Black vertical arrows above the diagram indicate endonucleolytic cleavage sites relevant to this study. Zhongxian3037, togr1-1 mutants (Wang et al., 2016), and 9311 belong to the indica rice subspecies (O. sativa ssp. 6). For each fragment, the number of clones obtained is indicated on the right. 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). All Rights Reserved. 2E; Supplemental Figs. 1E; Supplemental Fig. B, Northern blots to determine pre-rRNA processing in pre-60S LSU in Nipponbare (lane 1), Zhongxian3037 (ZX3037, lane 2), and togr1 mutants (lanes 3 and 4). A, Structure of pre-25S intermediates identified by a set of primers (in shaded box). Weis BL, Palm D, Missbach S, Bohnsack MT, Schleiff E. RNA. 1A; Supplemental Figs. © 2018 American Society of Plant Biologists. 1A; Supplemental 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). National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error. The 18S rRNAs identified by primers 18P1 were validated by sequencing of 20 independent clones. Similarly, the P-A3 intermediates were detected by four pairs of primers, 18P3, 18P4, 18P6, and 18P7 (Fig. It is a major constituent of the plant, representing up to 40% in its extracts. 1B). analyzed the data; R.H. and X.C. S8). Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. Mapping the cleavage sites on mammalian pre-rRNAs: where do we stand? Supplemental Figure S7. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. Two rice (Oryza sativa) subtypes were used in this work: Nipponbare belongs to the japonica subspecies (O. sativa ssp. Although the BoU3/NF-D complex has not been identified in Arabidopsis, systemic quantitative proteomic assays from subcellular fractionations identified plant-specific ribosome biogenesis factors in Arabidopsis (Palm et al., 2016). In the present study, we characterized an Arabidopsis Pumilio-encoding gene, APUM23. The relative intensities for 25S rRNA, 45S transcripts, and P-A3 intermediates are marked in black, blue, and red, respectively. 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). We further found that two pre-rRNA processing pathways, distinguished by the order of 5' ETS removal and ITS1 cleavage, coexist in vivo. 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. Chilling stress inhibits rRNA biogenesis mainly at pre-rRNAs processing levels. Ribosomal proteins (RPs) are essential structural components of ribosomes. To determine the steps of pre-18S rRNA processing in rice, we first performed cRT-PCR assays based on the canonical 18S rDNA annotation to identify specific 18S rRNA precursors in vivo (Supplemental Fig. P-A3, P′-A3, 18S-A3, and 18S-A2 belong to the pre-18S rRNAs. S9 and S10). The 18S-A2 intermediates identified by primers 18P1 and 18P8 were validated by sequencing of 33 independent clones (C). Our findings reveal that A. thalianaP5SM binds to ribosomal protein L5 in a directly analogous fashion to the eukaryotic 5S-L5 interaction. 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. Both P-A3 in the ITS1-first pathway and 27SA2 in the 5′ ETS-first pathway decreased under chilling stress in shoots (Fig. Oligonucleotides used in this article. Supplemental Figure S5. 7; Supplemental Figs. Published May 2018. Different rRNA precursors are marked. 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. 5B), but its definite 3′ extremities are still unclear (A). Here, we identified the rRNA intermediates and critical processing sites of the 5′ ETS and ITS1 regions in rice (Supplemental Figs. Alternative pre-rRNA processing is a conserved molecular characteristic in eukaryotes and has been well defined in budding yeast (Woolford and Baserga, 2013), mammalian cells (Bowman et al., 1981; Hadjiolova et al., 1993; Kent et al., 2009; Mullineux and Lafontaine, 2012; Henras et al., 2015), and Arabidopsis (Sikorski et al., 2015; Weis et al., 2015a; Tomecki et al., 2017). 2, B and D). 5B), but its definite 3′ extremities are still unclear (A). For seedlings in water (Supplemental Fig. To identify these primary transcripts and how they are processed in rice, we used the fixed forward primer 25R and reverse primers 18L and p23 to perform the cRT-PCR assay (Fig. B, The 32S and 35S(P) pre-rRNAs were determined in gel by cRT-PCR with primers 32P1 (18L/25R) and 32P2 (p23/25R). Then the 35S(P) transcript enters two alternative maturation pathways distinguished by the order of ITS1 cleavage and 5′ ETS removal. 2015 Mar-Apr;6(2):191-209. doi: 10.1002/wrna.1267. 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 3′-5.8S (7S and 6S) and 5′-5.8S are pre-5.8S rRNAs. The blots were washed and exposed to a storage phosphor screen (GE Healthcare), then detected with a Typhoon TRIO scanner (GE Healthcare). A, Structure…, Mapping of the 5′ and 3′ extremities of the pre-25S rRNAs. The 5′ ETS probe p23 between the P and P′ sites distinguished 35S(P) from 32S precursors in the 90S/SSU complex (Fig. Although Pumilio proteins have been characterized in many eukaryotes, their role in plants is unknown. Hereafter, we refer to this as the “5′ ETS-first” pathway (Supplemental Fig. 4, A, B, and D). Processing of ribosomal RNAs (rRNAs) is an essential step in ribosome biogenesis and begins with transcription of the rDNA. S6A, S7A, and S7B; Supplemental Table S1). The 7S rRNA marked with “?” was detected by probe S9 (Fig. 2014;42(17):11180-91. doi: 10.1093/nar/gku787. 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). 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). Therefore, our detection of a similar pre-rRNA pattern in vivo with RNA hybridization (Fig. Many RBFs are involved in the processing of the primary ribosomal (r)RNA transcript, in which three of the four rRNAs are imbedded. S4). Epub 2014 Oct 18. The S7 and p42 blots share the same loading control (D). 3A), similar to the 6S intermediates in Arabidopsis (Shanmugam et al., 2017). 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 … S6A and S7B). Moreover, functional studies of ribosome biogenesis mutants have identified the series of rRNA intermediates that occur during pre-rRNA processing (Lange et al., 2008, 2011; Abbasi et al., 2010; Zakrzewska-Placzek et al., 2010; Ohbayashi et al., 2011; Kumakura et al., 2013; Missbach et al., 2013; Hang et al., 2014; Weis et al., 2014, 2015b; Sikorski et al., 2015; Zhu et al., 2016). no. In many instances, RPs are encoded in small families of genes, suggesting possible neofunctionalization for some family members. S5C), and positive clones were selected for with a second PCR using the M13F and M13R primers. Processing of ribosomal RNAs (rRNAs) is an essential step in ribosome biogenesis and begins with transcription of the rDNA. After amplification for 35 cycles, bands obtained by cRT-PCR were subcloned into the pEasy-T vector (Transgene; CT101-02; Supplemental Fig. Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. 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). Northern blots to detect pre-rRNA processing in rice by probes p23 and S7A. 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. 1C) and the 5′ ends of 27SA2 (Fig. 1, E, and F; Supplemental Figs. Database searching was performed at NCBI. Nucleic Acids Res. Ribosome biogenesis is crucial for plant growth and environmental acclimation. 2018 Apr;64(2):393-404. doi: 10.1007/s00294-017-0764-x. Feedback regulation of ribosome assembly. Multiple alignments of DNA sequences were performed with ClustalX (Larkin et al., 2007) and were manually edited with the GeneDoc program. Plant J. 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. S6B, S7A, and S7B).  |  B, Pre-18S rRNA intermediates were determined in gel by cRT-PCR with primers 18P1 to 18P8. Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. Then, total RNA was extracted from the powder with TRNzol reagent (Tiangen; DP405-02) according to the manufacturer’s instructions. 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. Thus, alternative pre-rRNA processing events are generally believed to come from uncoupled processing for 5′ ETS removal and ITS1 cleavage mediated by the pre-ribosomal complex, the 90S/SSU processome, that was identified in budding yeast (Dragon et al., 2002; Grandi et al., 2002; Osheim et al., 2004; Phipps et al., 2011). Four (for short probes) or ten (for long probes) μg of total RNA was separated on a 1.2% (w/v) agarose/formaldehyde gel and then transferred to a Hybond N+ membrane (GE Healthcare; RPN1520B) by capillary elution. 5; Supplemental Table S1; Supplemental Fig. For circular RT-PCR assays (Figs. B, The 32S and 35S(P) pre-rRNAs were determined in gel by cRT-PCR with primers 32P1 (18L/25R) and 32P2 (p23/25R). S6 and S7). Circular RT-PCR assay to identify pre-rRNA precursors. Sequence identities were determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) in NCBI Global Alignment tool. Supplemental Figure S1. The 27SB intermediates identified by primers 25P2 and 27P1 were validated by sequencing of 51 independent clones (D). The 35S rRNA primary transcripts in budding yeast and Arabidopsis (Arabidopsis thaliana) are equivalent to the 47S rRNA transcripts in mammalian cells (Layat et al., 2012; Henras et al., 2015).