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ABSTRACT DEAD-box RNA helicases are vital for the regulation of various aspects of the RNA life cycle1, but the molecular underpinnings of their involvement, particularly in mammalian cells,

remain poorly understood. Here we show that the DEAD-box RNA helicase DDX21 can sense the transcriptional status of both RNA polymerase (Pol) I and II to control multiple steps of ribosome

biogenesis in human cells. We demonstrate that DDX21 widely associates with Pol I- and Pol II-transcribed genes and with diverse species of RNA, most prominently with non-coding RNAs

involved in the formation of ribonucleoprotein complexes, including ribosomal RNA, small nucleolar RNAs (snoRNAs) and 7SK RNA. Although broad, these molecular interactions, both at the

chromatin and RNA level, exhibit remarkable specificity for the regulation of ribosomal genes. In the nucleolus, DDX21 occupies the transcribed rDNA locus, directly contacts both rRNA and

snoRNAs, and promotes rRNA transcription, processing and modification. In the nucleoplasm, DDX21 binds 7SK RNA and, as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex,

is recruited to the promoters of Pol II-transcribed genes encoding ribosomal proteins and snoRNAs. Promoter-bound DDX21 facilitates the release of the positive transcription elongation

factor b (P-TEFb) from the 7SK snRNP in a manner that is dependent on its helicase activity, thereby promoting transcription of its target genes. Our results uncover the multifaceted role of

DDX21 in multiple steps of ribosome biogenesis, and provide evidence implicating a mammalian RNA helicase in RNA modification and Pol II elongation control. Access through your institution

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OTHERS DEAD-BOX ATPASE DBP2 IS THE KEY ENZYME IN AN MRNP ASSEMBLY CHECKPOINT AT THE 3’-END OF GENES AND INVOLVED IN THE RECYCLING OF CLEAVAGE FACTORS Article Open access 09 August 2024 THE

CONSERVED RNA-BINDING PROTEIN SEB1 PROMOTES COTRANSCRIPTIONAL RIBOSOMAL RNA PROCESSING BY CONTROLLING RNA POLYMERASE I PROGRESSION Article Open access 25 May 2023 THE NUCLEOLAR SHELL

PROVIDES ANCHORING SITES FOR DNA UNTWISTING Article Open access 23 January 2024 ACCESSION CODES PRIMARY ACCESSIONS GENE EXPRESSION OMNIBUS * GSE56802 DATA DEPOSITS All sequencing data have

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ACKNOWLEDGEMENTS We thank D. H. Price for the LARP7 antibody, K. Lane from M. Covert’s laboratory for metabolic inhibitors, K. Cimprich and members of the Chang and Wysocka laboratories for

discussions, and B. Zarnegar and P. Khavari for discussions regarding iCLIP. This work was supported by the Stanford Medical Scientist Training Program and T32CA09302 (R.A.F.), AP Giannini

Foundation (R.C.S.), National Institutes of Health grants R01-HG004361, R01-ES023168, P50-HG007735 (H.Y.C.) and R01-GM095555 (J.W.), W. M. Keck Foundation (J.W.), and Helen Hay Whitney

Foundation (E.C.). H.Y.C. is an Early Career Scientist of the Howard Hughes Medical Institute. AUTHOR INFORMATION Author notes * Eliezer Calo and Ryan A. Flynn: These authors contributed

equally to this work. AUTHORS AND AFFILIATIONS * Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, 94305, California, USA Eliezer Calo & 

Joanna Wysocka * Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, 94305, California, USA Ryan A. Flynn, Lance Martin, 

Robert C. Spitale & Howard Y. Chang * Department of Developmental Biology, Stanford University School of Medicine, Stanford, 94305, California, USA Joanna Wysocka Authors * Eliezer Calo

View author publications You can also search for this author inPubMed Google Scholar * Ryan A. Flynn View author publications You can also search for this author inPubMed Google Scholar *

Lance Martin View author publications You can also search for this author inPubMed Google Scholar * Robert C. Spitale View author publications You can also search for this author inPubMed 

Google Scholar * Howard Y. Chang View author publications You can also search for this author inPubMed Google Scholar * Joanna Wysocka View author publications You can also search for this

author inPubMed Google Scholar CONTRIBUTIONS H.Y.C. and J.W. supervised the project; E.C. and R.A.F. conceived and designed the study; E.C. and R.A.F. performed experiments and analysed

ChIP-seq data. R.A.F. performed iCLIP and L.M., R.C.S. and R.A.F. analysed iCLIP data; R.A.F., E.C., J.W. and H.Y.C. wrote the manuscript with input from all co-authors. CORRESPONDING

AUTHORS Correspondence to Howard Y. Chang or Joanna Wysocka. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. EXTENDED DATA FIGURES AND TABLES

EXTENDED DATA FIGURE 1 DDX21 ASSOCIATES WITH NON- AND PROTEIN-CODING RIBOSOMAL GENES. A, MEME analysis of DDX21-bound regions defined by DDX21 ChIP-seq. Motif logo, annotated transcription

factor, number of motif instances within the ChIP-seq regions, _Z_ score, and _P_ value for each motif are shown. B, DDX21 ChIP-qPCR from HeLa cell chromatin extracts with primers spanning a

representative number of loci found to be enriched in the DDX21 ChIP-seq analyses from HEK293 cells. Data are mean and s.d. of three independent experiments. C, Comparison of DDX21 (this

study) and H3K4me3 (publically available data, see Methods for accession numbers) ChIP-seq-bound regions. 2,863 regions are common between the data sets, 505 regions are unique to DDX21, and

11,403 regions are unique to the H3K4me3 data set. D, Gene Ontology terms for H3K4me3 regions that are either DDX21-bound (left) or not bound by DDX21 (right). E, Box plots representing the

expression levels of snoRNA-host genes whose promoter regions are either bound or not by DDX21. As shown, snoRNA-host gene promoters bound by DDX21 are, on average, more highly expressed

than those not occupied by DDX21. Fragments per kilobase of exon per million mapped reads (FPKM) values were taken from publically available HEK293 RNA-seq data (see Methods for accession

number). The _P_ value (_P_ ≤ 0.05) was calculated using the Wilcoxon signed-rank test. F, G, UCSC genome browser tracks depicting DDX21 ChIP-seq and iCLIP-seq, and RNA-seq enrichment

profiles at differentially expressed snoRNA-host genes in HEK293 cells. EXTENDED DATA FIGURE 2 DDX21 POSITIVELY REGULATES TRANSCRIPTION OF POL I- AND POL II-DEPENDENT RIBOSOMAL GENES. A,

siRNA-mediated knockdown of the DDX21 antibody used for ChIP. We transfected HEK293 cells with two different sets of siRNAs targeting endogenous _DDX21_ mRNA (siRNA1 and siRNA2 (3′ UTR)) and

performed western blots with the indicated antibodies. As shown, the DDX21-specific band is diminished in cells transfected with _DDX21_-targeting siRNAs, but not with control siRNAs. Actin

was used as a loading control for this experiment. B, RT–qPCR analysis assessing the RNA expression levels of the same genes analysed in Fig. 1h upon _DDX21_ knockdown by a second siRNA

that targets the 3′ UTR of _DDX21_ mRNA. Data are mean and s.d. of three independent experiments. For DDX21-target genes the difference between control and _DDX21_ siRNA is significant, _P_ 

≤ 0.05 (Student’s _t_-test). C, Diagram of DDX21 protein domains. The two conserved RecA-like (A and B) domains and the GUCT domains are shown in green and blue, respectively. Amino acids

targeted for mutation12 to convert DDX21WT into DDX21SAT, the ATP-hydrolysis mutant, are indicated with red and purple lines in the diagram. Specific amino acid changes are displayed below.

D, qRT–PCR analysis assessing nascent unspliced mRNA levels from additional DDX21-target and DDX21-non-target promoters. For a detailed description see Fig. 1j. Data are mean and s.d. of

three biological replicates. E, Nuclear rRNA abundance analysis by RNA BioAnalyzer of HEK293 cells depleted of DDX21 and rescued with DDX21WT, DDX21SAT, or DDX21DEV. For each analysis, total

RNA was isolated from 1,500,000 nuclei. Total nanogram amounts are shown for each of the two large rRNA subunits. EXTENDED DATA FIGURE 3 SELECTIVE INHIBITION OF POL I ALTERS DDX21 NUCLEAR

LOCALIZATION AND CHROMATIN ASSOCIATION. A, B, Immunofluorescence images of methanol-fixed HEK293 cells after 1 h incubation with either DMSO or 2 μM of the specific Pol I inhibitor CX-5461.

DDX21 (A) and fibrillarin (B) immuno-labellings are shown. Scale bars, 10 μm. C, D, ChIP-qPCR analyses from HEK293 sampling DDX21 genomic occupancy, at the rDNA locus (C) and at a

representative panel of Pol II-regulated gene promoters (D), after treatment with DMSO or CX-5461. Data are mean and s.d. of three independent experiments. As displayed, inhibition of Pol I

alters DDX21 nuclear localization and this coincides with nearly complete eviction of DDX21 from Pol I- and Pol II-regulated genes. E, F, ChIP-qPCR analyses from HEK293 cells treated with 50

 ng ml−1 of actinomycin-D for 1 h. Binding of the transcriptional repressor CTCF across the rDNA locus (E) and the c-MYC insulator element (MINE) (F) demonstrates that actinomycin-D

treatment does not effect CTCF binding to chromatin. Red arrow indicates relative location of the CTCF DNA-binding site (DBS) at the rDNA locus. Data are mean and s.d. of three independent

experiments. EXTENDED DATA FIGURE 4 DDX21 NUCLEAR RE-LOCALIZATION IS PREFERENTIALLY SENSITIVE TO ACUTE TRANSCRIPTIONAL INHIBITION OVER OTHER CELLULAR STRESSORS. Immunofluorescence analyses

of HEK293 cells after targeting different metabolic pathways. For inhibition of mitogen, cells were starved for 16 h in the absence of serum. For cellular respiration inhibition, cells were

treated for with either oligomycin (100 μM) or 2-deoxy-d-glucose (10 mM) for 1 h. To inhibit the mTOR pathway, cells were treated with 250 nM of either Torin 1 or rapamycin for 2 h. EXTENDED

DATA FIGURE 5 TANDEM AFFINITY ICLIP OF FH–DDX21WT. A, FH–DDX21WT iCLIP 32P-autoradiogram and western blots. All samples were loaded with constant input lysate amounts (actin loading).

FH–DDX21WT was isolated from HEK293 cells induced to express the transgene for 24 h and crosslinked with ultraviolet light (top panel same as Fig. 3a). B, Schematic of the modified iCLIP

procedure. To achieve high stringency and specificity Flag–HA–DDX21WT is first purified on anti-Flag–M2 agarose beads, washed with 1 M NaCl, 1% Triton X-100 and 1% sodium deoxycholate.

Complexes are specifically eluted with Flag peptide and recaptured with anti-HA agarose. Standard iCLIP steps were performed thereafter to generate deep sequencing libraries. C–E, Scatter

plot analysis of iCLIP reverse transcription stops on snoRNAs, rRNA and mRNAs within the FH–DDX21WT (this study) and hnRNP-C (ref. 16; publically available data) data sets. Little

concordance between the data sets is evident, suggesting specific transcriptome targets of these two RNA binding proteins (RBPs). F, DDX21WT ultraviolet RNA immunoprecipitation qRT–PCR of

FH–DDX21WT was performed in three conditions: native HEK293 cells crosslinked with ultraviolet light; FH–DDX21WT HEK293 cells without crosslinking; and FH–DDX21WT HEK293 cells with

ultraviolet crosslinking. snoRNAs, scaRNAs and TERC were validated targets identified in the sequencing data. Each experiment was performed in biological duplicates (rep1 and rep2) and error

bars represent s.d. of technical triplicates. EXTENDED DATA FIGURE 6 TANDEM AFFINITY ICLIP OF FH–DDX21SAT. A, FH–DDX21SAT was isolated from HEK293 cells induced to express the transgene for

24 h, at which point we did not observe significant dominant negative effects. iCLIP was performed as described for FH–DDX21WT, and biological duplicates of FH–DDX21SAT iCLIP

32P-autoradiogram and western blots (lanes 2 and 3) are shown. All samples were loaded with constant input lysate amounts (actin loading). FH–DDX21WT was loaded as a control. WB, western

blot. B, Left, DDX21SAT iCLIP reads annotated to known repetitive (rRNA and snRNAs) and non-repetitive (hg19 genome build: mRNAs and snoRNAs) regions of the human genome. Categories are

notes with their respective percentage of the total iCLIP experiment. Right, enriched Gene Ontology and KEGG pathway terms from DDX21SAT-bound mRNAs obtained using the DAVID tool. The _x_ 

axis values (in log scale) correspond to the negative Benjamini _P_ value. C, Distribution of all DDX21SAT-bound snoRNAs, representing C/D box, H/ACA box and scaRNAs. The number (_n_) and

fraction (per cent) of each snoRNA type is displayed. D, Comparison of the snoRNAs bound by DDX21WT and DDX21SAT, revealing significant overlap between the active and catalytically inactive

DDX21. E, DDX21SAT iCLIP reads mapped to the transcribed region of the rDNA. F, DDX21WT (left) and DDX21SAT (right) iCLIP reads mapped to the repetitive U3 snoRNA. Binding is represented as

reverse transcription stops per nucleotide normalized to the total number of reverse transcription stops mapping to the U3 snoRNA. Two strong binding sites are evident between bases 25–40

and 175–185 of U3 in DDX21WT iCLIP, whereas the 5′ binding site is reduced in DDX21SAT. nts, nucleotides. G, qRT–PCR analysis assessing the expression levels of several snoRNAs 24 h after

expression of either DDX21WT or DDX21SAT. This experiment was performed in biological duplicates. Data are mean and s.d. of technical triplicates. EXTENDED DATA FIGURE 7 DDX21 FUNCTIONALLY

INTERACTS WITH SNORNAS AND THE SNORNP. A, UCSC genome browser view of DDX21WT iCLIP reads across the snorD66 snoRNA. The C box [C] and D box [D] regions are highlighted in red. B, Same

visualization as in A but showing the snorA67 snoRNA with the H box [H] and ACA box [ACA] regions highlighted. C, Immunoprecipitation of NOP58 from HEK293 nuclear extracts confirms DDX21 as

a protein member of the snoRNP machinery. As a control for this experiment we performed western blots against FBL, a well-known NOP58-interacting partner and an essential factor of the

snoRNP machinery. D, DDX21 interacts with XRN2, a 5′–3′ exoribonuclease required for maturation and processing of snoRNAs. The DDX21–XRN2 interaction appears to be bridged by RNA, as

treatment of the nuclear lysates with RNaseA abolishes the interaction. E, Schematic of the site-directed RNaseH cleavage of RNA sensitive to 2′-_O_me. RNA of interest is hybridized to a

2′-_O_me/DNA chimaeric oligonucleotide in which the DNA nucleotides specifically target the ability of RNaseH to interrogate the 2′-_O_me status of a single nucleotide. 2′-_O_me will inhibit

RNaseH and leave intact RNA, while unmethylated RNA will be cleaved. F, UCSC genome browser view of DDX21WT and DDX21SAT iCLIP reads across the snoRNAs responsible for guiding the

modifications tested in Fig. 3f. EXTENDED DATA FIGURE 8 ASSOCIATION OF DDX21 WITH THE RNA AND PROTEIN COMPONENTS OF THE 7SK SNRNP. A, Comparison of DDX21WT ChIP-seq targets to DDX21WT

iCLIP-bound mRNAs. The numbers of unique and common genes are represented, revealing that most ChIP-seq-bound genes are not immunoprecipitated by iCLIP but that some are recovered in both

assays. B, iCLIP read distribution of DDX21WT-target mRNAs categorized by the regions within mRNAs that were bound. Most iCLIP reads fell outside the 5′ UTR. C, DDX21WT iCLIP reads mapping

to short repetitive RNAs of the human genome. Percentages of the top four short repetitive RNAs are shown. D, Secondary structure model of the 7SK snRNA annotated with iCLIP reverse

transcription stops identified from the DDX21WT (blue) and DDX21SAT (orange) experiments. Nucleotides commonly crosslinked are labelled in green. Known RNA binding protein sites: HEXIM1/2 is

highlighted in purple; P-TEFb is highlighted in red; and other P-TEFb ‘release’ factors in the centre are highlighted in green. E, Co-immunoprecipitation analysis of HEXIM1 as assayed by

western blotting for DDX21WT and 7SK snRNP components (CDK9 and LARP7). F, Immunoprecipitation of Flag–HA–DDX21WT from HEK293 nuclear extracts confirms DDX21 as a protein component of the

7SK snRNP through co-recovery of LARP7. The abundant protein actin, which is not part of the 7SK snRNP, was not recovered. EXTENDED DATA FIGURE 9 BINDING OF THE DDX21–7SK SNRNP AT RIBOSOMAL

GENE PROMOTERS. A, B, ChIP-qPCR of CDK9 (A) and HEXIM1 (B) in HEK293 cells at representative Pol II-regulated, DDX21-target and -non-target gene promoters, negative control regions, and the

rDNA locus. C, ChIP-qPCR of DDX21 in control or 3′-7SK-ASO-treated HEK293 cells at representative Pol II-regulated, DDX21-target gene promoters, negative control regions, and the rDNA locus.

For the promoter-associated genes, _P_ ≤ 0.05 (Student’s _t_-test) when compared to control ASO. D, Gene Ontology molecular function and cellular component analysis of publically available

CDK9 ChIP-seq data30. E, ChIP-qPCR of total Pol II in control or DDX21-targeting siRNA-treated HEK293 cells at representative TSSs of Pol II-regulated, DDX21-target gene promoters and

negative control regions. Data are mean and s.d. of three independent experiments. EXTENDED DATA FIGURE 10 CATALYTICALLY INACTIVE DDX21 IS STILL INCORPORATED INTO THE 7SK SNRNP. A, ChIP-qPCR

of DDX21WT (black) and DDX21SAT (green) in HEK293 cells at representative TSSs of Pol II-regulated, DDX21-target gene promoters and negative control regions. Data are mean and s.d. of three

independent experiments. B, Immunoprecipitation of DDX21WT, DDX21DEV or DDX21SAT from HEK293 nuclear extracts confirms DDX21 interacts with CDK9 (P-TEFb) regardless of its catalytic

activity. C, DDX21WT (blue) and DDX21SAT (orange) annotated iCLIP reads mapped across the 7SK snRNA. The four annotated stem–loops are marked below the graph. D, Model of multi-level control

of ribosomal pathway by DDX21. In the nucleolus, DDX21 associates with the chromatin across the transcribed region of the rDNA and is a component of the snoRNP. Furthermore, DDX21

functionally interacts with the rRNA, snoRNAs and snoRNP to control 2′-_O_me deposition on the rRNA in a helicase activity-dependent manner. In the nucleoplasm, DDX21 is bound to the

promoter regions of ribosomal Pol II-transcribed genes, many of which contain precursor snoRNA transcripts. Mechanistically, DDX21 activates transcription of its target genes through the

7SK–P-TEFb axis. As part of the 7SK snRNP, DDX21 can facilitate the release of P-TEFb from the inhibitory complex in a manner dependent on ATP hydrolysis, leading to productive Pol II

elongation and increased phosphorylation of Ser 2. Efficient transcription of its target genes enforces high expression of both snoRNAs and other ribosomal proteins critical for the rRNA

maturation process, placing DDX21 as a central operator of the ribosomal pathway. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TABLE I This fie contains oligonucleotides utilized in this study.

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ABOUT THIS ARTICLE CITE THIS ARTICLE Calo, E., Flynn, R., Martin, L. _et al._ RNA helicase DDX21 coordinates transcription and ribosomal RNA processing. _Nature_ 518, 249–253 (2015).

https://doi.org/10.1038/nature13923 Download citation * Received: 04 April 2014 * Accepted: 06 October 2014 * Published: 24 November 2014 * Issue Date: 12 February 2015 * DOI:

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