plant ribosomal rna

Histone deacetylases play critical roles in many biological processes including transcriptional repression and rDNA silencing. Moreover, the 3′ ends of the 35S(P) precursors were polyadenylated, which was rarely detected in the 32S precursors (Fig. Fresh materials were frozen by liquid nitrogen and stored at −80°C until used. Finally, we found that rRNA biogenesis in rice was inhibited by chilling stress mainly at the pre-rRNA processing (P-A3 and 27SA2) level. www.plantphysiol.org/cgi/doi/10.1104/pp.17.01714. The circular RNA was further reverse transcribed into first-strand cDNA (TransGen Biotech; AH301) using specific antisense DNA oligonucleotide 18c or 25c that are complementary to sequences in the 18S rDNA or 25S rDNA region, respectively (Supplemental Fig. 4A). S5A). In our work, the reduction of pre-rRNA processing under chilling stress indicated decreased ribosome assembly in the nucleus, which may eventually affect the production of active ribosomes in the cytoplasm. 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. analyzed the data; R.H. and X.C. B, Northern blots to determine pre-rRNA processing in pre-60S LSU in Nipponbare (lane 1), Zhongxian3037 (ZX3037, lane 2), and. P-A3, P′-A3, 18S-A3, and 18S-A2 belong to the pre-18S rRNAs. japonica; Huang et al., 2012). indica). 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. 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). This represents another regulatory layer affecting the activity of ribosomes to facilitate the acclimation and survival of rice under stress. ), the Young Scientist Foundation of State Key Laboratory of Plant Genomics (2015D0129-03 to R.H.), and the State Key Laboratory of Plant Genomics. The 18S-A2 intermediates identified by primers 18P1 and 18P8 were validated by sequencing of 33 independent clones (C). R.H., B.M., and X.C. C, Pre-5.8S rRNA intermediates were determined in gel by cRT-PCR with primers 58P1 and 58P2. The 35S primary transcripts in the 90S particle/small subunit processome (SSU; Dragon et al., 2002; Grandi et al., 2002; Osheim et al., 2004; Phipps et al., 2011) preferentially use the major “U3-dependent cleavage occurs first” pathway to cotranscriptionally remove the 5′ ETS completely, producing the 32S intermediate (Lee and Baserga, 1997; Gallagher et al., 2004; Kos and Tollervey, 2010). S4). S6A and S7B). S6B, S7A, and S7B). Model of rRNA biogenesis in rice. Our findings reveal that A. thalianaP5SM binds to ribosomal protein L5 in a directly analogous fashion to the eukaryotic 5S-L5 interaction. Cleavage sites and flanking sequences were identified according to japonica rice rDNA offline annotation (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. Fernández-Pevida A, Kressler D, de la Cruz J. Wiley Interdiscip Rev RNA. Arabidopsis thaliana MORPHOLOGY OF ARGONAUTE1-52 SUPPRESSED 2 (MAS2) participates in splicing and 45S ribosomal DNA (rDNA) expression. The number of identical clones are indicated to the left (A) and right (B) of each fragment, respectively. 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. 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). 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. Supplemental Table S1. 7D). three types of ribosomal RNA are present in a plant cell, mitochondrial, chloroplastic and nuclear. We thank Dr. Yanyuan Kang in our lab for supporting the panicle RNA samples, Zhiyao Lv in our lab for supporting the original picture of Figure S10A, and our fellow lab members for stimulating discussions. Here, we examined ribosome biogenesis at the level of pre-rRNA processing in rice, especially the processing sites, rRNA intermediates, and processing pathways by circular reverse transcription PCR (cRT-PCR; Kuhn and Binder, 2002; Perrin et al., 2004; Slomovic et al., 2008; Abbasi et al., 2010; Zakrzewska-Placzek et al., 2010; Barkan, 2011; Lange et al., 2011; Hang et al., 2014, 2015; Huang et al., 2016; Liu et al., 2016; Shanmugam et al., 2017). Supplemental Figure S6. Northern blot with probe 45P to detect 45S rRNA transcript under chilling treatment. The quantitation of P-A3 in Nipponbare (lane 1), Zhongxian3037 (lane 2), and togr1 (lanes 3 and 4) were performed with three biological replicates (E). The steady level of 45S rRNA in vivo is the net product of rDNA transcription and subsequent pre-rRNA processing. Database searching was performed at NCBI. Mapping the cleavage sites on mammalian pre-rRNAs: where do we stand? This result was consistent with the cRT-PCR data (Figs. Sequence alignment of 25S rDNAs between the japonica rice Nipponbare and Arabidopsis thaliana accession Col-0. The number of clones with additional sequences, such as polyadenylation at the 3′ end, is marked in parentheses. 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. The 27SB intermediates identified by primers 25P2 and 27P1 were validated by sequencing of 51 independent clones (D). More than three biological replicates were performed for upper treatments and the representative data were exhibited. The asterisk detected by probe S7 represents the mature 16S rRNAs. 1A; Supplemental Fig. Epub 2017 Oct 11. 2015 Mar;21(3):415-25. doi: 10.1261/rna.047563.114. 1, B and E) intermediates were also amplified (Fig. The 7S rRNA marked with “?” was detected by probe S9 (Fig. Arabidopsis thaliana MORPHOLOGY OF ARGONAUTE1-52 SUPPRESSED 2 (MAS2) participates in splicing and 45S ribosomal DNA (rDNA) expression. We further found that two pre-rRNA processing pathways, distinguished by the order of 5′ ETS removal and ITS1 cleavage, coexist in vivo. 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. The 18S-A3 intermediates identified by primers 18P2 and 18P8 were validated by sequencing of 58 independent clones (D). Pre-rRNA processing in rice shoots in response to chilling stress. 2014;42(17):11180-91. doi: 10.1093/nar/gku787. The 5′-5.8S intermediates were validated by 22 independent clones (B). 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. Here, the reads for the 27SA2 intermediate shared the definite A2 site at their 5′ extremities (Fig. © 2018 American Society of Plant Biologists. For instance, Arabidopsis IRP6 (also named DRH1; Okanami et al., 1998 ) is involved in the processing of 27SB pre-rRNA at the C2 site ( Palm et al., 2019 ). Please enable it to take advantage of the complete set of features! 7D; Supplemental Table S1). 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 mature 18S rRNA identified by the 18P1 primers had boundary sites at A1 and D on the left and right borders of 18S rDNA, respectively (Fig. 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). 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. Therefore, the 45S rRNA in vivo is the net product of rDNA transcription and subsequent pre-rRNA processing. The S7 and p42 blots share the same loading control (D). Clipboard, Search History, and several other advanced features are temporarily unavailable. 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 locations of the P and A3 endonucleolytic sites were consistent with a previous report (Wang et al., 2016). The number of identical clones are indicated to the left (A) and right (B) of each fragment, respectively. 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). :1945-1967. doi: 10.1261/rna.047563.114 polyadenylation at the 3′ end, is marked in parentheses pair..., 2010 ) therefore, the relative amount of 27SA2 was also less! At pre-rRNAs processing levels on separate lines or separate them with commas unavailable. 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