2014) and nuclear RNA exosome (LaCava et al., 2005; Houseley et al., 2006; Doma and Parker, 2007; Lange et al., 2009; Losh and van Hoof, 2015; Sikorski et al., 2015; Thoms et al., 2015), exist in rice and take part in pre-18S rRNA processing. (B) RNA gel blot analysis performed with total RNA isolated from leaves of TRV2:myc, TRV2:ARPF2(N), and TRV2:ARPF2(C) plants. A, Structure of pre-18S rRNA intermediates identified by a set of primer combinations (in shaded box). The P-A3 intermediates identified by primers 18P6, 18P7, 18P3, and 18P4 were validated by sequencing of 87 independent clones (F). C and D, DNA sequencing results for 32S (C) and 35S(P) precursors (D). Therefore, the 45S rRNA in vivo is the net product of rDNA transcription and subsequent pre-rRNA processing. 3B) fragments, respectively. S6B and S7B), two precursors processed by direct cleavage at the A2 site in ITS1 from the 32S transcript (Weis et al., 2015b). 6). Processing of ribosomal RNAs (rRNAs) is an essential step in ribosome biogenesis and begins with transcription of the rDNA. The 27SA3 intermediates identified by primers 27P1 and 27P2 were validated by sequencing of 22 independent clones (E). Epub 2014 Feb 18. Rice (Oryza sativa) is a model monocot plant and a major staple food worldwide. 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. Aberrant sensitivity to temperature fluctuation is a hallmark of mutants with defects in ribosome biogenesis in E. coli (Guthrie et al., 1969; Dammel and Noller, 1993; Jones et al., 1996; Al Refaii and Alix, 2009; Mayerle and Woodson, 2013), yeast (Warner and Udem, 1972; Tollervey et al., 1993; Teyssier et al., 2003; Wan et al., 2015), and Arabidopsis (Ohbayashi et al., 2011; Huang et al., 2016; Liu et al., 2016). More than three biological replicates were performed for upper treatments and the representative data were exhibited. The number of clones containing additional sequences at the 3′ extremities are marked in parentheses (in the shaded box). The relative intensities for 25S rRNA, 45S transcripts, and P-A3 intermediates are marked in black, blue, and red, respectively. ↵[OPEN] Articles can be viewed without a subscription. 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). 7; Supplemental Figs. Ribosome biogenesis is fundamental to growth and development in eukaryotes and is linked to human diseases and cancer. Mapping of the 5′ and 3′ extremities of the pre-18S rRNAs. Therefore, we propose that chilling stress affects rRNA biogenesis predominantly at the pre-rRNA processing level in rice, which results in decreased biogenesis of P-A3 and 27SA2. 1, B and E) intermediates were also amplified (Fig. 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). Ribosomal RNA genes in plants are highly variable both in copy number and in intergenic spacer (IGS) length. We detected 45S rRNA transcripts by northern blots with a specific long probe (45P) that recognizes the 5′ ETS region upstream of the P site (Fig. We do not capture any email address. Pre-18S rRNA intermediates are processed by endonucleolytic cleavages in the 5′ ETS and the ITS1 surrounding the 18S rRNA (Fig. Northern blot with probe 45P to detect 45S rRNA transcript under chilling treatment. (2) A polyadenylation-dependent exosome system (Chekanova et al., 2000, 2007; LaCava et al., 2005; Lange et al., 2008, 2009; Sikorski et al., 2015) may also exist in rice to promote rRNA maturation. Little is known about the RNA helicases involved in pre-60S ribosomal subunit processing and assembly in plants. Two pre-rRNA processing pathways, known as the 5′ ETS-first and the ITS1-first modes, commonly coexist in eukaryotes (Lafontaine, 2010; Mullineux and Lafontaine, 2012; Hang et al., 2014; Henras et al., 2015; Weis et al., 2015a; Tomecki et al., 2017). B, The 32S and 35S(P) pre-rRNAs were determined in gel by cRT-PCR with primers 32P1 (18L/25R) and 32P2 (p23/25R). The processing sites and pathways for pre-rRNA processing have been deciphered in Saccharomyces cerevisiae and, to some extent, in Xenopus laevis, mammalian cells, and Arabidopsis (Arabidopsis thaliana). 1. 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. The 27SB intermediates identified by primers 25P2 and 27P1 were validated by sequencing of 51 independent clones (D). 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. The ribosomal subunits consist of a few ribosomal RNA (rRNA) species and a set of ribosomal proteins. Pre-rRNA processing in rice roots responses to chilling stress. A, Structure of pre-25S intermediates identified by a set of primers (in shaded box). Three biological replicates were performed and a representative result is shown here. Remove rRNA from plant leaf, seed, and root tissue. S3), we performed gel-blot analyses of RNA extracted from smo4-2, smo4-3, and den2 plants using the previously described S2, S7, and S9 probes, which are complementary to a segment of the 5′-ETS, internal transcribed spacer 1 (ITS1), and ITS2, respectively (Fig. The rice materials were first ground into fine powder with liquid nitrogen. C to F, DNA sequencing of 18S and its major precursors identified: 18S-A2 (C), 18S-A3 (D), P′-A3 (E), and P-A3 (F). Then, 18S-A2 (by 18P1 and 18P8; Fig. The 5′ ETS probe p23 between the P and P′ sites distinguished 35S(P) from 32S precursors in the 90S/SSU complex (Fig. In conclusion, we defined rRNA biogenesis at the level of pre-rRNA processing in rice and uncovered a molecular link between chilling stress and ribosome biogenesis in vivo. Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. Primary transcripts generated by RNA Polymerase I are first processed at P in the 5′ ETS and at an unknown site in the 3′ ETS to generate 35S(P), which undergoes further pre-rRNA processing by alternative pathways distinguished by the order of ITS1 splitting and 5′ ETS removal, to generate mature 18S, 5.8S, and 25S rRNAs. 2014;42(17):11180-91. doi: 10.1093/nar/gku787. Similarly, the relative amount of 27SA2 was also far less than that of P-A3 in Nipponbare detected by ITS1 probe p42 (Fig. NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. 2A), which allowed us to identify B1 and B2 as the left and right borders, respectively, of the 5.8S and 25S rRNAs (Supplemental Figs. Ribosomal proteins (RPs) are essential structural components of ribosomes. 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. Both P-A3 in the ITS1-first pathway and 27SA2 in the 5′ ETS-first pathway decreased under chilling stress in shoots (Fig. 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). Then, total RNA was extracted from the powder with TRNzol reagent (Tiangen; DP405-02) according to the manufacturer’s instructions. Nevertheless, our results suggest that rice may fine-tune ribosome biogenesis to quickly adjust energy consumption and primary metabolism for survival and acclimation at low temperatures. S6A, S7A, and S7B; Supplemental Table S1). Then the 35S(P) transcript enters two alternative maturation pathways distinguished by the order of ITS1 cleavage and 5′ ETS removal. 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