More recently, the determination of 33S(P′) and 27SA2 rRNAs (Weis et al., 2015b) as the direct precursor and product of the 32S rRNA, respectively, further demonstrates the existence of the minor 5′ ETS-first pathway in Arabidopsis (Weis et al., 2015a). After amplification for 35 cycles, bands obtained by cRT-PCR were subcloned into the pEasy-T vector (Transgene; CT101-02; Supplemental Fig. 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). Therefore, our detection of a similar pre-rRNA pattern in vivo with RNA hybridization (Fig. Arabidopsis thaliana MORPHOLOGY OF ARGONAUTE1-52 SUPPRESSED 2 (MAS2) participates in splicing and 45S ribosomal DNA (rDNA) expression. The ribosome acts as a temperature sensor in Escherichia coli to coordinate metabolism and growth in response to the environment (VanBogelen and Neidhardt, 1990; Warner, 1999; Moss, 2004). In rice, temperature fluctuations such as heat and chilling stresses adversely affect the vegetative and reproductive stages (Zhou et al., 2012, 2014; Fan and Zhang, 2014), which eventually affect yields (Cruz et al., 2013; Ray et al., 2015). The 5′ ETS probe p23 between the P and P′ sites distinguished 35S(P) from 32S precursors in the 90S/SSU complex (Fig. Similarly, the 35S(P) fragment was further identified by primer combination 32P2 (p23/25R; Fig. 5D; Supplemental Fig. The remaining seedlings were transferred to precooled water and treated in a dark growth chamber at 4°C. Chilling stress inhibits rRNA biogenesis mainly at pre-rRNAs processing levels. Here, the reads for the 27SA2 intermediate shared the definite A2 site at their 5′ extremities (Fig. The 18S-A2 intermediates identified by primers 18P1 and 18P8 were validated by sequencing of 33 independent clones (C). A more definitive demonstration of the role of plant RNase T2 enzymes in tsRNA production was recently presented by Megel et al. Moreover, the abundance of P-A3 in the indica cultivar Zhongxian3037 was less than in the japonica cultivar Nipponbare (Fig. Sequence alignments of 5′ ETS, ITS1, ITS2, and partial 3′ ETS rDNAs between the japonica rice Nipponbare and the Arabidopsis thaliana accession Col-0. Pre-rRNA processing in rice shoots in response to chilling stress. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. This variability exists not only between distantly related species, but among members of the same genus and also among members of the same population of a single species. 1A; Supplemental Fig. Here, we propose a working model for rRNA biogenesis in rice (Fig. In many instances, RPs are encoded in small families of genes, suggesting possible neofunctionalization for some family members. Hammond MC(1), Wachter A, Breaker RR. three types of ribosomal RNA are present in a plant cell, mitochondrial, chloroplastic and nuclear. Arabidopsis protein arginine methyltransferase 3 is required for ribosome biogenesis by affecting precursor ribosomal RNA processing Runlai Hanga,b, Chunyan Liua, Ayaz Ahmada,b,1, Yong Zhanga,b,2, Falong Lua,b,3, and Xiaofeng Caoa,c,4 aState Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Also, constitutive expression of TOGR1 enhanced the tolerance of rice to heat stress (Wang et al., 2016). Compared with the 18S, 5.8S, and 25S rDNAs (Supplemental Figs. 5B), but its definite 3′ extremities are still unclear (A). The 27SB intermediates identified by primers 25P2 and 27P1 were validated by sequencing of 51 independent clones (D). Hybridization was performed overnight at 45°C (for short probes) or 65°C (for long probes) as previously described (Hang et al., 2014). 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). Then, total RNA was extracted from the powder with TRNzol reagent (Tiangen; DP405-02) according to the manufacturer’s instructions. The 18S-A2 intermediates identified by primers 18P1 and 18P8 were validated by sequencing of 33 independent clones (C). Circular RT-PCR assay to identify pre-rRNA precursors. 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). A, Pre-rRNA processing intermediates detected…, Model of rRNA biogenesis in rice. 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 35S(P) and 27SA2 could be specifically detected by probes p23 and p42, respectively. Environmental signals affect plant growth and crop yield. Ribosome biogenesis is fundamental to growth and development in eukaryotes and is linked to human diseases and cancer. The P′-A3 intermediates identified by primers 18P2 and 18P8 were validated by sequencing of 21 independent clones (E). Author information: (1)Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. ↵1 This work was supported by grants from the National Natural Science Foundation of China (grants 91540203, 31788103, and 31330020 to X.C., 31770874 and 31370770 to C.L., and 31571332 to B.M. The 18S-A3 intermediates identified by primers 18P2 and 18P8 were validated by sequencing of 58 independent clones (D). Supplemental Figure S4. For seedlings in water (Supplemental Fig. 7D). 3B; Henry et al., 1994; Zakrzewska-Placzek et al., 2010). S5, A and B). As an essential, complicated, energy-intensive process (Warner, 1999), ribosome biogenesis is strictly regulated by endogenous signals (Lykke-Andersen et al., 2009; Lafontaine, 2010; Sanchez et al., 2016) and environmental stimuli (Sinturel et al., 2017) such as ambient temperature (Warner and Udem, 1972; Tollervey et al., 1993; Al Refaii and Alix, 2009; Ohbayashi et al., 2011). Cannabidiol Cannabidiol (CBD) is a cannabinoid found in cannabis. S6A and S7B). The 45S rRNA, transcribed by RNA Pol I from rDNAs, undergoes pre-rRNA processing to release mature rRNAs. 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. S6A, S7A, and S7B; Supplemental Table S1). First, RNA polymerase I (Pol I) transcribes the tandem repeated rDNA units into polycistronic primary transcripts, where the 18S, 5.8S, and 25S/28S rRNAs are separated by the internal transcribed spacer 1 (ITS1) and ITS2, and flanked by 5′ and 3′ external transcribed spacers (5′ ETS and 3′ ETS, respectively; Henras et al., 2015). D, Simplified model that the inhibition of rRNA biogenesis in rice by chilling stress predominantly occurs at posttranscriptional level. HHS For cold treatment of seedlings in soil (Fig. Matured rRNAs stained with MB serve as the loading control. Rice (Oryza sativa) is a model monocot plant and a major staple food worldwide. Mapping of the 5′ and 3′ extremities of the pre-18S rRNAs. Notably, we found that P-A3, P′-A3, and 18S-A3 in rice were highly polyadenylated (Fig. S5A). Ribosome biogenesis is fundamental to growth and development in eukaryotes and is linked to human diseases and cancer. 5; Supplemental Fig. The number of clones containing additional sequences at the 3′ extremities are marked in parentheses (in the shaded box). Fernández-Pevida A, Kressler D, de la Cruz J. Wiley Interdiscip Rev RNA. 27SA2, 27SA3, and 27SB belong to the 27S rRNA, the common precursor of 5.8S and 25S rRNAs. We do not capture any email address. 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). B, The 32S and 35S(P) pre-rRNAs were determined in gel by cRT-PCR with primers 32P1 (18L/25R) and 32P2 (p23/25R). For circular RT-PCR assays (Figs. 5B). rRNA intermediates and primer combinations used in cRT-PCR assays. For each fragment, the number of clones obtained is indicated on the right. Fresh materials were frozen by liquid nitrogen and stored at −80°C until used. Hereafter, we refer to this as the “5′ ETS-first” pathway (Supplemental Fig. Then, 1 μg of purified 45P fragment was subjected to labeling with [α-32P]dCTP (Perkin-Elmer; NEG513H) using a commercial Random Primer DNA Labeling kit (TaKaRa; cat. 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. The 27SA2 intermediates identified by primers 27P2 were validated by sequencing of 21 independent clones (F). 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. More than three biological replicates were performed for upper treatments and the representative data were exhibited. The ribosomal subunits consist of a few ribosomal RNA (rRNA) species and a set of ribosomal proteins. The numbers below each lane represent the intensity ratio of each signal relative to the 0 h sample. For northern-blot assays (Figs. Matured rRNAs stained with MB serve as the loading control. S10C), indicating reduced pre-rRNA processing. The number of identical clones is indicated to the right of each fragment. The 32S pre-rRNAs were validated by sequencing of 20 independent clones (D). Representative DNA sequencing results for the identified pre-rRNAs. Overall, our study identified the pre-rRNA processing pathway in rice and showed that ribosome biogenesis is quickly inhibited by low temperatures, which may shed light on the link between ribosome biogenesis and environmental acclimation in crop plants. USA.gov. The RNA components of the plant cytoplasmic ribosomes consist of the 17/18S rRNA (ribosomal RNA) in the 40S ribosome subunit and the 5S, 5.8S, and 25S/26S rRNA in the 60S ribosome subunit. Precursors with partial transparency indicate putative intermediates in these pathways. The 45S pre-rRNA transcribed from the rDNA clusters by Pol I is quickly processed into 35S(P) by cleavage at the P site in the 5′ ETS and an unknown site in the 3′ ETS and then undergoes pre-rRNA processing to release mature rRNAs (Fig. 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). B, Northern blots to determine pre-rRNA processing in pre-60S LSU in Nipponbare (lane 1), Zhongxian3037 (ZX3037, lane 2), and. Both probes S7 and p42 detect 35S(P), 32S, P-A3, and 18S-A3. Moreover, the ITS1 probe p42 between A2 and A3 sites detected 18S-A3 and 27SA2 specifically (Fig. Epub 2014 Feb 18. The ITS1 locus matched by the 3′ ends of these clones are indicated by black triangles as well as the number of clones. 2, B, E, and F), in which the left primer p4 was in the ITS1 region adjacent to the left boundary of 5.8S rRNA (Fig. 6). 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. The 3′ ends of the 35S(P) fragments were not uniform, harboring two to seven nucleotides of extra sequence downstream of the B2 site in the 3′ ETS (Fig. The 27SA2 intermediates identified by primers 27P2 were validated by sequencing of 21 independent clones (F). 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). 2, B and C), which defined its boundary sites, C1 and B2 on the left and right borders of the 25S rRNA, respectively (Supplemental Figs. Cleavage sites and flanking sequences were identified according to japonica rice rDNA offline annotation (Supplemental Fig. S5E), 0.10 g of panicles of 1 to 2 mm in length were harvested from Nipponbare grown in the paddy fields under natural conditions for RNA extraction. S6B, S7A, and S7B). To this end, the DNA oligonucleotide 18c (Fig. The DNA sequencing reads for P-A3 intermediates (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. The S7 and p42 blots share the same loading control (D).  |  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Xiaofeng Cao (xfcao{at}genetics.ac.cn). C to E, Northern blots to determine pre-rRNA processing in pre-40S SSU by probes p23 (C), S7, and p42 (D) in rice. The P-A3 intermediates identified by primers 18P6, 18P7, 18P3, and 18P4 were validated by sequencing of 87 independent clones (F). 6). 5, B–E; Supplemental Fig. 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). 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. 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). The 35S(P) pre-rRNAs were validated by sequencing of 25 independent clones (D). The 5.8S-3′ intermediates were validated by 70 independent clones (A). Total RNA was dissolved in DEPC-treated deionized water and quantified with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific; ND-1000). Learn about the structure and function of ribosomal RNA. Supplemental Figure S6. 5A) were designed to detect the pre-18S rRNAs in the pre-40S SSU (Fig. S8). 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. In the major ITS1-first pathway, the 35SP transcript is split at ITS1 endonucleolytic site A3 into P-A3 and 27SA3 precursors. Epub 2015 Jan 20. 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, Pre-rRNA processing intermediates detected by northern blots with specific probes, which are indicated by horizontal arrows. 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