Ribosomal RNA Biogenesis and Its Response to Chilling Stress in Oryza sativa^,

Pre-rRNA processing in rice involves two coexisting pathways and responds to chilling stress.

Abstract

Ribosome biogenesis is crucial for plant growth and environmental acclimation. Processing of ribosomal RNAs (rRNAs) is an essential step in ribosome biogenesis and begins with transcription of the rDNA. 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). 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). 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 identified the rRNA precursors produced during rRNA biogenesis and the critical endonucleolytic cleavage sites in the transcribed spacer regions of pre-rRNAs in rice. We further found that two pre-rRNA processing pathways, distinguished by the order of 5′ ETS removal and ITS1 cleavage, coexist in vivo. Moreover, exposing rice to chilling stress resulted in the inhibition of rRNA biogenesis mainly at the pre-rRNA processing level, suggesting that these energy-intensive processes may be reduced to increase acclimation and survival at lower temperatures. 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 ribosome translates the genetic information from messenger RNAs (mRNAs) into functional proteins (Crick, 1970; Yusupova and Yusupov, 2014; Browning and Bailey-Serres, 2015). In eukaryotes, the mature 80S ribosome in the cytoplasm comprises the 40S small subunit and the 60S large subunit. 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). Ribosome biogenesis involves transcription of the ribosomal DNA (rDNA), precursor-rRNA (pre-rRNA) processing, RNA modifications, as well as assembly of the rRNAs with ribosomal proteins and assembly factors (Brown and Shaw, 1998; Venema and Tollervey, 1999; Lin et al., 2011; Woolford and Baserga, 2013). 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). In eukaryotic cells, aberrant rRNA biogenesis activates RNA quality control in the nucleus, which triggers higher polyadenylation of certain rRNA intermediates and by-products catalyzed by the Trf/Air/Mtr4 polyadenylation complex (TRAMP; Jia et al., 2011; Lange et al., 2014). 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). 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).

Eukaryotic ribosome biogenesis is coupled with rRNA biogenesis, which starts in the nucleolus. 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). 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. The processing sites and rRNA intermediates have been well defined in budding yeast (Saccharomyces cerevisiae), revealing the detailed mechanism of ribosome biogenesis and pre-rRNA processing in eukaryotes (Venema and Tollervey, 1999; Henras et al., 2015). In general, budding yeast pre-rRNA has two major endonucleolytic sites in the 5′ ETS (A0 and A1), five in ITS1 (D, A2, A3, B1L, and B1S), three in ITS2 (E, C2, and C1), and two in the 3′ ETS (B2 and B0; Mullineux and Lafontaine, 2012; Woolford and Baserga, 2013; Henras et al., 2015; Tomecki et al., 2017). 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). Hereafter, we refer to this as the “5′ ETS-first” pathway (Supplemental Fig. S1A). 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). 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). However, in contrast to budding yeast (Gallagher et al., 2004), metazoan (including mammalian) cells preferentially use the “ITS1-first” mechanism to split the ITS1 before the complete removal of the 5′ ETS (Mullineux and Lafontaine, 2012; Sloan et al., 2013; Henras et al., 2015).

The major pre-rRNA endonucleolytic cleavage sites have been determined in Arabidopsis. Three sites (P, P′, and A1 [P2]) exist in the 5′ ETS, four (D, A2, A3, and B1) in ITS1, three (E, C2, and C1) in ITS2, and two in the 3′ ETS (B2 and B0; Sáez-Vasquez et al., 2004a, 2004b; Zakrzewska-Placzek et al., 2010; Lange et al., 2011; Weis et al., 2015a, 2015b; Sikorski et al., 2015; Tomecki et al., 2017). 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). The high abundance of the P-A3 intermediate, which is easily detected in vivo, defines the major ITS1-first pathway in Arabidopsis, in which ITS1 cleavage at A3 occurs before complete removal of the 5′ ETS in the 35S(P) primary transcript (Zakrzewska-Placzek et al., 2010; Lange et al., 2011; Sikorski et al., 2015). 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). 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).

However, in contrast to the model dicot species Arabidopsis, rRNA maturation in monocot crops remains unexplored. Rice (Oryza sativa) is a model monocot plant and a major staple food worldwide. Recent work showed that the DEAD-box RNA helicase TOGR1 (Thermo-tolerant Growth Required 1), the rice homolog of Rrp3 (rRNA processing protein 3) in S. cerevisiae (O’Day et al., 1996) and DDX47 in Homo sapiens (Sekiguchi et al., 2006), is required for rice thermo-tolerant growth, acting as a key chaperone for rRNA homeostasis by fine-tuning pre-rRNA processing (Wang et al., 2016). This highlights the importance of ribosome biogenesis in rice development and temperature acclimation.

Rice rDNAs mainly occur as a cluster on chromosome 9 in Nipponbare, the well-annotated japonica rice genome (Goff et al., 2002; Kawahara et al., 2013; Sakai et al., 2013). Compared with the 18S, 5.8S, and 25S rDNAs (Supplemental Figs. S1B, S2, and S3), the DNA sequences for ETS and ITS spacers are much more variable in both length and sequence between Nipponbare and Arabidopsis accession Col-0 (Supplemental Fig. S4). Therefore, independently determining the pathway of rRNA biogenesis in rice, especially the precise processing sites in the ETS and ITS1 during pre-rRNA processing, is essential. 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). 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. Finally, we found that rRNA biogenesis in rice was inhibited by chilling stress mainly at the pre-rRNA processing (P-A3 and 27SA2) level.

RESULTS

Identification of Pre-18S rRNA Intermediates for the Pre-40S Small Subunit

Biogenesis and maturation of the 18S rRNA, the only structural RNA in the 40S SSU, are essential for ribosome biogenesis (Karbstein, 2011; Zhang et al., 2016). Pre-18S rRNA intermediates are processed by endonucleolytic cleavages in the 5′ ETS and the ITS1 surrounding the 18S rRNA (Fig. 1A). The processing order of 5′ ETS removal and ITS1 splitting is always uncoupled, resulting in various 18S precursors during 18S rRNA biogenesis. 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. S5). To this end, the DNA oligonucleotide 18c (Fig. 1A) in the 18S rDNA region was used for specific reverse transcription of circularized rRNA intermediates (Supplemental Fig. S5, A and B). These intermediates were then amplified by pairs of PCR primers, and the resulting amplification products were verified by sequencing (Supplemental Fig. S5, C and D). The locations of primer pairs (18P1 to 18P8) are shown in Figure 1A and summarized in Supplemental Tables S1 and S2. We also validated the efficiency of cRT-PCR with primer pairs 18P1 to 18P8, all of which could amplify specific bands with cDNAs reverse-transcribed from ligated RNAs (Supplemental Fig. S5E). The mature 18S rRNA was detected by the 18P1 primer pair (Fig. 1B). Then, 18S-A2 (by 18P1 and 18P8; Fig. 1, B and C), 18S-A3 (by 18P2 and 18P8; Fig. 1, B and D), and P′-A3 (by 18P2 and 18P5; Fig. 1, B and E) intermediates were also amplified (Fig. 1A; Supplemental Tables S1 and S2). Similarly, the P-A3 intermediates were detected by four pairs of primers, 18P3, 18P4, 18P6, and 18P7 (Fig. 1, B and F). We identified P-A3, P′-A3, 18S-A3, and 18S-A2 as the major pre-18S rRNAs in maturation of rice pre-40S (Fig. 1A; Supplemental Fig. S6A; Supplemental Table S2).

Figure 1.

Mapping of the 5′ and 3′ extremities of the pre-18S rRNAs. A, Structure of pre-18S rRNA intermediates identified by a set of primer combinations (in shaded box). Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. For each fragment, the number of clones obtained is indicated on the right. The number of clones with additional sequences, such as polyadenylation at the 3′ end, is marked in parentheses. 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). B, Pre-18S rRNA intermediates were determined in gel by cRT-PCR with primers 18P1 to 18P8. 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). The 18S rRNAs identified by primers 18P1 were validated by sequencing of 20 independent clones. The 18S-A2 intermediates identified by primers 18P1 and 18P8 were validated by sequencing of 33 independent clones (C). The 18S-A3 intermediates identified by primers 18P2 and 18P8 were validated by sequencing of 58 independent clones (D). The P′-A3 intermediates identified by primers 18P2 and 18P8 were validated by sequencing of 21 independent clones (E). The P-A3 intermediates identified by primers 18P6, 18P7, 18P3, and 18P4 were validated by sequencing of 87 independent clones (F). The ITS1 locus matched by the 3′ ends of these clones are indicated by black triangles as well as the number of clones. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. The numbers of identical clones are indicated to the right of each fragment.

We next used the sequences at the 5′ and 3′ extremities of the identified processing intermediates to define the processing sites. 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. 1A; Supplemental Figs. S6A, S7A, and S7B; Supplemental Table S1). Similarly, the P′ site of P′-A3 was at G¹⁶³⁴/A¹⁶³⁵ of TCGGAAGACGACAG in the 5′ ETS (Fig. 1E; Supplemental Fig. S7B). The DNA sequencing reads for P-A3 intermediates (Fig. 1F) defined the P site as between C¹¹⁶⁰/T¹¹⁶¹ of “ACACCTCTCCCACG” in the 5′ ETS region (Supplemental Fig. S7B). The P-A3, P′-A3, and 18S-A3 intermediates further confirmed the A3 site to be between G³⁶⁶⁰/A³⁶⁶¹ in “GTCAAGGAACACAG” in the ITS1 region (Supplemental Figs. S6A and S7B). The locations of the P and A3 endonucleolytic sites were consistent with a previous report (Wang et al., 2016). Notably, we found that P-A3, P′-A3, and 18S-A3 in rice were highly polyadenylated (Fig. 1, A and D–F; Supplemental Fig. S6A), similar to results reported in Arabidopsis (Abbasi et al., 2010; Lange et al., 2011; Hang et al., 2014; Sikorski et al., 2015; Shanmugam et al., 2017). 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.

Identification of rRNA Intermediates for the Pre-60S Large Subunit

The mature 25S and 5.8S rRNAs are the structural RNAs in the 60S large subunit (LSU) (Anger et al., 2013). To identify the pre-25S rRNA intermediates and processing sites in rice, we performed cRT-PCR assays with the 25c primer for specific reverse transcription (25c_cDNA) followed by PCR with primer combinations 25P1 (25L/25R), 25P2 (p44/25R), 27P1 (58L/25R), and 27P2 (p4/25R) (Fig. 2A). The intact 25S rRNA was identified efficiently by the 25P1 primers within the 25S rDNA (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. S6B, S7A, and S7B). When the reverse primers were switched to p44 in 25P2 or 58L in 27P1 (Fig. 2A), the intact 27SB intermediate was identified (Fig. 2, B and D). The 27SB intermediate covers the 5.8S, ITS2, and 25S rRNA (Fig. 2A), which allowed us to identify B1 and B2 as the left and right borders, respectively, of the 5.8S and 25S rRNAs (Supplemental Figs. S6B, S7A, and S7B). Similarly, the 27SA3 and 27SA2 sites were detected by primer combination 27P2 (Fig. 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. 2A). 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).

Figure 2.

Mapping of the 5′ and 3′ extremities of the pre-25S rRNAs. A, Structure of pre-25S intermediates identified by a set of primers (in shaded box). Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. Four pairs of primers were used for pre-25S rRNAs: 25P1 (25L/25R), 25P2 (p44/25R), 27P1 (58L/25R), and 27P2 (p4/25R). For each fragment, the number of clones obtained is indicated on the right. The number of clones containing additional sequences at the 3′ extremities is marked in parentheses. B, Pre-25S rRNA intermediates were determined in gel by cRT-PCR with primers 25P1, 25P2, 27P1, and 27P2. C to F, The DNA sequencing results for 25S (C) and its major precursors identified: 27SB (D), 27SA3 (E), and 27SA2 (F). The 25S rRNA identified by primers 25P1 were validated by sequencing of 20 independent clones (C). The 27SB intermediates identified by primers 25P2 and 27P1 were validated by sequencing of 51 independent clones (D). The 27SA3 intermediates identified by primers 27P1 and 27P2 were validated by sequencing of 22 independent clones (E). The 27SA2 intermediates identified by primers 27P2 were validated by sequencing of 21 independent clones (F). 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. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. The number of identical clones is indicated to the right of each fragment.

Among the pre-25S rRNA intermediates identified, 27SA3 exhibited uniform 5′ extremities at A³⁶⁶¹ in “GTCAAGGAACACAG” in the ITS1 region (Fig. 2E; Supplemental Figs. 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, E, and F; Supplemental Figs. S6A and S7B). Moreover, the A2 endonucleolytic site was deduced to be between A³⁵⁶⁰/C³⁵⁶¹ in “ACCAAAACAGACCG” by comparing the 3′ ends of 18S-A2 (Fig. 1C) and the 5′ ends of 27SA2 (Fig. 2F; Supplemental Figs. S6B and S7B), two precursors processed by direct cleavage at the A2 site in ITS1 from the 32S transcript (Weis et al., 2015b). Here, the reads for the 27SA2 intermediate shared the definite A2 site at their 5′ extremities (Fig. 2F), but the 3′ extremities of the 18S-A2 fragments we identified were much more heterogeneous (Fig. 1C), indicating that the putative fast 3′→5′ exonucleolytic trimming occurs in the processing of this precursor.

Similarly, the 58c oligonucleotide was used for specific reverse transcription of the pre-5.8S rRNAs (Fig. 3). PCR amplification with primer pairs 58P1 (58L1/58R1) and 58P2 (58L2/58R2; Fig. 3, A and B) was performed to obtain both 5.8S-3′ (6S) (Fig. 3A) and 5′-5.8S (Fig. 3B) fragments, respectively. The 6S intermediates exhibited heterogeneous 3′ ends, part of which contained additional polyadenylation sequences (Fig. 3A), similar to the 6S intermediates in Arabidopsis (Shanmugam et al., 2017). 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 5′→3′ exonucleolytic trimming may contribute more than endonucleolytic cleavage to the 5′-5.8S processing (Fig. 3B; Henry et al., 1994; Zakrzewska-Placzek et al., 2010).

Figure 3.

Mapping of the 5′ and 3′ extremities of the pre-5.8S rRNAs. A and B, Structure of 3′-5.8S identified by 58P1 (58L1/58R1; A) and 5′-5.8S by 58P2 (58L2/58R2; B), respectively. Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. For each fragment, the number of clones obtained is indicated on the right. The number of clones containing additional sequences at the 3′ extremities are marked in parentheses (in the shaded box). The 5.8S-3′ intermediates were validated by 70 independent clones (A). The 5′-5.8S intermediates were validated by 22 independent clones (B). 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. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. The number of identical clones are indicated to the left (A) and right (B) of each fragment, respectively. C, Pre-5.8S rRNA intermediates were determined in gel by cRT-PCR with primers 58P1 and 58P2.

Identification of rRNA Intermediates in the 90S/SSU Processome

The pre-40S SSU and pre-60S LSU derive from the split of the 90S/SSU processome at the ITS1 region of the nascent primary transcripts (Kornprobst et al., 2016; Zhang et al., 2016; Johnson et al., 2017; Sun et al., 2017). 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. 4A). The 32S transcript from A1 to B2 sites was detected using primer pair 32P1 (18L/25R; Fig. 4, A–C) and contained the intact 18S, ITS1, 5.8S, ITS2, and 25S rRNA sequences. Similarly, the 35S(P) fragment was further identified by primer combination 32P2 (p23/25R; Fig. 4, A, B, and D).

Figure 4.

Mapping of the 5′ and 3′ extremities of the 35S(P) and 32S transcripts. A, Structure of early pre-rRNA intermediates identified (in shaded box) by two pairs of primers: 32P1 and 32P2. Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. For each fragment, the number of clones obtained is indicated on the right. The number of clones with additional sequences at the 3′ end is marked in parentheses. B, The 32S and 35S(P) pre-rRNAs were determined in gel by cRT-PCR with primers 32P1 (18L/25R) and 32P2 (p23/25R). C and D, DNA sequencing results for 32S (C) and 35S(P) precursors (D). The 32S pre-rRNAs were validated by sequencing of 20 independent clones (D). The 35S(P) pre-rRNAs were validated by sequencing of 25 independent clones (D). 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. Additional sequences in the 3′ extremities of these clones are marked in red lowercase letters. The number of identical clones is indicated to the right of each fragment.

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. 4D). Moreover, the 3′ ends of the 35S(P) precursors were polyadenylated, which was rarely detected in the 32S precursors (Fig. 4C). 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. (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.

Determination of Pre-rRNA Processing in Rice in Vivo

The identification of P-A3, 32S, and 27SA2 by cRT-PCR indicates that conserved modes of pre-rRNA processing could coexist in rice. To further determine the pre-rRNA processing pattern in vivo in rice, we set up a northern-blot assay with a series of short oligonucleotide probes (Fig. 5; Supplemental Table S1; Supplemental Fig. S8). Probes p4 and S9 that are adjacent to the left and right borders of 5.8S rDNAs, respectively (Fig. 5A), recognized pre-5.8S rRNAs and 27S rRNAs in the pre-60S LSU (Fig. 5B). This result was consistent with the cRT-PCR data (Figs. 2 and 3). The probes p23, S7, and p42 (Fig. 5A) were designed to detect the pre-18S rRNAs in the pre-40S SSU (Fig. 5, C and D; Supplemental Fig. S8). The 5′ ETS probe p23 between the P and P′ sites distinguished 35S(P) from 32S precursors in the 90S/SSU complex (Fig. 5A). Moreover, the ITS1 probe p42 between A2 and A3 sites detected 18S-A3 and 27SA2 specifically (Fig. 5, A and D), compared with the probes p23 and S7 recognizing 18S-A3, or p4 and S9 recognizing 27SA2 (Fig. 5A).

Figure 5.

Northern blots to detect pre-rRNA processing in rice. A, Pre-rRNA processing intermediates detected by northern blots with specific probes, which are indicated by horizontal arrows. Black vertical arrows above the diagram indicate endonucleolytic cleavage sites relevant to this study. Different rRNA precursors are marked. P-A3, P′-A3, 18S-A3, and 18S-A2 belong to the pre-18S rRNAs. 27SA2, 27SA3, and 27SB belong to the 27S rRNA, the common precursor of 5.8S and 25S rRNAs. The 3′-5.8S (7S and 6S) and 5′-5.8S are pre-5.8S rRNAs. The 7S rRNA marked with “?” was detected by probe S9 (Fig. 5B), but its definite 3′ extremities are still unclear (A). The 35S(P) and 27SA2 could be specifically detected by probes p23 and p42, respectively. Both probes S7 and p42 detect 35S(P), 32S, P-A3, and 18S-A3. 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. 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). The togr1 mutant is a positive control that accumulates the 35S pre-rRNA and P-A3 intermediates, when compared with its wild type, Zhongxian3037 (Wang et al., 2016). Probes p4 and S9 were used. Methylene blue staining (MB stain) of the membrane is shown as the loading control. 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 S7 and p42 blots share the same loading control (D). 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). Matured rRNAs stained with MB serve as the loading control. The relative intensities for P-A3 intermediate in each lane are normalized to Zhongxian3037. Error bars represent sd. Data are given as means and sd of three independent biological replicates.

We used the togr1-1 mutant as the positive control (Wang et al., 2016), which exhibited an aberrant accumulation of 35S(P) and P-A3 compared with the wild type, Zhongxian3037 (Fig. 5, B–E; Supplemental Fig. S8). The P-A3 intermediate and its direct precursor 35S(P) were both readily detected by the 5′ ETS probe p23 (Fig. 5C; Supplemental Fig. S8), as well as ITS1 probes S7A (Supplemental Fig. S8), S7, and p42 (Fig. 5D). By contrast, we observed much less of the 32S intermediate than the 35S(P), when probed with S7A (Supplemental Fig. S8), p4, and S9 (Fig. 5B). Similarly, the relative amount of 27SA2 was also far less than that of P-A3 in Nipponbare detected by ITS1 probe p42 (Fig. 5D). Moreover, the abundance of P-A3 in the indica cultivar Zhongxian3037 was less than in the japonica cultivar Nipponbare (Fig. 5E), as detected by probes p23 (Fig. 5C), S7, and p42 (Fig. 5D; Supplemental Fig. S8). This variation in pre-rRNA processing between these two rice subspecies may come from genome variation during evolution (Huang et al., 2012), a possibility that will require further examination in the future.

Alternative rRNA Biogenesis Pathways in Rice

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). These pre-rRNA processing modes are distinguished by the order of 5′ ETS removal and ITS1 splitting and reflect ribosome assembly dynamics during ribosome biogenesis (Mullineux and Lafontaine, 2012; Weis et al., 2015a; Tomecki et al., 2017). 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).

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). 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 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). Therefore, our detection of a similar pre-rRNA pattern in vivo with RNA hybridization (Fig. 5; Supplemental Fig. S8) suggests that similar alternative rRNA maturation pathways may coexist in rice in vivo.

Here, we propose a working model for rRNA biogenesis in rice (Fig. 6). 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. Then the 35S(P) transcript enters two alternative maturation pathways distinguished by the order of ITS1 cleavage and 5′ ETS removal. 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. As the diagnostic marker for major pathway, P-A3 in the pre-40S SSU can be further processed into P′-A3, 18S-A3, 18S-A2 (predicted) sequentially, and eventually matures into the 18S rRNA (Fig. 6). In the minor 5′ ETS-first pathway in rice, marked by the 32S and 27SA2 intermediates, the primary 35S(P) transcript is first shortened at its 5′ end by complete removal of the 5′ ETS resulting in 32S rRNAs. 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. 6).

Figure 6.

Model of rRNA biogenesis in rice. 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. In the major ITS1-first pathway, the 35SP transcript is split at ITS1 endonucleolytic site A3 into P-A3 and 27SA3 precursors. 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. Both endo- and exonucleolytic processing occur sequential and coordinately in this progress. Precursors with partial transparency indicate putative intermediates in these pathways.

Inhibition of rRNA Biogenesis under Chilling Stress

To test for a potential relationship between chilling stress and ribosome biogenesis at the level of pre-rRNA processing in rice, we performed northern-blot assays with rice shoots after a time-course chilling treatment (Fig. 7, A and B; Supplemental Figs. S9 and S10). Both P-A3 in the ITS1-first pathway and 27SA2 in the 5′ ETS-first pathway decreased under chilling stress in shoots (Fig. 7, A and B; Supplemental Figs. S9A, S9B, and S10B) and roots (Supplemental Fig. S10C), indicating reduced pre-rRNA processing.

Figure 7.

Chilling stress inhibits rRNA biogenesis mainly at pre-rRNAs processing levels. 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). Matured rRNAs stained with MB serve as the loading control. The numbers below each lane represent the intensity ratio of each signal relative to the 0 h sample. The relative intensities for 25S rRNA, P-A3, and 27SA2 intermediates are marked in black, red, and blue, respectively. The asterisk detected by probe S7 represents the mature 16S rRNAs. Three biological replicates were performed and a representative result is shown here. 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. Matured rRNAs stained with MB serve as the loading control. The numbers below each lane represent the intensity ratio of each signal relative to the 0 h sample. The relative intensities for 25S rRNA, 45S transcripts, and P-A3 intermediates are marked in black, blue, and red, respectively. RNA samples from two biological replicates were loaded and detected in parallel. D, Simplified model that the inhibition of rRNA biogenesis in rice by chilling stress predominantly occurs at posttranscriptional level. The 45S rRNA, transcribed by RNA Pol I from rDNAs, undergoes pre-rRNA processing to release mature rRNAs. The steady level of 45S rRNA in vivo is the net product of rDNA transcription and subsequent pre-rRNA processing. Chilling stress inhibits pre-rRNA processing, shown by the time-course reduction of P-A3 and 27SA2 in both ITS1-first and 5′ ETS-first processing pathways, respectively (A and B). 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. The long probe 45P could distinguish the 45S rRNA from its product 35S(P).

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. 6). Therefore, the 45S rRNA in vivo is the net product of rDNA transcription and subsequent pre-rRNA processing. 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. The 5′ ETS region from the transcription initiation site (TIS) to the P site is unique to the 45S pre-rRNA with the exception of 35S(P) (Fig. 6). We detected 45S rRNA transcripts by northern blots with a specific long probe (45P) that recognizes the 5′ ETS region upstream of the P site (Fig. 7D; Supplemental Table S1). The steady state of 45S pre-rRNA increased under chilling stress, which was inversely correlated with the abundance of P-A3 (Fig. 7C; Supplemental Fig. S11). 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. Then, decreased pre-rRNA processing may negatively affect the processing dynamics of 45S transcript, resulting in its accumulation (Fig. 7D). Although the transcriptional activity of RNA Pol I would provide direct evidence to illustrate the transcription of 45S pre-rRNA (Ream et al., 2015), the appropriate antibodies or transgenic materials in rice are not currently available. Besides, de novo characterization of nascent transcripts under chilling treatments using unbiased global nuclear run-on sequencing will provide us with more information at the transcriptional level (Hetzel et al., 2016). Nevertheless, our results suggest that rice may fine-tune ribosome biogenesis to quickly adjust energy consumption and primary metabolism for survival and acclimation at low temperatures. Moreover, compared with both the mature rRNAs and other rRNA precursors, the P-A3 precursor readily detected by northern blots could be a reliable marker for rRNA biogenesis under chilling stress.

DISCUSSION

Two pre-rRNA processing pathways, known as the 5′ ETS-first and the ITS1-first modes, commonly coexist in eukaryotes (Lafontaine, 2010; Mullineux and Lafontaine, 2012; Hang et al., 2014; Henras et al., 2015; Weis et al., 2015a; Tomecki et al., 2017). Such conserved molecular characters have been well deciphered in budding yeast (Gallagher et al., 2004; Lamanna and Karbstein, 2011; Mullineux and Lafontaine, 2012) and to some extent in animal systems, such as Xenopus laevis oocytes (Savino and Gerbi, 1990; Borovjagin and Gerbi, 1999), Drosophila melanogaster (Long and Dawid, 1980), and mammalian cells (Bowman et al., 1981; Hadjiolova et al., 1993; Kent et al., 2009). Here, we identified the rRNA intermediates and critical processing sites of the 5′ ETS and ITS1 regions in rice (Supplemental Figs. S6 and S7). These findings ultimately uncovered the rice alternative pre-rRNA processing pathways with the ITS1-first mode as the major pathway (Fig. 6). 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). 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. S7C). This observation suggests that conserved cis-elements shared by both species contribute to the selection of these endonucleolytic cleavage sites. In contrast, the sequences flanking the P site in the 5′ ETS are highly variable between rice and Arabidopsis (Supplemental Fig. S7C). 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.

Ribosome assembly and rRNA maturation include a series of rRNA conformational changes and protein-binding events (Marmier-Gourrier et al., 2011; Phipps et al., 2011). 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). Using cryo-electron microscopy, the assembly of the 90S/SSU processome (Kornprobst et al., 2016; Zhang et al., 2016; Johnson et al., 2017; Sun et al., 2017) and pre-60S LSU (Gamalinda et al., 2014; Greber, 2016; Greber et al., 2016; Wu et al., 2016; Ma et al., 2017) were further resolved. 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). The BoU3/NF-D complex is recruited by a conserved A¹²³B [A(1), A(2), A(3), and B motifs] to mediate P-site cleavage in the 5′ ETS (Caparros‐Ruiz et al., 1997; Sáez-Vasquez et al., 2004a, 2004b; Samaha et al., 2010). 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 rice, TOGR1 was the first well-defined RNA helicase essential for ribosome biogenesis during rice growth and development (Wang et al., 2016). It will be interesting to decipher the functional complexes that form during rice ribosome biogenesis in the future.

Environmental signals affect plant growth and crop yield. Such signals include environmental factors such as photoperiod (Ding et al., 2012; Fan et al., 2016) and ambient temperature fluctuations (Gong et al., 2002, 2005; da Cruz et al., 2013; Challinor et al., 2014; Ray et al., 2015; Shi et al., 2015). Rice originated from tropical and subtropical regions (Huang et al., 2012); therefore, rice cultivated in temperate zones can exhibit more sensitivity to chilling stress than other crops such as barley (Hordeum vulgare) and wheat (Triticum aestivum; Zhang et al., 2014). 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). 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). Effective ribosomal biogenesis is tightly fine-tuned by cellular status (Lempiäinen and Shore, 2009) and variations in environmental conditions (Planta, 1997; Mayer and Grummt, 2006), such as ambient temperature (Kaczanowska and Rydén-Aulin, 2007; Al Refaii and Alix, 2009; Baliga et al., 2016). 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). 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). 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. Also, constitutive expression of TOGR1 enhanced the tolerance of rice to heat stress (Wang et al., 2016). Therefore, an understanding of ribosome biogenesis and its response to ambient temperature in rice can benefit basic scientific research and facilitate efforts to improve thermo-tolerance (Chen et al., 2009; Song et al., 2012a, 2012b; Zhou et al., 2014; Li et al., 2015; Shen et al., 2015; Wang et al., 2016; Yu et al., 2018) and chilling tolerance (Fan and Zhang, 2014; Lu et al., 2014; Ma et al., 2015; Li and Lin, 2016; Zhang et al., 2017b) in agricultural applications.

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). 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. 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). Here, we found that rRNA biogenesis is down-regulated by chilling stress at the posttranscriptional levels, potentially for the adjustment of energy consumption and primary metabolism to adapt to cold stress (Fig. 7C). In addition, the translational activity of ribosomes in the cytoplasm could be directly and dynamically fine-tuned by various environmental signals (Bailey-Serres et al., 2009; Browning and Bailey-Serres, 2015), such as dehydration stress (Kawaguchi et al., 2004), hypoxia (Branco-Price et al., 2008; Mustroph et al., 2009; Juntawong et al., 2014), heat stress (Zhang et al., 2017a), and light signals (Liu et al., 2012, 2013). This represents another regulatory layer affecting the activity of ribosomes to facilitate the acclimation and survival of rice under stress.

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. It will be intriguing to determine the molecular mechanism of temperature sensing in ribosome biogenesis in rice in the future.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Two rice (Oryza sativa) subtypes were used in this work: Nipponbare belongs to the japonica subspecies (O. sativa ssp. japonica; Huang et al., 2012). Zhongxian3037, togr1-1 mutants (Wang et al., 2016), and 9311 belong to the indica rice subspecies (O. sativa ssp. indica). For circular RT-PCR assays (Figs. 1–4; 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. For northern-blot assays (Figs. 5 and 7; Supplemental Figs. 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. 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. For cold treatment of seedlings in soil (Fig. 7; Supplemental Figs. 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. Then 0.15 to ∼0.20 g of shoots were harvested every 2 h for two or three intervals. For seedlings in water (Supplemental Fig. S10A), after 2 h in the dark, 0.15 to ∼0.20 g samples of shoots and roots were harvested separately as 0-h controls. The remaining seedlings were transferred to precooled water and treated in a dark growth chamber at 4°C. Then, 0.15 to ∼0.20 g of shoots and roots were harvested in the same way every 2 h for two or three intervals. The water was changed every two days during growth. Fresh materials were frozen by liquid nitrogen and stored at −80°C until used. More than three biological replicates were performed for upper treatments and the representative data were exhibited.

RNA Extraction

The rice materials were first ground into fine powder with liquid nitrogen. Then, total RNA was extracted from the powder with TRNzol reagent (Tiangen; DP405-02) according to the manufacturer’s instructions. Total RNA was dissolved in DEPC-treated deionized water and quantified with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific; ND-1000).

cRT-PCR

cRT-PCR analysis was performed as previously described (Slomovic et al., 2008; Barkan, 2011; Hang et al., 2015), with slight modification (Supplemental Fig. S5). 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. S5A). 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. S5B). Then a series of primers (Supplemental Table S1) around the 18c or 25c RT primer were used to amplify the flanking sequences of precursors with 2× Phanta Master Mix (Vazyme Biotech; P511-01). After amplification for 35 cycles, bands obtained by cRT-PCR were subcloned into the pEasy-T vector (Transgene; CT101-02; Supplemental Fig. S5C), and positive clones were selected for with a second PCR using the M13F and M13R primers. Finally, target products exhibiting sharp bands at the proper molecular weight were excised for DNA sequencing and further analyzed by BLAST from the National Center for Biotechnology Information (NCBI), choosing the organism (O. sativa, japonica group; taxid:39947) and database (reference genomic sequences [refseq_genomic]) using Megablast (optimized for highly similar sequences). Cleavage sites and flanking sequences were identified according to japonica rice rDNA offline annotation (Supplemental Fig. S5D).

Northern-Blot Analysis

The northern-blot assays were performed as described (Hang et al., 2014), with slight modification. 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. For short DNA probes, oligonucleotides labeled with [γ-³²P]ATP (Perkin-Elmer; BLU002A001MC) by T4 polynucleotide kinase (New England Biolabs; M0201) were used to detect precursor RNAs. For long DNA probes, 45P was first amplified by primers 45P-F1 and 45P-F2 (Supplemental Table S1) using genomic DNA. 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. no. 6045). Hybridization was performed overnight at 45°C (for short probes) or 65°C (for long probes) as previously described (Hang et al., 2014). The blots were washed and exposed to a storage phosphor screen (GE Healthcare), then detected with a Typhoon TRIO scanner (GE Healthcare). A complete list of probes is included in Supplemental Table S1. Image J was used to quantify band intensity (Schneider et al., 2012).

Sequence Alignment

Multiple alignments of DNA sequences were performed with ClustalX (Larkin et al., 2007) and were manually edited with the GeneDoc program. Sequence identities were determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970) in NCBI Global Alignment tool.

Accession Numbers

Database searching was performed at NCBI. The GenBank accession numbers for rDNA sequences of rice and Arabidopsis are AP008225 (region: 1,069 to ∼8,996) and CP002686 (region: 14,195,840 to ∼14,203,859), respectively.

Supplemental Data

The following supplemental materials are available.

• Supplemental Figure S1. Simplified pre-rRNA processing in eukaryotes and comparison of rDNA components between the japonica rice Nipponbare and Arabidopsis thaliana accession Col-0.

• Supplemental Figure S2. Sequence alignments of 18S and 5.8S rDNAs between the japonica rice Nipponbare and Arabidopsis thaliana accession Col-0.

• Supplemental Figure S3. Sequence alignment of 25S rDNAs between the japonica rice Nipponbare and Arabidopsis thaliana accession Col-0.

• Supplemental Figure S4. Sequence alignments of 5′ ETS, ITS1, ITS2, and partial 3′ ETS rDNAs between the japonica rice Nipponbare and the Arabidopsis thaliana accession Col-0.

• Supplemental Figure S5. Circular RT-PCR assay to identify pre-rRNA precursors.

• Supplemental Figure S6. Representative DNA sequencing results for the identified pre-rRNAs.

• Supplemental Figure S7. Summary of endonucleolytic sites identified during pre-rRNA processing in rice.

• Supplemental Figure S8. Northern blots to detect pre-rRNA processing in rice by probes p23 and S7A.

• Supplemental Figure S9. Pre-rRNA processing in rice shoots in response to chilling stress.

• Supplemental Figure S10. Pre-rRNA processing in rice roots responses to chilling stress.

• Supplemental Figure S11. Northern blot with probe 45P to detect 45S rRNA transcript under chilling treatment.

• Supplemental Table S1. Oligonucleotides used in this article.

• Supplemental Table S2. rRNA intermediates and primer combinations used in cRT-PCR assays.