ribosomal rna biogenesis and its response to chilling ...and its2, and ﬂanked by 59 and 39...

Ribosomal RNA Biogenesis and Its Response to ChillingStress in Oryza sativa1[OPEN]

Runlai Hang,a,b Zhen Wang,b,c Xian Deng,b Chunyan Liu,b Bin Yan,b,c Chao Yang,b,c Xianwei Song,b

Beixin Mo,a and Xiaofeng Caob,c,2

aGuangdong Provincial Key Laboratory for Plant Epigenetics, College of Life Sciences and Oceanography,Shenzhen University, Shenzhen 518060, Guangdong Province, ChinabState Key Laboratory of Plant Genomics and National Center for Plant Gene Research, CAS Center forExcellence in Molecular Plant Sciences, Institute of Genetics and Developmental Biology, Chinese Academy ofSciences, Beijing 100101, ChinacCollege of Life Sciences, University of Chinese Academy of Sciences, Beijing, 100039, China

ORCID IDs: 0000-0002-2981-6121 (R.H.); 0000-0003-0215-9077 (Z.W.); 0000-0001-7279-9788 (B.M.); 0000-0001-9871-0753 (X.C.).

Ribosome biogenesis is crucial for plant growth and environmental acclimation. Processing of ribosomal RNAs (rRNAs) is anessential 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 externaland internal transcribed spacers (ETS and ITS). The processing sites and pathways for pre-rRNA processing have beendeciphered in Saccharomyces cerevisiae and, to some extent, in Xenopus laevis, mammalian cells, and Arabidopsis (Arabidopsisthaliana). 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 duringrRNA biogenesis and the critical endonucleolytic cleavage sites in the transcribed spacer regions of pre-rRNAs in rice. We furtherfound that two pre-rRNA processing pathways, distinguished by the order of 59 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 quicklyinhibited by low temperatures, which may shed light on the link between ribosome biogenesis and environmentalacclimation in crop plants.

The ribosome translates the genetic information frommessenger RNAs (mRNAs) into functional proteins(Crick, 1970; Yusupova and Yusupov, 2014; Browning

and Bailey-Serres, 2015). In eukaryotes, the mature 80Sribosome in the cytoplasm comprises the 40S smallsubunit and the 60S large subunit. The small subunitcontains 18S ribosomal RNAs (rRNAs) and more than30 ribosomal proteins, while the large subunit containsthe 25S/28S, 5.8S, and 5S rRNAs and more than 40 ri-bosomal proteins (Yusupova and Yusupov, 2014). Ri-bosome biogenesis involves transcription of theribosomal DNA (rDNA), precursor-rRNA (pre-rRNA)processing, RNA modifications, as well as assembly ofthe rRNAs with ribosomal proteins and assembly fac-tors (Brown and Shaw, 1998; Venema and Tollervey,1999; Lin et al., 2011; Woolford and Baserga, 2013). Asan essential, complicated, energy-intensive process(Warner, 1999), ribosome biogenesis is strictly regu-lated by endogenous signals (Lykke-Andersen et al.,2009; Lafontaine, 2010; Sanchez et al., 2016) and envi-ronmental stimuli (Sinturel et al., 2017) such as ambienttemperature (Warner and Udem, 1972; Tollervey et al.,1993; Al Refaii andAlix, 2009; Ohbayashi et al., 2011). Ineukaryotic cells, aberrant rRNA biogenesis activatesRNA quality control in the nucleus, which triggershigher polyadenylation of certain rRNA intermediatesand by-products catalyzed by the Trf/Air/Mtr4 poly-adenylation complex (TRAMP; Jia et al., 2011; Lange

1 This work was supported by grants from the National NaturalScience Foundation of China (grants 91540203, 31788103, and31330020 to X.C., 31770874 and 31370770 to C.L., and 31571332 toB.M.), the National Key Research and Development Program ofChina (2016YFD0100904 to X.C.), the Strategic Priority Research Pro-grams (grants XDA08010202 and XDPB0403 to X.C.), the ChinaPostdoctoral Science Foundation (2015M570169 and 2017T100113 toR.H.), the Key Research Program of Frontier Sciences of ChineseAcademy of Sciences (grant QYZDY-SSW-SMC022 to X.C.), theYoung Scientist Foundation of State Key Laboratory of Plant Ge-nomics (2015D0129-03 to R.H.), and the State Key Laboratory of PlantGenomics.

2 Address correspondence to [email protected]., B.M., and X.C. designed the research; R.H., Z.W., C.L., B.Y.,

C.Y., X.S., and X.D. performed the experiments; R.H. and X.C. ana-lyzed the data; R.H. and X.C. wrote the article.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Xiaofeng Cao ([email protected]).

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et al., 2014). These intermediates are degraded se-quentially by the nuclear exosome complex (LaCavaet 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 biogen-esis (Gallagher et al., 2004; Ferreira-Cerca et al., 2005,2007; Tafforeau et al., 2013) results in severe develop-mental defects in higher plants (Byrne, 2009; Fujikuraet 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; McCannand Baserga, 2013; Sondalle and Baserga, 2014; Baiet al., 2016).

Eukaryotic ribosome biogenesis is coupled withrRNA biogenesis, which starts in the nucleolus. First,RNA polymerase I (Pol I) transcribes the tandem re-peated rDNA units into polycistronic primary tran-scripts, where the 18S, 5.8S, and 25S/28S rRNAs areseparated by the internal transcribed spacer 1 (ITS1)and ITS2, and flanked by 59 and 39 external transcribedspacers (59 ETS and 39 ETS, respectively; Henras et al.,2015). Then, multiple endonucleolytic and exonucleo-lytic processing steps sequentially and coordinatelyremove the ETS and ITS regions to release mature 18S,5.8S, and 25S/28S rRNAs. The processing sites andrRNA intermediates have beenwell defined in buddingyeast (Saccharomyces cerevisiae), revealing the detailedmechanism of ribosome biogenesis and pre-rRNAprocessing in eukaryotes (Venema and Tollervey,1999; Henras et al., 2015). In general, budding yeastpre-rRNA has two major endonucleolytic sites in the 59ETS (A0 and A1), five in ITS1 (D, A2, A3, B1L, and B1S),three in ITS2 (E, C2, and C1), and two in the 39 ETS (B2and B0; Mullineux and Lafontaine, 2012; Woolford andBaserga, 2013; Henras et al., 2015; Tomecki et al., 2017).The 35S primary transcripts in the 90S particle/smallsubunit processome (SSU; Dragon et al., 2002; Grandiet al., 2002; Osheim et al., 2004; Phipps et al., 2011)preferentially use the major “U3-dependent cleavageoccurs first” pathway to cotranscriptionally remove the59 ETS completely, producing the 32S intermediate (Leeand Baserga, 1997; Gallagher et al., 2004; Kos andTollervey, 2010). Hereafter, we refer to this as the “59ETS-first” pathway (Supplemental Fig. S1A). Then,endonucleolytic cleavage at the A2 site in ITS1 splits the90S processome/SSU into pre-40S and pre-60S parti-cles, which further undergo a series of endo- and exo-nucleolytic processing events and finally matureinto the 40S and 60S subunits, respectively (Venemaand Tollervey, 1999; Woolford and Baserga, 2013;Fernández-Pevida et al., 2015; Henras et al., 2015). The35S rRNA primary transcripts in budding yeast andArabidopsis (Arabidopsis thaliana) are equivalent to the47S rRNA transcripts in mammalian cells (Layat et al.,2012; Henras et al., 2015). However, in contrast tobudding yeast (Gallagher et al., 2004), metazoan (in-cluding mammalian) cells preferentially use the “ITS1-first” mechanism to split the ITS1 before the completeremoval of the 59 ETS (Mullineux and Lafontaine, 2012;Sloan et al., 2013; Henras et al., 2015).

The major pre-rRNA endonucleolytic cleavage siteshave been determined in Arabidopsis. Three sites (P, P9,and A1 [P2]) exist in the 59 ETS, four (D, A2, A3, and B1)in ITS1, three (E, C2, and C1) in ITS2, and two in the 39ETS (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; Tomeckiet al., 2017). Moreover, functional studies of ribosomebiogenesis mutants have identified the series of rRNAintermediates that occur during pre-rRNA process-ing (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; Zhuet al., 2016). The high abundance of the P-A3 interme-diate, which is easily detected in vivo, defines the majorITS1-first pathway in Arabidopsis, in which ITS1cleavage at A3 occurs before complete removal of the 59ETS 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, theintact 18S-ITS1-5.8S-ITS2-25S intermediate rangingfrom site A1 to B2, defines the minor 59 ETS-firstpathway, which coexists in Arabidopsis and involvesITS1 cleavage after complete removal of the 59-ETS(Hang et al., 2014; Weis et al., 2014, 2015b). More re-cently, the determination of 33S(P9) and 27SA2 rRNAs(Weis et al., 2015b) as the direct precursor and productof the 32S rRNA, respectively, further demonstrates theexistence of the minor 59 ETS-first pathway in Arabi-dopsis (Weis et al., 2015a).

However, in contrast to the model dicot speciesArabidopsis, rRNA maturation in monocot crops re-mains unexplored. Rice (Oryza sativa) is a modelmonocot plant and a major staple food worldwide.Recent work showed that the DEAD-box RNA helicaseTOGR1 (Thermo-tolerant Growth Required 1), the ricehomolog of Rrp3 (rRNA processing protein 3) inS. cerevisiae (O’Day et al., 1996) and DDX47 in Homosapiens (Sekiguchi et al., 2006), is required for ricethermo-tolerant growth, acting as a key chaperone forrRNAhomeostasis by fine-tuning pre-rRNAprocessing(Wang et al., 2016). This highlights the importance ofribosome biogenesis in rice development and temper-ature acclimation.

Rice rDNAs mainly occur as a cluster on chromo-some 9 in Nipponbare, the well-annotated japonicarice genome (Goff et al., 2002; Kawahara et al., 2013;Sakai et al., 2013). Compared with the 18S, 5.8S, and25S rDNAs (Supplemental Figs. S1B, S2, and S3), theDNA sequences for ETS and ITS spacers are muchmore variable in both length and sequence betweenNipponbare and Arabidopsis accession Col-0(Supplemental Fig. S4). Therefore, independently de-termining the pathway of rRNA biogenesis in rice, es-pecially the precise processing sites in the ETS and ITS1during pre-rRNA processing, is essential. Here, weexamined ribosome biogenesis at the level of pre-rRNAprocessing in rice, especially the processing sites, rRNAintermediates, and processing pathways by circular

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reverse transcription PCR (cRT-PCR; Kuhn and Binder,2002; Perrin et al., 2004; Slomovic et al., 2008; Abbasiet al., 2010; Zakrzewska-Placzek et al., 2010; Barkan,2011; Lange et al., 2011; Hang et al., 2014, 2015; Huanget al., 2016; Liu et al., 2016; Shanmugam et al., 2017).Furthermore, northern-blot assays showed that themajor ITS1-first and the minor 59 ETS-first processingpathways coexist in vivo to ensure rRNAmaturation inrice. Finally, we found that rRNA biogenesis in rice wasinhibited by chilling stress mainly at the pre-rRNAprocessing (P-A3 and 27SA2) level.

RESULTS

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

Biogenesis and maturation of the 18S rRNA, the onlystructural RNA in the 40S SSU, are essential for ribo-some biogenesis (Karbstein, 2011; Zhang et al., 2016).Pre-18S rRNA intermediates are processed by endo-nucleolytic cleavages in the 59 ETS and the ITS1 sur-rounding the 18S rRNA (Fig. 1A). The processing orderof 59 ETS removal and ITS1 splitting is always uncou-pled, resulting in various 18S precursors during 18SrRNA biogenesis. To determine the steps of pre-18SrRNA processing in rice, we first performed cRT-PCRassays based on the canonical 18S rDNA annotationto identify specific 18S rRNA precursors in vivo(Supplemental Fig. S5). To this end, the DNA oligonu-cleotide 18c (Fig. 1A) in the 18S rDNA region was usedfor specific reverse transcription of circularized rRNAintermediates (Supplemental Fig. S5, A and B). Theseintermediates were then amplified by pairs of PCRprimers, and the resulting amplification products wereverified by sequencing (Supplemental Fig. S5, C andD).The locations of primer pairs (18P1 to 18P8) are shownin Figure 1A and summarized in Supplemental TablesS1 and S2. We also validated the efficiency of cRT-PCRwith primer pairs 18P1 to 18P8, all of which couldamplify specific bands with cDNAs reverse-transcribedfrom ligated RNAs (Supplemental Fig. S5E). The ma-ture 18S rRNA was detected by the 18P1 primer pair(Fig. 1B). Then, 18S-A2 (by 18P1 and 18P8; Fig. 1, B andC), 18S-A3 (by 18P2 and 18P8; Fig. 1, B and D), andP9-A3 (by 18P2 and 18P5; Fig. 1, B and E) intermediateswere also amplified (Fig. 1A; Supplemental Tables S1and S2). Similarly, the P-A3 intermediates were detec-ted by four pairs of primers, 18P3, 18P4, 18P6, and 18P7(Fig. 1, B and F).We identified P-A3, P9-A3, 18S-A3, and18S-A2 as the major pre-18S rRNAs in maturationof rice pre-40S (Fig. 1A; Supplemental Fig. S6A;Supplemental Table S2).We next used the sequences at the 59 and 39 extremities

of the identified processing intermediates to define theprocessing sites. The mature 18S rRNA identified by the18P1 primers had boundary sites at A1 and D on the leftand right borders of 18S rDNA, respectively (Fig. 1A;Supplemental Figs. S6A, S7A, and S7B; SupplementalTable S1). Similarly, the P9 site of P9-A3was at G1634/A1635

of TCGGAAGACGACAG in the 59 ETS (Fig. 1E;Supplemental Fig. S7B). The DNA sequencing reads forP-A3 intermediates (Fig. 1F) defined the P site as be-tween C1160/T1161 of “ACACCTCTCCCACG” in the 59ETS region (Supplemental Fig. S7B). The P-A3, P9-A3,and 18S-A3 intermediates further confirmed the A3 siteto be between G3660/A3661 in “GTCAAGGAACACAG”in the ITS1 region (Supplemental Figs. S6A and S7B). Thelocations of the P and A3 endonucleolytic sites wereconsistent with a previous report (Wang et al., 2016).Notably, we found that P-A3, P9-A3, and 18S-A3 in ricewere highly polyadenylated (Fig. 1, A and D–F;Supplemental Fig. S6A), similar to results reported inArabidopsis (Abbasi et al., 2010; Lange et al., 2011; Hanget al., 2014; Sikorski et al., 2015; Shanmugam et al., 2017).The results suggested that active polyadenylation-dependent RNA processing systems, such as those me-diated 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; Langeet al., 2009; Losh and vanHoof, 2015; Sikorski et al., 2015;Thoms et al., 2015), exist in rice and take part in pre-18SrRNA processing.

Identification of rRNA Intermediates for the Pre-60SLarge Subunit

The mature 25S and 5.8S rRNAs are the structuralRNAs in the 60S large subunit (LSU) (Anger et al.,2013). To identify the pre-25S rRNA intermediates andprocessing sites in rice, we performed cRT-PCR assayswith the 25c primer for specific reverse transcription(25c_cDNA) followed by PCR with primer combina-tions 25P1 (25L/25R), 25P2 (p44/25R), 27P1 (58L/25R),and 27P2 (p4/25R) (Fig. 2A). The intact 25S rRNA wasidentified efficiently by the 25P1 primers within the 25SrDNA (Fig. 2, B and C), which defined its boundarysites, C1 and B2 on the left and right borders of the 25SrRNA, respectively (Supplemental Figs. S6B, S7A, andS7B). When the reverse primers were switched to p44 in25P2 or 58L in 27P1 (Fig. 2A), the intact 27SB interme-diate was identified (Fig. 2, B and D). The 27SB inter-mediate covers the 5.8S, ITS2, and 25S rRNA (Fig. 2A),which allowed us to identify B1 and B2 as the left andright borders, respectively, of the 5.8S and 25S rRNAs(Supplemental Figs. S6B, S7A, and S7B). Similarly, the27SA3 and 27SA2 sites were detected by primer com-bination 27P2 (Fig. 2, B, E, and F), in which the leftprimer p4 was in the ITS1 region adjacent to the leftboundary of 5.8S rRNA (Fig. 2A). Thus, we identified27SA2, 27SA3, and 27SB precursors as major pre-25SrRNAs, as well as 6S and 59-5.8S rRNAs during the60S LSU maturation in rice, consistent with results inbudding yeast (Woolford and Baserga, 2013) andArabidopsis (Weis et al., 2015a).

Among the pre-25S rRNA intermediates identified,27SA3 exhibited uniform 59 extremities at A3661 in“GTCAAGGAACACAG” in the ITS1 region (Fig. 2E;Supplemental Figs. S6B and S7B), which further

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Figure 1. Mapping of the 59 and 39 extremities of the pre-18S rRNAs. A, Structure of pre-18S rRNA intermediates identified by aset of primer combinations (in shaded box). Forward and reverse PCR primers for cDNA amplification aremarked in red and blue,respectively. For each fragment, the number of clones obtained is indicated on the right. The number of clones with additional


Hang et al.



confirmed the A3 site in rice to be between G3660/A3661

detected by P-A3, P9-A3, and 18S-A3 (Fig. 1, E, and F;Supplemental Figs. S6A and S7B). Moreover, the A2endonucleolytic sitewasdeduced to be betweenA3560/C3561

in “ACCAAAACAGACCG” by comparing the 39 endsof 18S-A2 (Fig. 1C) and the 59 ends of 27SA2 (Fig. 2F;Supplemental Figs. S6B and S7B), two precursors pro-cessed by direct cleavage at the A2 site in ITS1 from the32S transcript (Weis et al., 2015b). Here, the reads forthe 27SA2 intermediate shared the definite A2 site attheir 59 extremities (Fig. 2F), but the 39 extremities of the18S-A2 fragments we identified were much more het-erogeneous (Fig. 1C), indicating that the putative fast39→59 exonucleolytic trimming occurs in the processingof this precursor.Similarly, the 58c oligonucleotide was used for spe-

cific 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 performedto obtain both 5.8S-39 (6S) (Fig. 3A) and 59-5.8S (Fig. 3B)fragments, respectively. The 6S intermediates exhibitedheterogeneous 39 ends, part of which contained addi-tional polyadenylation sequences (Fig. 3A), similar tothe 6S intermediates in Arabidopsis (Shanmugam et al.,2017). This result indicates that 39→59 exonucleolytictrimming promotes 5.8S-39 rRNAs processing (Mitchellet 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 59→39exonucleolytic trimming may contribute more thanendonucleolytic cleavage to the 59-5.8S processing (Fig.3B; Henry et al., 1994; Zakrzewska-Placzek et al., 2010).

Identification of rRNA Intermediates in the90S/SSU Processome

The pre-40S SSU and pre-60S LSU derive from thesplit of the 90S/SSU processome at the ITS1 region ofthe 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 areprocessed in rice, we used the fixed forward primer 25Rand reverse primers 18L and p23 to perform the cRT-PCR assay (Fig. 4A). The 32S transcript from A1 to B2sites 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 combination32P2 (p23/25R; Fig. 4, A, B, and D).

The 39 ends of the 35S(P) fragments were not uni-form, harboring two to seven nucleotides of extra se-quence downstream of the B2 site in the 39 ETS (Fig.4D). Moreover, the 39 ends of the 35S(P) precursorswere polyadenylated, which was rarely detected in the32S precursors (Fig. 4C). This observation indicatedthat (1) the complete trimming of the 39 ETS regionoccurred from 35S(P) to 32S in rice, in 39→59 exonu-cleolytic processing (Lange and Gagliardi, 2010) bypresently 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 promoterRNA 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-rRNAprocessing could coexist in rice. To further determinethe pre-rRNA processing pattern in vivo in rice, we setup a northern-blot assay with a series of short oligo-nucleotide probes (Fig. 5; Supplemental Table S1;Supplemental Fig. S8). Probes p4 and S9 that are adja-cent to the left and right borders of 5.8S rDNAs, re-spectively (Fig. 5A), recognized pre-5.8S rRNAs and27S rRNAs in the pre-60S LSU (Fig. 5B). This result wasconsistent with the cRT-PCR data (Figs. 2 and 3). Theprobes p23, S7, and p42 (Fig. 5A) were designed todetect the pre-18S rRNAs in the pre-40S SSU (Fig. 5, Cand D; Supplemental Fig. S8). The 59 ETS probe p23between the P and P9 sites distinguished 35S(P) from32S precursors in the 90S/SSU complex (Fig. 5A).Moreover, the ITS1 probe p42 between A2 and A3 sitesdetected 18S-A3 and 27SA2 specifically (Fig. 5, A andD), compared with the probes p23 and S7 recognizing18S-A3, or p4 and S9 recognizing 27SA2 (Fig. 5A).

We used the togr1-1 mutant as the positive control(Wang et al., 2016), which exhibited an aberrant accu-mulation of 35S(P) and P-A3 compared with the wildtype, Zhongxian3037 (Fig. 5, B–E; Supplemental Fig. S8).

Figure 1. (Continued.)sequences, such as polyadenylation at the 39 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 se-quencing of 18S and its major precursors identified: 18S-A2 (C), 18S-A3 (D), P9-A3 (E), and P-A3 (F). The 18S rRNAs identified byprimers 18P1 were validated by sequencing of 20 independent clones. The 18S-A2 intermediates identified by primers 18P1 and18P8were validated by sequencing of 33 independent clones (C). The 18S-A3 intermediates identified by primers 18P2 and 18P8were validated by sequencing of 58 independent clones (D). The P9-A3 intermediates identified by primers 18P2 and 18P8 werevalidated by sequencing of 21 independent clones (E). The P-A3 intermediates identified by primers 18P6, 18P7, 18P3, and 18P4were validated by sequencing of 87 independent clones (F). The ITS1 locus matched by the 39 ends of these clones are indicatedby black triangles as well as the number of clones. Additional sequences in the 39 extremities of these clones are marked in redlowercase letters. The numbers of identical clones are indicated to the right of each fragment.











The P-A3 intermediate and its direct precursor 35S(P)were both readily detected by the 59 ETS probe p23 (Fig.5C; Supplemental Fig. S8), as well as ITS1 probes S7A(Supplemental Fig. S8), S7, and p42 (Fig. 5D). By con-trast, we observed much less of the 32S intermediatethan the 35S(P), when probed with S7A (SupplementalFig. S8), p4, and S9 (Fig. 5B). Similarly, the relativeamount of 27SA2 was also far less than that of P-A3 inNipponbare detected by ITS1 probe p42 (Fig. 5D).Moreover, the abundance of P-A3 in the indica cultivarZhongxian3037 was less than in the japonica cultivarNipponbare (Fig. 5E), as detected by probes p23 (Fig.

5C), S7, and p42 (Fig. 5D; Supplemental Fig. S8). Thisvariation in pre-rRNAprocessing between these two ricesubspecies may come from genome variation duringevolution (Huang et al., 2012), a possibility that will re-quire further examination in the future.

Alternative rRNA Biogenesis Pathways in Rice

Uncoupled processing of 59 ETS removal and ITS1cleavage during the processing of early transcriptsresulted in alternative rRNA biogenesis pathways

Figure 2. Mapping of the 59 and 39 extremities of the pre-25S rRNAs. A, Structure of pre-25S intermediates identified by a set ofprimers (in shaded box). Forward and reverse PCR primers for cDNA amplification are marked in red and blue, respectively. Fourpairs of primers were used for pre-25S rRNAs: 25P1 (25L/25R), 25P2 (p44/25R), 27P1 (58L/25R), and 27P2 (p4/25R). For eachfragment, the number of clones obtained is indicated on the right. The number of clones containing additional sequences at the 39extremities 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 27SBintermediates identified by primers 25P2 and 27P1 were validated by sequencing of 51 independent clones (D). The 27SA3intermediates identified by primers 27P1 and 27P2 were validated by sequencing of 22 independent clones (E). The 27SA2 in-termediates identified by primers 27P2 were validated by sequencing of 21 independent clones (F). The ITS1 and ITS2 locusmatched by the 59 and 39 ends of these DNA sequences, respectively, are indicated by black triangles as well as the number ofclones. Additional sequences in the 39 extremities of these clones are marked in red lowercase letters. The number of identicalclones is indicated to the right of each fragment.


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(Hang et al., 2014; Weis et al., 2015a, 2015b; Tomeckiet al., 2017). These pre-rRNA processing modes aredistinguished by the order of 59 ETS removal and ITS1splitting and reflect ribosome assembly dynamicsduring ribosome biogenesis (Mullineux and Lafon-taine, 2012; Weis et al., 2015a; Tomecki et al., 2017).Alternative pre-rRNA processing is a conserved mo-lecular characteristic in eukaryotes and has been welldefined in budding yeast (Woolford and Baserga,2013), mammalian cells (Bowman et al., 1981;Hadjiolova et al., 1993; Kent et al., 2009; Mullineuxand Lafontaine, 2012; Henras et al., 2015), and Ara-bidopsis (Sikorski et al., 2015; Weis et al., 2015a;Tomecki et al., 2017).The definition of major and minor pathways in eu-

karyotes is based on the amount of marker pre-rRNAtranscripts in wild type by northern-blot or pulse-chaselabeling (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 tothe situation in unicellular budding yeast (Kos andTollervey, 2010), the pulse-chase labeling approach forstudying rRNA synthesis remains technically difficultin higher plants (Weis et al., 2015a). The northern-blotapproach is reliable (Barkan, 2011) and has shown thatArabidopsis preferentially uses the ITS1-first mode asthe major pathwaymarked 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 thanthe minor 59 ETS-first mode marked by 33S(P9), 32S,and 27SA2 (Hang et al., 2014; Weis et al., 2015a, 2015b;Tomecki et al., 2017). Therefore, our detection of asimilar pre-rRNA pattern in vivo with RNA hybridi-zation (Fig. 5; Supplemental Fig. S8) suggests thatsimilar alternative rRNA maturation pathways maycoexist in rice in vivo.

Figure 3. Mapping of the 59 and 39 extremities of the pre-5.8S rRNAs. A and B, Structure of 39-5.8S identified by 58P1 (58L1/58R1; A) and 59-5.8S by 58P2 (58L2/58R2; B), respectively. Forward and reverse PCR primers for cDNA amplification are markedin red and blue, respectively. For each fragment, the number of clones obtained is indicated on the right. The number of clonescontaining additional sequences at the 39 extremities are marked in parentheses (in the shaded box). The 5.8S-39 intermediateswere validated by 70 independent clones (A). The 59-5.8S intermediates were validated by 22 independent clones (B). The ITS1and ITS2 locusmatched by the 59 and 39 ends of theseDNA sequences, respectively, are indicated by black triangles aswell as thenumber of clones. Additional sequences in the 39 extremities of these clones are marked in red lowercase letters. The number ofidentical clones are indicated to the left (A) and right (B) of each fragment, respectively. C, Pre-5.8S rRNA intermediates weredetermined in gel by cRT-PCR with primers 58P1 and 58P2.






Here, we propose a working model for rRNA bio-genesis in rice (Fig. 6). After rDNA transcription byRNA Pol I, the 45S rRNA transcripts undergo primarycleavages at the P site in the 59 ETS and an unknown sitein the 39 ETS to generate the 35S(P) intermediate. Thenthe 35S(P) transcript enters two alternative maturationpathways distinguished by the order of ITS1 cleavageand 59 ETS removal. In the major ITS1-first pathway,the 35S(P) transcript is first split into P-A3 and 27SA3 byendonucleolytic cleavage at the A3 site in the ITS1. Asthe diagnostic marker for major pathway, P-A3 in thepre-40S SSU can be further processed into P9-A3, 18S-A3, 18S-A2 (predicted) sequentially, and eventuallymatures into the 18S rRNA (Fig. 6). In the minor 59 ETS-first pathway in rice, marked by the 32S and 27SA2intermediates, the primary 35S(P) transcript is firstshortened at its 59 end by complete removal of the 59

ETS resulting in 32S rRNAs. Then, cleavage at the A2site splits the 32S rRNA into the 18S-A2 and 27SA2intermediates, which undergo further endo- and exo-nucleolytic processing into mature 18S, 5.8S, and 25SrRNAs (Fig. 6).

Inhibition of rRNA Biogenesis under Chilling Stress

To test for a potential relationship between chill-ing stress and ribosome biogenesis at the level ofpre-rRNA processing in rice, we performed northern-blot assays with rice shoots after a time-course chill-ing treatment (Fig. 7, A and B; Supplemental Figs.S9 and S10). Both P-A3 in the ITS1-first pathwayand 27SA2 in the 59 ETS-first pathway decreasedunder chilling stress in shoots (Fig. 7, A and B;

Figure 4. Mapping of the 59 and 39 extremities of the 35S(P) and 32S transcripts. A, Structure of early pre-rRNA intermediatesidentified (in shaded box) by two pairs of primers: 32P1 and 32P2. Forward and reverse PCR primers for cDNA amplification aremarked in red and blue, respectively. For each fragment, the number of clones obtained is indicated on the right. The number ofcloneswith additional sequences at the 39 end is marked in parentheses. B, The 32S and 35S(P) pre-rRNAswere determined in gelby cRT-PCRwith primers 32P1 (18L/25R) and 32P2 (p23/25R). C andD, 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 bysequencing of 25 independent clones (D). The ITS1 and ITS2 locus matched by the 59 and 39 ends of these DNA sequences,respectively, are indicated by black triangles and the number of clones. Additional sequences in the 39 extremities of these clonesare marked in red lowercase letters. The number of identical clones is indicated to the right of each fragment.


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Supplemental Figs. S9A, S9B, and S10B) and roots(Supplemental Fig. S10C), indicating reduced pre-rRNAprocessing.

The 45S pre-rRNA transcribed from the rDNA clus-ters by Pol I is quickly processed into 35S(P) by cleavageat the P site in the 59 ETS and an unknown site in the 39

Figure 5. Northern blots to detect pre-rRNA processing in rice. A, Pre-rRNA processing intermediates detected by northern blotswith specific probes, which are indicated by horizontal arrows. Black vertical arrows above the diagram indicate endonucleolyticcleavage sites relevant to this study. Different rRNA precursors are marked. P-A3, P9-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 39-5.8S (7S and6S) and 59-5.8S are pre-5.8S rRNAs. The 7S rRNAmarkedwith “?”was detected by probe S9 (Fig. 5B), but its definite 39 extremitiesare still unclear (A). The 35S(P) and 27SA2 could be specifically detected by probes p23 and p42, respectively. Both probes S7 andp42 detect 35S(P), 32S, P-A3, and 18S-A3. Although 18S-A2 could be detected by S7, its low abundance inwild-type ricemakes itharder to distinguish from 18S-A3 by northern-blot assay. B, Northern blots to determine pre-rRNA processing in pre-60S LSU inNipponbare (lane 1), Zhongxian3037 (ZX3037, lane 2), and togr1mutants (lanes 3 and 4). The togr1mutant is a positive controlthat 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 blotsshare the same loading control (D). The quantitation of P-A3 in Nipponbare (lane 1), Zhongxian3037 (lane 2), and togr1 (lanes3 and 4) were performed with three biological replicates (E). Matured rRNAs stained with MB serve as the loading control. Therelative intensities for P-A3 intermediate in each lane are normalized to Zhongxian3037. Error bars represent SD. Data are given asmeans and SD of three independent biological replicates.







ETS and then undergoes pre-rRNA processing to re-lease mature rRNAs (Fig. 6). Therefore, the 45S rRNAin vivo is the net product of rDNA transcription andsubsequent pre-rRNA processing. The decreased bio-genesis of P-A3 and 27SA2 under chilling treatmentprompted us to investigate whether this inhibition be-gan with the 45S transcript or at pre-rRNA processingstages. The 59 ETS region from the transcription initia-tion site (TIS) to the P site is unique to the 45S pre-rRNAwith the exception of 35S(P) (Fig. 6). We detected 45SrRNA transcripts by northern blots with a specific longprobe (45P) that recognizes the 59 ETS region upstreamof the P site (Fig. 7D; Supplemental Table S1). Thesteady state of 45S pre-rRNA increased under chilling

stress, which was inversely correlated with the abun-dance of P-A3 (Fig. 7C; Supplemental Fig. S11). There-fore, we propose that chilling stress affects rRNAbiogenesis predominantly at the pre-rRNA processinglevel in rice, which results in decreased biogenesis ofP-A3 and 27SA2. Then, decreased pre-rRNAprocessingmay negatively affect the processing dynamics of 45Stranscript, resulting in its accumulation (Fig. 7D). Al-though the transcriptional activity of RNA Pol I wouldprovide direct evidence to illustrate the transcription of45S pre-rRNA (Ream et al., 2015), the appropriate an-tibodies or transgenic materials in rice are not currentlyavailable. Besides, de novo characterization of nascenttranscripts under chilling treatments using unbiased

Figure 6. Model of rRNA biogenesis in rice. Primary transcripts generated by RNA Polymerase I are first processed at P in the 59ETS and at an unknown site in the 39 ETS to generate 35S(P), which undergoes further pre-rRNA processing by alternativepathways distinguished by the order of ITS1 splitting and 59 ETS removal, to generate mature 18S, 5.8S, and 25S rRNAs. In themajor ITS1-first pathway, the 35SP transcript is split at ITS1 endonucleolytic site A3 into P-A3 and 27SA3 precursors. In the minor59 ETS-first pathway, the removal of the 59 ETS in the 35S(P) transcript occurs first to generate the 32S intermediate before its split atthe ITS1 cleavage site A2. Both endo- and exonucleolytic processing occur sequential and coordinately in this progress. Pre-cursors with partial transparency indicate putative intermediates in these pathways.


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global nuclear run-on sequencing will provide us withmore information at the transcriptional level (Hetzelet al., 2016). Nevertheless, our results suggest that ricemay fine-tune ribosome biogenesis to quickly adjustenergy consumption and primary metabolism for

survival and acclimation at low temperatures. More-over, compared with both the mature rRNAs and otherrRNA precursors, the P-A3 precursor readily detectedby northern blots could be a reliable marker for rRNAbiogenesis under chilling stress.

Figure 7. Chilling stress inhibits rRNA biogenesis mainly at pre-rRNAs processing levels. A and B, Northern blots to detectpre-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 signalrelative 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 arepresentative result is shownhere. C,Northern blots to detect the 45S rRNA transcript by probe 45P inNipponbare under 4°C treatmentfor 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 loadingcontrol. The numbers beloweach lane represent the intensity ratio of each signal relative to the 0h sample. The relative intensities for 25SrRNA, 45S transcripts, and P-A3 intermediates are marked in black, blue, and red, respectively. RNA samples from two biologicalreplicates were loaded and detected in parallel. D, Simplified model that the inhibition of rRNA biogenesis in rice by chilling stresspredominantly occurs at posttranscriptional level. The 45S rRNA, transcribed byRNAPol I from rDNAs, undergoes pre-rRNAprocessingto release mature rRNAs. The steady level of 45S rRNA in vivo is the net product of rDNA transcription and subsequent pre-rRNAprocessing. Chilling stress inhibits pre-rRNAprocessing, shown by the time-course reduction of P-A3 and 27SA2 in both ITS1-first and 59ETS-first processing pathways, respectively (A and B). Although it remains unknown whether and how chilling treatment affect rDNAtranscription, the increased45S rRNA (C) couldmainlyoriginate from reducedpre-rRNAprocessing under chilling stress. The longprobe45P could distinguish the 45S rRNA from its product 35S(P).





DISCUSSION

Two pre-rRNA processing pathways, known as the59 ETS-first and the ITS1-first modes, commonly coexistin eukaryotes (Lafontaine, 2010; Mullineux and Lafon-taine, 2012; Hang et al., 2014; Henras et al., 2015; Weiset al., 2015a; Tomecki et al., 2017). Such conservedmolecular characters have been well deciphered inbudding yeast (Gallagher et al., 2004; Lamanna andKarbstein, 2011; Mullineux and Lafontaine, 2012) andto some extent in animal systems, such asXenopus laevisoocytes (Savino and Gerbi, 1990; Borovjagin and Gerbi,1999), Drosophila melanogaster (Long and Dawid, 1980),and mammalian cells (Bowman et al., 1981; Hadjiolovaet al., 1993; Kent et al., 2009). Here, we identified therRNA intermediates and critical processing sites of the59 ETS and ITS1 regions in rice (Supplemental Figs. S6and S7). These findings ultimately uncovered the ricealternative pre-rRNA processing pathways with theITS1-first mode as the major pathway (Fig. 6). The ITS1-first mode in rice resembles that in Arabidopsis (Hanget al., 2014; Weis et al., 2015a, 2015b) and mammaliansystems, rather than the unicellular budding yeast, inwhich the 59 ETS-first mode is the dominant pathway(Mullineux and Lafontaine, 2012; Henras et al., 2015).Moreover, we found that rice and Arabidopsis havesimilar flanking sequences around the A2 and A3endonucleolytic sites in the ITS1 and the P9 in the 59ETS, respectively (Supplemental Fig. S7C). This obser-vation suggests that conserved cis-elements shared byboth species contribute to the selection of these endo-nucleolytic cleavage sites. In contrast, the sequencesflanking the P site in the 59 ETS are highly variablebetween rice and Arabidopsis (Supplemental Fig. S7C).We propose that currently unknown transacting factorsor 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 inboth species in vivo.

Ribosome assembly and rRNA maturation include aseries of rRNA conformational changes and protein-binding events (Marmier-Gourrier et al., 2011; Phippset al., 2011). Thus, alternative pre-rRNA processingevents are generally believed to come from uncoupledprocessing for 59 ETS removal and ITS1 cleavage me-diated by the pre-ribosomal complex, the 90S/SSUprocessome, 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 micros-copy, the assembly of the 90S/SSU processome(Kornprobst et al., 2016; Zhang et al., 2016; Johnsonet 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 re-solved. In higher plants, the U3 small nucleolar ribo-nucleoprotein (U3 snoRNP) was first purified fromcauliflower inflorescences as the Nuclear Factor Dcomplex (Sáez-Vasquez et al., 2004a, 2004b) and fromBrassica 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 Bmotifs] to mediate P-site cleavage in the 59 ETS(Caparros‐Ruiz et al., 1997; Sáez-Vasquez et al., 2004a,2004b; Samaha et al., 2010). Although the BoU3/NF-Dcomplex has not been identified in Arabidopsis, sys-temic quantitative proteomic assays from subcellularfractionations identified plant-specific ribosome bio-genesis factors in Arabidopsis (Palm et al., 2016). In rice,TOGR1 was the first well-defined RNA helicase es-sential for ribosome biogenesis during rice growth anddevelopment (Wang et al., 2016). It will be interesting todecipher the functional complexes that form during riceribosome biogenesis in the future.

Environmental signals affect plant growth and cropyield. Such signals include environmental factors suchas photoperiod (Ding et al., 2012; Fan et al., 2016) andambient temperature fluctuations (Gong et al., 2002,2005; da Cruz et al., 2013; Challinor et al., 2014; Rayet al., 2015; Shi et al., 2015). Rice originated from trop-ical and subtropical regions (Huang et al., 2012);therefore, rice cultivated in temperate zones can exhibitmore sensitivity to chilling stress than other crops suchas barley (Hordeum vulgare) and wheat (Triticum aestivum;Zhang et al., 2014). Accordingly, rice has evolvedmechanisms to adapt to heat stress (Li et al., 2015; Shenet al., 2015;Wang et al., 2016) and cold temperature (Maet al., 2015; Zhang et al., 2017 b). The ribosome acts as atemperature sensor in Escherichia coli to coordinatemetabolism 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 ambienttemperature (Kaczanowska and Rydén-Aulin, 2007; AlRefaii and Alix, 2009; Baliga et al., 2016). Aberrantsensitivity to temperature fluctuation is a hallmark ofmutants with defects in ribosome biogenesis in E. coli(Guthrie et al., 1969; Dammel and Noller, 1993; Joneset al., 1996; Al Refaii and Alix, 2009; Mayerle andWoodson, 2013), yeast (Warner and Udem, 1972;Tollervey et al., 1993; Teyssier et al., 2003; Wan et al.,2015), and Arabidopsis (Ohbayashi et al., 2011; Huanget al., 2016; Liu et al., 2016). In rice, temperature fluc-tuations such as heat and chilling stresses adverselyaffect the vegetative and reproductive stages (Zhouet al., 2012, 2014; Fan and Zhang, 2014), which even-tually affect yields (Cruz et al., 2013; Ray et al., 2015).Likewise, dysfunction of the ribosome biogenesis factorTOGR1 affected pre-rRNA processing, which resultedin severe developmental defects and hypersensitivity toheat stress in rice. Also, constitutive expression ofTOGR1 enhanced the tolerance of rice to heat stress(Wang et al., 2016). Therefore, an understanding of ri-bosome biogenesis and its response to ambient tem-perature in rice can benefit basic scientific research andfacilitate efforts to improve thermo-tolerance (Chenet al., 2009; Song et al., 2012a, 2012b; Zhou et al., 2014; Liet al., 2015; Shen et al., 2015; Wang et al., 2016; Yu et al.,2018) and chilling tolerance (Fan and Zhang, 2014; Lu


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et al., 2014;Ma et al., 2015; Li and Lin, 2016; Zhang et al.,2017 b) in agricultural applications.rRNA biogenesis at the level of pre-rRNA processing

is an ideal and reliable molecular diagnostic reflectingribosome biogenesis and ribosome assembly statusin vivo (Mullineux and Lafontaine, 2012; Tomecki et al.,2017). In our work, the reduction of pre-rRNA pro-cessing under chilling stress indicated decreased ribo-some assembly in the nucleus, which may eventuallyaffect the production of active ribosomes in the cyto-plasm. Ribosome biogenesis in vivo is highly energy-consuming and strictly orchestrated by internal andexternal signals to meet the demand for mature ribo-somes in mRNA translation (Warner, 1999; Woolfordand Baserga, 2013). Here, we found that rRNA bio-genesis is down-regulated by chilling stress at theposttranscriptional levels, potentially for the adjust-ment of energy consumption and primary metabolismto adapt to cold stress (Fig. 7C). In addition, the trans-lational activity of ribosomes in the cytoplasm could bedirectly and dynamically fine-tuned by various envi-ronmental signals (Bailey-Serres et al., 2009; Browningand 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), heatstress (Zhang et al., 2017a), and light signals (Liu et al.,2012, 2013). This represents another regulatory layeraffecting the activity of ribosomes to facilitate the ac-climation and survival of rice under stress.In conclusion, we defined rRNA biogenesis at the

level of pre-rRNA processing in rice and uncovered amolecular link between chilling stress and ribosomebiogenesis in vivo. It will be intriguing to determine themolecular mechanism of temperature sensing in ribo-some 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 be-longs to the japonica subspecies (O. sativa ssp. japonica; Huang et al., 2012).Zhongxian3037, togr1-1 mutants (Wang et al., 2016), and 9311 belong to theindica 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 har-vested fromNipponbare grown in the paddy fields under natural conditions forRNA 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 mmol quanta m22 s21 and 80% hu-midity, unless otherwise specified) at 28°C for 10 d after germination. In Figure5 and Supplemental Figure S8, 0.15 to ;0.20 g of shoots (from around three tofour plants) were harvested for RNA extraction. For cold treatment of seedlingsin 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 seedlingswere treated in dark growth chamber at 4°C. Then 0.15 to ;0.20 g of shootswere 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 shootsand roots were harvested separately as 0-h controls. The remaining seedlingswere transferred to precooled water and treated in a dark growth chamber at4°C. Then, 0.15 to ;0.20 g of shoots and roots were harvested in the same wayevery 2 h for two or three intervals. The water was changed every two daysduring growth. Fresh materials were frozen by liquid nitrogen and stored at280°C until used. More than three biological replicates were performed forupper treatments and the representative data were exhibited.

RNA Extraction

The rice materials were first ground into fine powder with liquid nitrogen.Then, total RNAwas extracted from the powderwith TRNzol reagent (Tiangen;DP405-02) according to the manufacturer’s instructions. Total RNA was dis-solved in DEPC-treated deionized water and quantified with a NanoDrop1000 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 (SupplementalFig. S5). Briefly, 10 mg of total RNA extracted from Nipponbare panicles wasself-ligated into circular RNA by T4 RNA ligase 1 (New England Biolabs;M0204S; Supplemental Fig. S5A). The circular RNA was further reverse tran-scribed into first-strand cDNA (TransGen Biotech; AH301) using specific anti-sense DNA oligonucleotide 18c or 25c that are complementary to sequences inthe 18S rDNAor 25S rDNA region, respectively (Supplemental Fig. S5B). Then aseries of primers (Supplemental Table S1) around the 18c or 25c RT primer wereused to amplify the flanking sequences of precursors with 23 Phanta MasterMix (Vazyme Biotech; P511-01). After amplification for 35 cycles, bandsobtained by cRT-PCR were subcloned into the pEasy-T vector (Transgene;CT101-02; Supplemental Fig. S5C), and positive clones were selected for with asecond PCR using the M13F and M13R primers. Finally, target productsexhibiting sharp bands at the proper molecular weight were excised for DNAsequencing and further analyzed by BLAST from the National Center for Bio-technology Information (NCBI), choosing the organism (O. sativa, japonicagroup; taxid:39947) and database (reference genomic sequences [refseq_ge-nomic]) using Megablast (optimized for highly similar sequences). Cleavagesites and flanking sequences were identified according to japonica rice rDNAoffline 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) mg oftotal RNA was separated on a 1.2% (w/v) agarose/formaldehyde gel and thentransferred to aHybondN+membrane (GEHealthcare; RPN1520B) by capillaryelution. For short DNA probes, oligonucleotides labeled with [g-32P]ATP(Perkin-Elmer; BLU002A001MC) by T4 polynucleotide kinase (New EnglandBiolabs; 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 mg of purified 45P fragment was subjected tolabeling with [a-32P]dCTP (Perkin-Elmer; NEG513H) using a commercialRandom Primer DNA Labeling kit (TaKaRa; cat. no. 6045). Hybridization wasperformed overnight at 45°C (for short probes) or 65°C (for long probes) aspreviously described (Hang et al., 2014). The blots were washed and exposed toa storage phosphor screen (GEHealthcare), then detectedwith a Typhoon TRIOscanner (GE Healthcare). A complete list of probes is included in SupplementalTable 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 num-bers for rDNA sequences of rice andArabidopsis 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 eukaryotesand comparison of rDNA components between the japonica rice Nippon-bare and Arabidopsis thaliana accession Col-0.





















Supplemental Figure S2. Sequence alignments of 18S and 5.8S rDNAsbetween the japonica rice Nipponbare and Arabidopsis thaliana accessionCol-0.

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

Supplemental Figure S4. Sequence alignments of 59 ETS, ITS1, ITS2, andpartial 39 ETS rDNAs between the japonica rice Nipponbare and theArabidopsis thaliana accession Col-0.

Supplemental Figure S5. Circular RT-PCR assay to identify pre-rRNAprecursors.

Supplemental Figure S6. Representative DNA sequencing results for theidentified pre-rRNAs.

Supplemental Figure S7. Summary of endonucleolytic sites identified dur-ing pre-rRNA processing in rice.

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

Supplemental Figure S9. Pre-rRNA processing in rice shoots in responseto chilling stress.

Supplemental Figure S10. Pre-rRNA processing in rice roots responses tochilling stress.

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

Supplemental Table S1. Oligonucleotides used in this article.

Supplemental Table S2. rRNA intermediates and primer combinationsused in cRT-PCR assays.

ACKNOWLEDGMENTS

We thank Dr. Yanyuan Kang in our lab for supporting the panicle RNAsamples, Zhiyao Lv in our lab for supporting the original picture of FigureS10A, and our fellow lab members for stimulating discussions. We thankDr. Yongbiao Xue at the Institute of Genetics and Developmental Biology,Chinese Academy of Sciences, for providing Zhongxian3037 and togr1-1 seeds.

Received November 30, 2017; accepted March 2, 2018; published March 19,2018.

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ribosomal rna biogenesis and its response to chilling ...and its2, and ﬂanked by 59 and 39...

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