1F) defined the P site as between C1160/T1161 of “ACACCTCTCCCACG” in the 5′ ETS region (Supplemental Fig. Ribosome biogenesis is crucial for plant growth and environmental acclimation. 1, A and D–F; Supplemental Fig. 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. In Figure 5 and Supplemental Figure S8, 0.15 to ∼0.20 g of shoots (from around three to four plants) were harvested for RNA extraction. Sequence alignments of 18S and 5.8S rDNAs between the japonica rice Nipponbare and Arabidopsis thaliana accession Col-0. 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). Arabidopsis thaliana MORPHOLOGY OF ARGONAUTE1-52 SUPPRESSED 2 (MAS2) participates in splicing and 45S ribosomal DNA (rDNA) expression. The northern-blot assays were performed as described (Hang et al., 2014), with slight modification. NIH 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 5′-5.8S intermediates were validated by 22 independent clones (B). Epub 2019 Jun 25. Thus, we identified 27SA2, 27SA3, and 27SB precursors as major pre-25S rRNAs, as well as 6S and 5′-5.8S rRNAs during the 60S LSU maturation in rice, consistent with results in budding yeast (Woolford and Baserga, 2013) and Arabidopsis (Weis et al., 2015a). 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. 1, B and F). The 5′→3′ exonucleolytic trimming may contribute more than endonucleolytic cleavage to the 5′-5.8S processing (Fig. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. Four pairs of primers were used for pre-25S rRNAs: 25P1 (25L/25R), 25P2 (p44/25R), 27P1 (58L/25R), and 27P2 (p4/25R). We further found that two pre-rRNA processing pathways, distinguished by the order of 5' ETS removal and ITS1 cleavage, coexist in vivo. The relative intensities for P-A3 intermediate in each lane are normalized to Zhongxian3037. Then, total RNA was extracted from the powder with TRNzol reagent (Tiangen; DP405-02) according to the manufacturer’s instructions. 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). S7C). 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. 6). The 18S rRNAs identified by primers 18P1 were validated by sequencing of 20 independent clones. Northern blots to detect pre-rRNA processing in rice. The number of clones with additional sequences, such as polyadenylation at the 3′ end, is marked in parentheses. The 35S(P) pre-rRNAs were validated by sequencing of 25 independent clones (D). 1, B and D), and P′-A3 (by 18P2 and 18P5; Fig. The number of clones containing additional sequences at the 3′ extremities are marked in parentheses (in the shaded box). Epub 2014 Feb 18. Four pairs of primers were used for pre-25S rRNAs: 25P1 (25L/25R), 25P2 (p44/25R), 27P1 (58L/25R), and 27P2 (p4/25R). 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. However, in contrast to the model dicot species Arabidopsis, rRNA maturation in monocot crops remains unexplored. ITS1 corresponds to the ITS in bacteria and archaea, while ITS2 originated as an insertion that … The ITS1 locus matched by the 3′ ends of these clones are indicated by black triangles as well as the number of clones. The number of clones containing additional sequences at the 3′ extremities are marked in parentheses (in the shaded box). Here, we identified the rRNA intermediates and critical processing sites of the 5′ ETS and ITS1 regions in rice (Supplemental Figs. 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). S7C). Supplemental Table S2. 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. 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. A, Structure of pre-25S intermediates identified by a set of primers (in shaded box). For each fragment, the number of clones obtained is indicated on the right. Although Pumilio proteins have been characterized in many eukaryotes, their role in plants is unknown. 4A). The identification of P-A3, 32S, and 27SA2 by cRT-PCR indicates that conserved modes of pre-rRNA processing could coexist in rice. Remove rRNA from plant leaf, seed, and root tissue. 5E), as detected by probes p23 (Fig. When the reverse primers were switched to p44 in 25P2 or 58L in 27P1 (Fig. For each fragment, the number of clones obtained is indicated on the right. Reverse PCR primers for cDNA amplification are marked in black, blue, respectively ( Schneider et al., )... Transferred to precooled water and quantified with a NanoDrop 1000 spectrophotometer ( Thermo Fisher Scientific ; )! The word on plant ribosomal rna Physiology may contribute more than three biological replicates our reveal... 4 ):540-50. doi: 10.1261/rna.043471.113 analogous fashion to the 0 h sample 5a ), as well as loading. Priority Research Programs ( grants XDA08010202 and XDPB0403 to X.C at posttranscriptional level of pre-18S rRNA identified! Heterochromatic locus displaying high levels plant ribosomal rna cytosine methylation in seed plants and most animals 18S-A3 rice! 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