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. Both probes S7 and p42 detect 35S(P), 32S, P-A3, and 18S-A3. Epub 2019 Jun 25. 1A; Supplemental Tables S1 and S2). The 18S-A3 intermediates identified by primers 18P2 and 18P8 were validated by sequencing of 58 independent clones (D). Plant Cell. S9A, S9B, and S10B) and roots (Supplemental Fig. Fresh materials were frozen by liquid nitrogen and stored at −80°C until used. 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). Matured rRNAs stained with MB serve as the loading control. The northern-blot assays were performed as described (Hang et al., 2014), with slight modification. 5D; Supplemental Fig. A plant 5S ribosomal RNA mimic regulates alternative splicing of transcription factor IIIA pre-mRNAs. Supplemental Figure S2. Then the 35S(P) transcript enters two alternative maturation pathways distinguished by the order of ITS1 cleavage and 5′ ETS removal. 3B; Henry et al., 1994; Zakrzewska-Placzek et al., 2010). Accordingly, rice has evolved mechanisms to adapt to heat stress (Li et al., 2015; Shen et al., 2015; Wang et al., 2016) and cold temperature (Ma et al., 2015; Zhang et al., 2017b). 7C; Supplemental Fig. The fungal ribonuclease-like effector protein CSEP0064/BEC1054 represses plant immunity and interferes with degradation of host ribosomal RNA Author summary Powdery mildews are common plant diseases which affect important crop plants including cereals such as wheat and barley. Supplemental Figure S6. USA.gov. Mapping of the 5′ and 3′ extremities of the pre-5.8S rRNAs. Arabidopsis thaliana MORPHOLOGY OF ARGONAUTE1-52 SUPPRESSED 2 (MAS2) participates in splicing and 45S ribosomal DNA (rDNA) expression. S10C), indicating reduced pre-rRNA processing. RNA samples from two biological replicates were loaded and detected in parallel. 3A) and 5′-5.8S (Fig. 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. In higher plants, the U3 small nucleolar ribonucleoprotein (U3 snoRNP) was first purified from cauliflower inflorescences as the Nuclear Factor D complex (Sáez-Vasquez et al., 2004a, 2004b) and from Brassica oleracea as BoU3 (B. oleracea U3) complex (Samaha et al., 2010). Ribosome biogenesis in vivo is highly energy-consuming and strictly orchestrated by internal and external signals to meet the demand for mature ribosomes in mRNA translation (Warner, 1999; Woolford and Baserga, 2013). A, Pre-rRNA processing intermediates detected by northern blots with specific probes, which are indicated by horizontal arrows. In rice, TOGR1 was the first well-defined RNA helicase essential for ribosome biogenesis during rice growth and development (Wang et al., 2016). PCR amplification with primer pairs 58P1 (58L1/58R1) and 58P2 (58L2/58R2; Fig. 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 2F), but the 3′ extremities of the 18S-A2 fragments we identified were much more heterogeneous (Fig. 1, B and D), and P′-A3 (by 18P2 and 18P5; Fig. 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. Supplemental Figure S3. The numbers of identical clones are indicated to the right of each fragment. Although it remains unknown whether and how chilling treatment affect rDNA transcription, the increased 45S rRNA (C) could mainly originate from reduced pre-rRNA processing under chilling stress. Hereafter, we refer to this as the “5′ ETS-first” pathway (Supplemental Fig. The number of identical clones are indicated to the left (A) and right (B) of each fragment, respectively. 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). Different rRNA precursors are marked. The 18S rRNAs identified by primers 18P1 were validated by sequencing of 20 independent clones. A and B, Structure of 3′-5.8S identified by 58P1 (58L1/58R1; A) and 5′-5.8S by 58P2 (58L2/58R2; B), respectively. 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. 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 water was changed every two days during growth. 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. S8). 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). The number of identical clones is indicated to the right of each fragment. Two rice (Oryza sativa) subtypes were used in this work: Nipponbare belongs to the japonica subspecies (O. sativa ssp. S7C). (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. For circular RT-PCR assays (Figs. The 5′→3′ exonucleolytic trimming may contribute more than endonucleolytic cleavage to the 5′-5.8S processing (Fig. 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 … Moreover, we found that rice and Arabidopsis have similar flanking sequences around the A2 and A3 endonucleolytic sites in the ITS1 and the P′ in the 5′ ETS, respectively (Supplemental Fig. A complete list of probes is included in Supplemental Table S1. 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. Both endo- and exonucleolytic processing occur sequential and coordinately in this progress. Eukaryotic ribosome biogenesis is coupled with rRNA biogenesis, which starts in the nucleolus. Little is known about the RNA helicases involved in pre-60S ribosomal subunit processing and assembly in plants. In the present study, we characterized an Arabidopsis Pumilio-encoding gene, APUM23. Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and the charophyte Chara corallina. 7; Supplemental Figs. The 35S(P) and 27SA2 could be specifically detected by probes p23 and p42, respectively. However, their function in pre-rRNA processing remains poorly understood. C, Northern blots to detect the 45S rRNA transcript by probe 45P in Nipponbare under 4°C treatment for 0, 2, 4, and 6 h. Both blots of 45P and p42 came from the same membrane. 2015 Mar;21(3):415-25. doi: 10.1261/rna.047563.114. Epub 2017 Feb 14. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. S5). 2, B and D). Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. Error bars represent sd. In a screen for MAS2 interactors, we identified RIBOSOMAL RNA PROCESSING 7 (RRP7), an ortholog of yeast … The number of clones containing additional sequences at the 3′ extremities are marked in parentheses (in the shaded box). The asterisk detected by probe S7 represents the mature 16S rRNAs. 2015 Mar-Apr;6(2):191-209. doi: 10.1002/wrna.1267. The 35S(P) pre-rRNAs were validated by sequencing of 25 independent clones (D). Feedback regulation of ribosome assembly. The 6S intermediates exhibited heterogeneous 3′ ends, part of which contained additional polyadenylation sequences (Fig. 2017 Mar;89(5):1020-1030. doi: 10.1111/tpj.13442. Rice (Oryza sativa) is a model monocot plant and a major staple food worldwide. TruSeq Stranded Total RNA with Ribo-Zero Plant | For plant transcriptome studies Capture a comprehensive view of the plant transcriptome with this RNA-Seq library prep workflow. Probes p4 and S9 that are adjacent to the left and right borders of 5.8S rDNAs, respectively (Fig. The DNA sequencing reads for P-A3 intermediates (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). 8. The number of clones with additional sequences, such as polyadenylation at the 3′ end, is marked in parentheses. This highlights the importance of ribosome biogenesis in rice development and temperature acclimation. Processing of ribosomal RNAs (rRNAs) is an essential step in ribosome biogenesis and begins with transcription of the rDNA. 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. S9 and S11), after 2 h in the dark, 0.15 to ∼0.20 g of shoots were harvested as 0-h controls and the remaining seedlings were treated in dark growth chamber at 4°C. To this end, the DNA oligonucleotide 18c (Fig. 2019 Sep 19;47(16):8649-8661. doi: 10.1093/nar/gkz679. 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). 2A), the intact 27SB intermediate was identified (Fig. S6B, S7A, and S7B). The number of clones containing additional sequences at the 3′ extremities is marked in parentheses. 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. 2F; Supplemental Figs. The P-A3 intermediates identified by primers 18P6, 18P7, 18P3, and 18P4 were validated by sequencing of 87 independent clones (F). S6A, S7A, and S7B; Supplemental Table S1). Therefore, our detection of a similar pre-rRNA pattern in vivo with RNA hybridization (Fig. Here, the reads for the 27SA2 intermediate shared the definite A2 site at their 5′ extremities (Fig. The relative intensities for 25S rRNA, 45S transcripts, and P-A3 intermediates are marked in black, blue, and red, respectively. COVID-19 is an emerging, rapidly evolving situation. Mapping of the 5′ and 3′ extremities of the pre-25S rRNAs. S5). Ribosomal proteins (RPs) are essential structural components of ribosomes. 1. The P′-A3 intermediates identified by primers 18P2 and 18P8 were validated by sequencing of 21 independent clones (E). 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. 3A; Lange et al., 2011). 5C), S7, and p42 (Fig. This result indicates that 3′→5′ exonucleolytic trimming promotes 5.8S-3′ rRNAs processing (Mitchell et al., 1996; Chekanova et al., 2000; LaCava et al., 2005; Lange et al., 2009, 2011; Lange and Gagliardi, 2010; Kumakura et al., 2013; Sikorski et al., 2015). The number of identical clones is indicated to the right of each fragment. 5B), but its definite 3′ extremities are still unclear (A). S11). 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). 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). Mapping of the 5′ and 3′ extremities of the 35S(P) and 32S transcripts. 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/. 3, A and B) was performed to obtain both 5.8S-3′ (6S) (Fig. 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. © 2018 American Society of Plant Biologists. The 18S-A2 intermediates identified by primers 18P1 and 18P8 were validated by sequencing of 33 independent clones (C). 6045). Similarly, the 35S(P) fragment was further identified by primer combination 32P2 (p23/25R; Fig. wrote the article. Fernández-Pevida A, Kressler D, de la Cruz J. Wiley Interdiscip Rev RNA. The 3′-5.8S (7S and 6S) and 5′-5.8S are pre-5.8S rRNAs. The northern-blot approach is reliable (Barkan, 2011) and has shown that Arabidopsis preferentially uses the ITS1-first mode as the major pathway marked by P-A3 (Abbasi et al., 2010; Zakrzewska-Placzek et al., 2010; Lange et al., 2011; Huang et al., 2016; Shanmugam et al., 2017), rather than the minor 5′ ETS-first mode marked by 33S(P′), 32S, and 27SA2 (Hang et al., 2014; Weis et al., 2015a, 2015b; Tomecki et al., 2017). 5, C and D; Supplemental Fig. The ribosome translates the genetic information from messenger RNAs (mRNAs) into functional proteins (Crick, 1970; Yusupova and Yusupov, 2014; Browning and Bailey-Serres, 2015). 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. Mapping of the 5′ and 3′ extremities of the pre-18S rRNAs. The ITS1 locus matched by the 3′ ends of these clones are indicated by black triangles as well as the number of clones. 1B). 6). 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, 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). 5B). 4C). 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). Author information: (1)Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA. 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. 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. 1A; Supplemental Fig. Alternatively, the identification of 32S rRNA, the intact 18S-ITS1-5.8S-ITS2-25S intermediate ranging from site A1 to B2, defines the minor 5′ ETS-first pathway, which coexists in Arabidopsis and involves ITS1 cleavage after complete removal of the 5′-ETS (Hang et al., 2014; Weis et al., 2014, 2015b). 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). The 7S rRNA marked with “?” was detected by probe S9 (Fig. 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. ↵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. For each fragment, the number of clones obtained is indicated on the right. In the major ITS1-first pathway, the 35S(P) transcript is first split into P-A3 and 27SA3 by endonucleolytic cleavage at the A3 site in the ITS1. The 25S rRNA identified by primers 25P1 were validated by sequencing of 20 independent clones (C). Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. 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Transcript under plant ribosomal rna treatment indicated by horizontal arrows blots with specific probes, starts.
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