Zygotic Expression of the Double-Stranded RNA Binding ... · Similarly, the entire PDD1 coding sequence was ampliﬁed from genomic DNA (primers are listed in Table 1) and cloned

EUKARYOTIC CELL, Dec. 2011, p. 1648–1659 Vol. 10, No. 121535-9778/11/$12.00 doi:10.1128/EC.05216-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Zygotic Expression of the Double-Stranded RNA Binding MotifProtein Drb2p Is Required for DNA Elimination in the

Ciliate Tetrahymena thermophila�

Jason A. Motl and Douglas L. Chalker*Department of Biology, Washington University in St. Louis, Campus Box 1137,

One Brookings Dr., St. Louis, Missouri 63130-4899

Received 19 August 2011/Accepted 13 October 2011

Double-stranded RNA binding motif (DSRM)-containing proteins play many roles in the regulation of genetranscription and translation, including some with tandem DSRMs that act in small RNA biogenesis. Wereport the characterization of the genes for double-stranded RNA binding proteins 1 and 2 (DRB1 and DRB2),two genes encoding nuclear proteins with tandem DSRMs in the ciliate Tetrahymena thermophila. Both proteinsare expressed throughout growth and development but exhibit distinct peaks of expression, suggesting differentbiological roles. In support of this, we show that expression of DRB2 is essential for vegetative growth whileDRB1 expression is not. During conjugation, Drb1p and Drb2p localize to distinct nuclear foci. Cells lackingall DRB1 copies are able to produce viable progeny, although at a reduced rate relative to wild-type cells. Incontrast, cells lacking germ line DRB2 copies, which thus cannot express Drb2p zygotically, fail to produceprogeny, arresting late into conjugation. This arrest phenotype is accompanied by a failure to organize theessential DNA rearrangement protein Pdd1p into DNA elimination bodies and execute DNA elimination andchromosome breakage. These results implicate zygotically expressed Drb2p in the maturation of these nuclearstructures, which are necessary for reorganization of the somatic genome.

Proteins containing a double-stranded RNA (dsRNA) bind-ing motif (DSRM) participate in diverse biological pathways ina wide range of organisms. This motif was first identified in thedevelopmentally essential gene Staufen of Drosophila melano-gaster and has since been recognized to be encoded in thegenomes in all three domains of living organisms, as well as inviruses (63; reviewed in references 20 and 67). DSRM proteinscommonly act in developmental pathways (e.g., RNA localiza-tion by the Staufen family and developmental transcriptionalregulation by the DIP1 family) (5, 18, 62, 68) but also haveubiquitous roles in transcriptional and translational regulation(e.g., PKR family and PKR-associated proteins) (26, 45, 55,58). Proteins vital for RNA interference (RNAi) also containDSRMs. These include members of the RNase III family (e.g.,Dicer and Drosha family proteins) and their tandem DSRM-containing partner proteins (e.g., RDE-4 of Caenorhabditiselegans, Pasha, R2D2, and Loqs in D. melanogaster, and theirhomologues in Homo sapiens) (4, 12, 17, 23, 27, 36, 37, 57, 65).

In the ciliate Tetrahymena thermophila, the DSRM-contain-ing protein Dicer-like 1 (DCL1) has been shown to play apivotal role in a process linking RNAi to heterochromatinformation and developmentally regulated DNA elimination(42, 49). Like all ciliates, T. thermophila is unicellular yet con-tains two distinct types of nuclei, the somatic macronucleusand the germ line micronucleus (reviewed in references 46 and56). The polyploid micronucleus (�50C) acts as a transcrip-tionally active somatic nucleus during vegetative growth, while

the diploid, germ line micronucleus is transcriptionally silent(19, 70; reviewed in references 46 and 56). Under optimalgrowth conditions T. thermophila undergoes asexual, binaryfission; however, when starved T. thermophila reproducesthrough the sexual process of conjugation, generating newmicronuclei and macronuclei from the parental germ line mi-cronucleus (reviewed in references 46 and 56). During thematuration of the zygotic macronuclei, the macronuclear chro-mosomes are fragmented at �180 sites, lose �15% of theiroverall genomic content, and are amplified to �50C (1, 7, 14,19, 29, 69, 70). The loss of genome complexity is the result ofprogrammed DNA rearrangements that remove specific DNAsequences, called internal eliminated sequences (IESs), fromthousands of chromosomal sites (46, 56).

DNA elimination has been shown to be guided by an RNAi-related mechanism (11, 42, 47, 49). Bidirectional transcriptionof the germ line genome in meiotic micronuclei provides anabundant source of IES-specific dsRNA (11, 44). The resultingnoncoding RNAs (ncRNAs) are processed into 27- to 30-nucleotide (nt) sRNA species, called scan RNAs (scnRNAs),by Dcl1p in the meiotic micronucleus (42, 49). These scnRNAsare exported into the cytoplasm, where they are bound by aPIWI homologue, Twi1p (47). Twi1p/scnRNA complexes aretransported into the parental macronucleus, where these com-plexes scan macronuclear ncRNAs, and possibly mRNAs. TheTwi1p/scnRNA complexes homologous to the parental macro-nucleus are removed from the pool of active complexes, andthe remaining complexes are transported to the zygotic macro-nuclei upon their emergence, where they guide H3K9 andH3K27 methylation of IES-associated histones by the E(z)homologue Ezl1p (38, 47, 48). Methylated histones in zygoticmacronuclei are bound by the chromo domain-containing pro-teins Pdd1p and Pdd3p, which along with other associated

* Corresponding author. Mailing address: Washington University inSt. Louis, Campus Box 1137, One Brookings Dr., St. Louis, MO 63130-4899. Phone: (314) 935-8838. Fax: (314) 935-4432. E-mail: [email protected].

� Published ahead of print on 21 October 2011.

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proteins form large nuclear structures called DNA eliminationbodies late in conjugation (38, 40, 51, 66). DNA elimination inthese bodies is catalyzed by the domesticated PiggyBac trans-posase Tpb2p, resulting in removal of IESs from zygotic ma-cronuclei (13).

A second endogenous RNAi pathway that acts to silencegenes and/or pseudogenes is evidenced by a class of 23- to24-nt sRNAs that accumulate during vegetative growth (33).These sRNAs are homologous to loci clustered at �12genomic positions and exhibit biased polarity, mapping to onlyone strand. They are produced by the essential Dicer proteinDcr2p in a coupled reaction with an RNA-dependent RNApolymerase, Rdr1p (34). This coupling likely accounts for thestrand specificity observed.

As dsRNA has clear roles in regulating genome structureand activity, we characterized the two putative tandem DSRM-containing proteins, double-stranded RNA binding proteins 1and 2 (Drb1p and Drb2p), encoded in the T. thermophilagenome (21, 64). We show that both are nuclear proteins thatexhibit distinct subnuclear organization. By knocking out thegene for each, we found that Drb2p is essential both duringvegetative growth and also late in conjugation, where it facil-itates DNA elimination body formation and subsequent RNAi-dependent DNA elimination. Drb1p, in contrast, is dispens-able but is nonetheless important for efficient prezygoticdevelopment. Our data do not support that either protein actsas an essential Dicer partner protein as do tandem DSRMproteins in other eukaryotes, but instead our data suggest thatthese proteins have diverse roles during the T. thermophila lifecycle and expose a role for dsRNA late in macronuclear de-velopment (4, 12, 17, 23, 27, 37, 57, 65).

MATERIALS AND METHODS

Tetrahymena strains and growth conditions. Standard wild-type, laboratory T.thermophila strains CU427 (Chx/Chx [VI, cy-s]), CU428 (Mpr/Mpr [VII, mp-s]),B2086 (II), and micronucleus-defective strains B*VI (VI) and B*VII (VII) wereoriginally obtained from Peter Bruns (Cornell University, Ithaca, NY).BVIICU427/CU427 (Chx/Chx [VII, cy-s] was generated through genomic exclusionmating between CU427 and B*VII. These strains or their transformed progenywere used for expression studies, biolistic transformations, and subsequent anal-yses. �DCL1 homozygous knockout strains were described earlier (42). Cellswere grown and maintained as previously described (25, 52). Strains were starved6 h to overnight in 10 mM Tris (pH 7.5) prior to mixing to initiate conjugation.Optical densities of cell populations were used to estimate cell numbers prior tomixing equal numbers of mating-compatible strains.

Identification of DRB1 and DRB2 sequences. DRB1 (TTHERM_00078870;NCBI Gene ID 7837033) and DRB2 (TTHERM_00825510; NCBI Gene ID7836999) sequences were identified by BLAST search of the T. thermophilamacronuclear genome (http://www.ciliate.org) and the D. melanogaster R2D2(accession number CG7138) and Loqs (accession number CG6866) DNA se-quences. DSRMs of DRB1 and DRB2 were initially identified on the T. thermo-phila macronuclear genome by using InterProScan (72). Further sequence anal-ysis included Pfam analysis of the DRB1 and DRB2 coding sequences (http://pfam.janelia.org) and alignment of DSRM sequences of DRB1 and DRB2 withthose of tandem DSRM-containing proteins R2D2 (accession numberNP_609152.1) and Loqs (accession number NP_609646.1) from D. melanogaster,RDE-4 (accession number NP_499265.1) from C. elegans, and TRBP2 (accessionnumber NP_599150.1) from H. sapiens by using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and Boxshade (http://www.fr33.net/boxshadeprotein.php)for better visualization. Identification of additional homologous regions of theDRB1 and DRB2 protein sequences was carried out by protein alignment usingEmboss Needle (http://www.ebi.ac.uk).

RT-PCR expression analysis. RNA was isolated from growing, starved, andconjugating T. thermophila (CU428 � B2086) at 2-h intervals from 2 h to 14 h byRNAsol extraction (22). RNA isolation from DRB1 knockouts and DRB2 mi-

cronucleus (mic) knockouts at 4 h and 12 h after mixing was also done byRNAsol extraction. Reverse transcription-PCR (RT-PCR) of wild-type andknockout matings was done to determine expression and show loss or decreaseof expression, respectively, as previously described (Table 1 provides a list of theprimers used) (42).

Cloning of T. thermophila genes for protein localization. Oligonucleotide prim-ers (Table 1) were used to amplify the entire DRB1 or DRB2 coding sequencesfrom genomic DNA by PCR. The resulting products were cloned into the Gate-way recombination-compatible pENTR-D (Invitrogen) to create pENTR-D-DRB1 and pENTR-D-DRB2, respectively. Plasmids containing the DRB1 andDRB2 coding sequences were sequenced to verify coding sequence integrity. LRrecombination of pENTR-D-DRB1 with pICY-GTW and of pENTR-D-DRB2with pICC-GTW using LR clonase II (Invitrogen) fused the coding regions toyellow fluorescent protein (YFP) and cyan fluorescent protein (CFP) in pICY-DRB1 and pICC-DRB2, respectively (71). Plasmids pICY-DRB1 and pICC-DRB2 were introduced into mating wild-type cells (CU427 � CU428) by con-jugative electroporation (24).

Similarly, the entire PDD1 coding sequence was amplified from genomic DNA(primers are listed in Table 1) and cloned into pENTR-D to create donorplasmid pENTR-D-PDD1. LR recombination of pENTR-D-PDD1 with pICY-GTW using LR clonase II (Invitrogen) created pICY-PDD1, which was thenintroduced into cells by conjugative electroporation (24).

For Pdd1p-YFP localization in DRB2 mic knockout strains, pENTR-D-PDD1was recombined with pBS2-ICY-GTW by using LR clonase II (Invitrogen) tocreate pBS2-ICY-PDD1. BclI- and SalI-digested pBS-ICY-PDD1 was trans-formed into starved, homozygous micronuclear DRB2 knockout strains(B*VI�D2/�D2 1 and 6 and B*VII�D2/�D2 1 and 2) by using the PDS-1000/Heparticle bombardment system (Bio-Rad) as previously described (6, 8). Trans-formants were identified by their resistance to 25 �g/ml cycloheximide.

To visualize localization, starved transformed cells were mixed to begin con-jugation in 0.08 �g/ml CdCl2 to induce expression of the fusion protein. Live cellswere harvested by low-speed centrifugation (1,000 � g) at 4 h, 10 h, and 14 hpostmixing, stained with 4�,6-diamidino-2-phenylindole (DAPI; 1 �g/ml) andimmobilized in 5 �l 2% methylcellulose. DIC, CFP fluorescence, YFP fluores-cence, and DAPI fluorescence images were captured using a QimagingRetigaEX charge-coupled-device camera (Burnaby, British Columbia, Canada)and Openlab software (PerkinElmer). Images were cropped and their brightnessand contrast uniformly adjusted using Adobe Photoshop CS3.

Generation of DRB1, DRB2, and PDD1 knockout strains. Genomic sequencesupstream and downstream of each gene’s coding region were amplified by PCRand recombined into pDONR-P4-P1R (upstream) and pDONR-P2R-P3 (down-stream) by using BP Clonase (Invitrogen) (Table 1). The resulting donor plas-mids containing up- and downstream regions were mixed with equal amounts ofpENTR-D-MTT1/NEO3 and the multisite destination vector pDEST-R4-R3,along with LR Clonase Plus II (Invitrogen) to create the DRB knockout plasmidspDEST-B4-DRB1Up-B1-MTT1/NEO3-B2-DRB1Down-B3 and pDEST-B4-DRB2UpN1-B1-MTT1/NEO3-B2-DRB2Down-B3. DRB1 and DRB2 knockoutconstructs were linearized by digestion with KpnI and transformed into conju-gating wild-type cells (CU428 � B2086) between 2 and 3 h after mixing by usinga PDS-1000/He particle bombardment system (Bio-Rad) as previously described(6, 8). Heterozygous micronuclear transformants were identified by their resis-tance to 80 �g/ml paromomycin with 1 �g/ml CdCl2 and 15 �g/ml 6-methylpu-rine. Heterozygous micronuclear transformants were verified through matingswith CU427 by monitoring segregation of paromomycin resistance conferred bythe MTT1-neomycin (NEO3) paromomycin resistance cassette among cyclohex-imide-resistant progeny (61), as well as through PCR screening of T. thermophilacrude cell lysates (Table 1 provides sequences of the primers) (10). Homozygousmicronuclear knockout heterokaryons were generated by crossing heterozygousmicronuclear transformants with B*VI or B*VII star strains. Homozygous mi-cronuclear knockout heterokaryons were identified by paromomycin/CdCl2 sen-sitivity and verified through crosses with CU427, which produced progeny resis-tant to 100 �g/ml paromomycin with 1 �g/ml CdCl2 and 25 �g/ml cycloheximide.Complete (micro- and macronuclear) DRB1 knockout strains were generated bycrossing homozygous micronuclear knockouts of compatible mating types,screening for progeny resistant to paromomycin/CdCl2, and verifying by PCRdetection of the knockout allele (10).

To generate �PDD1 strains, the NEO3 cassette (61), cloned between genomicsequences upstream and downstream of the PDD1 coding region (obtained fromYifan Liu, University of Michigan), was introduced into conjugating B2086 andCU428 cells by biolistic transformation (6, 8). Homologous recombination of thiscassette into the genome removed 1,714 bp, including the entire PDD1 codingsequence (nucleotides 76,907 to 78,620 of scaffold CH445650.1; accessiongi�62422296�gb� CH445650.1). Germ line (micronuclear) knockouts were identi-

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TABLE 1. Oligonucleotides used in the course of this study

Primer purpose and name Sequence (5�–3�)

For RT-PCR to determine expressionDRB1

1688-Loq1-836.................................................CGAAAAGGGGTTAGGGTTTTCTAGC1689-Loq1-1325r .............................................CCCTTATCCCATCGTTTTCAG

DRB21692-Loq3-1875...............................................GCAATAGCCAAACACAAAGAGTGTAGC1879-Loq3-2230r .............................................GCATCAATAAGGCTACAACATCC

ATU13364-ATU1-2391r ...........................................GTGGCAATAGAAGCGTTGACA3365-ATU1-1997 ............................................TGCTCGATAACGAAGCCATCT

For gene amplification of coding sequenceDRB1

1701-Loq1X.....................................................CACCCTCGAGAAAATGAATTCTTAGCAAG1732-Loq1rH-Short ........................................AAGCTTTAGACTTATACTTTTCATGAAAG

DRB21887-Loq3X-Modified....................................CACCCTCGAGAAAATGGCGCAATCTTTTAGATTTATAG1911-Loq3rP-Full length................................CTGCAGCCCATTACAAATAATTATTAAGTTATCATAAGC

For knockout cassette generationDRB1 upstream

1761-Loq1-2321AattB4 ..................................GGGGACAACTTTGTATAGAAAAGTTGGTACCGGGATTACATAAAGATTTGATTCC1762-Loq1-3366rattB1 Downstream ............GGGGACTGCTTTTTTGTACAAACTTGCACAATTCAATCAAAAGTGCG1763-Loq1-5746attB2 .....................................GGGGACAGCTTTCTTGTACAAAGTGGCACTCTCATTAATGCCCCC1764-Loq1-7093AattB3 ..................................GGGGACAACTTTGTATAATAAAGTTGGTACCAGTAAAGAGCCTAAATCAAGG

DRB2 upstream2370-DRB2-1226AattB4 ................................GGGGACAACTTTGTATAGAAAAGTTGGTACCGAAAGCCTATGGGAGAGCAAG2372-DRB2-2595rattB1..................................GGGGACTGCTTTTTTGTACAAACTTGCACTTTTAGGAAATAATGAATGTGTCAC

DRB2 downstream2322-DRB2-6530attB2 ...................................GGGGACAGCTTTCTTGTACAAAGTGGGTTGTGTTTAAAAAGAAGGTGTGTGTTATG2323-DRB2-7708ArattB3Ext.........................GGGGACAACTTTGTATAATAAAGTTGGTACCTTCACTTAAACCGCACCCAG

For knockout PCR screeningDRB1

5� 1679-MTT1-11484r ....................................ATTTGGAATTAAGTACTTATTTCCAAAC1946-DRB1-1086 ............................................CGCGCACTTTTGATTGAATTGTG3� 1866-Neo KO 2 ..........................................CGTGATATTGCTGAAGAGCTTG1867-Loq1-5353...............................................CAGGGGAAGATATATTTTATGAAGC1868-Loq1-5764r .............................................GGGGGCATTAATGAGAGTG

DRB25� 2477-DRB2-2195........................................CAATTTATCTATTAAAATACCTTTACTTAC2478-DRB2-2805r...........................................AAAATCTGTAATTGAGAAGAAACAAAAAC3001-LIA4MTTLR.........................................AACATTCAAACATTGTGCACTAAATA3� 2367-p4T2-3351 ..........................................TCGCCTTCTTGACGAGTTCT2391-DRB2-6323 ............................................GCTTAGATGATATTACACATGATAATC2392-DRB2-6721r...........................................AAAGAGAGTGAGTTTTTCTTTTTGG

For assay rearrangement of IESsM

1439-M808 .......................................................ATATTGTGTGGTACAATAGGTTGTCGTAG3111-M002 .......................................................AGCTTAAACAAATGCCATATTGAG3114-M1194 .....................................................GTGGGGAGGGAGAAGGATTCAAC

B3246-IES7_MDSL-112 ...................................GGATTGATTGGCATAAATGGA3247-IES7_MDSR-158...................................AAGCCCAGAATACCGCAGTTC

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fied by their resistance to both paromomycin/CdCl2 and 6-methylpurine (resis-tance gene from the micronucleus of CU428). Genomic exclusion crosses ofheterozygous germ line transformants with star strains B*VI or B*VII generatedhomozygous mutants that were subsequently crossed to produce complete PDD1knockouts �PDD1 39.1 and �PDD1 W3.3, missing all copies of the gene fromboth the micro- and macronucleus.

Southern blotting and PCR analyses. T. thermophila genomic DNA was iso-lated using a Wizard genomic DNA purification kit (Promega). Gel electropho-resis, blotting, and hybridization were performed as previously described, exceptblots were washed with 0.5� SSC (1� SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate–1% SDS after hybridization (42). The probe for DRB2 was a radiolabeledKpnI and BsrGI restriction fragment of pDONR-R2-DRB2Down-L3. The DRB1probe was a labeled BsrGI and XmnI restriction fragment from pDONR-R2-DRB1Down-L3. Genomic DNA from heterozygous, homozygous, and homozy-gous micronuclear DRB1 knockouts was digested with XmnI and separated on a1.0% agarose gel. Genomic DNA from heterozygous DRB2 knockouts was di-gested with ClaI and SacI and fractionated on a 0.8% agarose gel prior toblotting.

Chromosome breakage was assayed in DRB2 mic knockouts by using genomicDNA from CU428 � B2086, �PDD1 39.1 � �PDD1 W3.3, �DCL1 1.8.6 ��DCL1 4.2.4, and B*VI�D2/�D2 1 � B*VII�D2/�D2 1 30 h after mixing and thendigestion with EcoRI and separated on a 0.8% agarose gel. The Southernblotting probe for chromosome breakage was created using a 0.8-kbp probefragment that spans the EcoRI site at position 335013 of chromosomal scaffoldCH445662 (GenBank accession number gi62422284). DNA rearrangement ofIES B and the M IES was assayed by PCR using CU428 � B2086, �DCL11.8.6 � �DCL1 4.2.4, B*VI�D2/�D2 1 � B*VII�D2/�D2 1, and B*VI�D2/�D2 6 �B*VII�D2/�D2 2 30-h genomic DNA and primers flanking each IES (Table 1).

Analysis of nuclear morphology postconjugation. Wild-type or the indicatedknockout cells 30 h into conjugation were harvested by low-speed centrifugation(1,000 � g), DAPI stained (1 �g/ml), and immobilized in 5 �l 2% methylcellu-lose. Differential interference contrast (DIC) and DAPI fluorescence imageswere captured using a Qimaging RetigaEX charge-coupled-device camera(Burnaby, British Columbia, Canada) and Openlab software (PerkinElmer). Theconjugation stage of each mating at 30 h was determined by comparison of

images with previously described wild-type stages of conjugation (44). Imageswere cropped and their brightness and contrast uniformly adjusted using AdobePhotoshop CS3.

RESULTS

The T. thermophila macronuclear genome encodes two pro-teins with tandem DSRMs. For optimal sRNA production andprotein localization, Dicer and Drosha homologues in C. el-egans, D. melanogaster, and H. sapiens require association witha tandem DSRM-containing protein (4, 12, 17, 23, 27, 37, 53,57, 65). Bioinformatic analysis (BLAST, Pfam, and ClustalW)of the T. thermophila macronuclear genome identified twogenes, DRB1 and DRB2, encoding tandem DSRM-containingproteins (Fig. 1A). Alignment of their putative DSRMs withother DSRM-containing proteins indicated conservation in theregions where key residues known to be important for DSRMstructure and function are located. The homologies of theseproteins with other tandem DSRM proteins did not extendbeyond these domains (Fig. 1A and data not shown). However,alignment of full-length DRB1 and DRB2 revealed additionalregions of similarity outside the DSRMs; one in the N-terminalregion (NTR) and two in the C-terminal regions (CT1 andCT2) of each protein (data not shown). In the ciliate Parame-cium, only DRB1 homologues are evident, which suggests thatthe duplication and diversification of these proteins occurredafter these two ciliates diverged.

RT-PCR and Northern blot analysis demonstrated thatDRB1 and DRB2 are both expressed throughout much of the

FIG. 1. T. thermophila contains two predicted tandem double-stranded RNA binding motif proteins. (A)Genomic locus, conserved motifs, andlength of the putative tandem double-stranded RNA binding motif proteins Drb1p and Drb2p. Splice sites are indicated by small connected gapsin the gene. aa, amino acids. (B) ClustalW alignment of DSRMs from Dicer family tandem DSRM-containing partner proteins. #, sites determinedto be essential for structure and function of DSRMs; ^, sites that have been mutated in DSRM-containing proteins and shown to cause loss ofRNA binding. Conserved aa (white text) are shaded in black (identical aa) or gray (similar aa). (C and D) RT-PCR analysis of DRB1 and DRB2expression relative to �-tubulin (ATU1). RNA samples were isolated from CU428 cells growing vegetatively (V), after an 18-h starvation (S), andfrom CU428 � B2086 conjugating cells at 2-h intervals and were used to monitor the expression of each gene.



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T. thermophila life cycle (Fig. 1C and D and data not shown).DRB1 mRNA levels are low in growing and starved cells butincrease significantly during meiosis (2 to 4 h into conjugation,when scnRNA production occurs) and again after the appear-ance of the zygotic macronuclei (8 h), a pattern that parallelsDCL1 expression (Fig. 1C) (42, 49). Its decrease in expressionat 6 h coincides with the drop in ATU1 RNA levels, which maysimply reflect the switch between parental and zygotic expres-sion. DRB2 expression is higher during vegetative growth butalso shows less dramatic induction during conjugation relativeto DRB1. After decreased expression during starvation, DRB2is induced starting at 2 h of conjugation and peaks at 8 h,shortly after the appearance of the zygotic macronuclei (Fig.1D). This profile suggests possible roles for Drb2p during bothgrowth and development.

DRB1 and DRB2 encode nuclear proteins that localize todistinct structures. Ectopic expression of Drb1p and Drb2ptagged with YFP or CFP, respectively, on their C terminishowed that both are nuclear proteins visible in small focithroughout the macronucleus during vegetative growth (datanot shown), whereas green fluorescent protein (GFP) alone

expressed in cells is uniformly distributed (42, 43). Duringearly conjugation, both proteins localize to the parental ma-cronucleus in distinct foci (Fig. 2). Later, at the beginning ofzygotic macronuclear differentiation (10 h), all Drb1p-YFPand most Drb2p-CFP disappeared from the parental macro-nucleus and then appeared in zygotic macronuclei (Fig. 2A andB, middle rows). Whether the foci seen in the parental macro-nucleus are functionally related to those observed in zygoticmacronuclei could not be determined (Fig. 2A and B, comparetop and middle rows). Near completion of zygotic macronu-clear development (14 h into conjugation), Drb1p-YFP local-ization was primarily diffuse (Fig. 2A, bottom row). In contrast,the small Drb2p-CFP foci coalesced into larger foci, althoughlow-level diffuse localization remained throughout the zygoticmacronucleus as well (Fig. 2B, bottom row).

Upon initial inspection, the size and number of nuclear fociof Drb1p and Drb2p in parental macronuclei appeared ratherdifferent. To better compare their localizations, Drb1p-YFPand Drb2p-CFP were coexpressed and visualized 4 h into con-jugation. Their nuclear foci were distinct, with only a smalldegree of overlapping localization (Fig. 2C). DRB1 and DRB2

FIG. 2. Nuclear localization of Drb1p and Drb2p during conjugation. (A and B) Nuclear localization of Drb1p-YFP (A) and Drb2p-CFP (B) at4, 10, and 14 h into conjugation. White arrowheads, micronuclei; black arrowheads, parental macronuclei; white arrows, zygotic macronuclei.(C) Simultaneous localization of Drb1p-YFP and Drb2p-CFP in the parental macronucleus 4 h into conjugation. (Top) Drb1p-YFP andDrb2p-CFP foci are predominantly distinct in the macronucleus early during conjugation. Yellow arrowheads, Drb1p-YFP foci only; bluearrowheads, Drb2p-CFP foci only; green arrowheads, Drb1p-YFP and Drb2p-CFP foci. (Bottom) The number of Drb1p-YFP foci, Drb2p-CFPfoci, and Drb1p-YFP/Drb2p-CPF colocalization foci, and the total number of foci in the parental macronucleus above.



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were best reciprocal hits in a BLASTp analysis of the CT2regions, which could explain the small overlap in localizationthrough partially redundant protein function. Despite this, itseems that both Drb1p and Drb2p have distinct primary func-tions based on their localizations and divergent protein se-quences outside their DSRMs and CT2.

In addition to its abundant macronuclear localization,Drb1p-YFP also localized to the micronucleus just prior to andduring crescent formation (prophase meiosis I) (Fig. 2A, toprow, and data not shown). Drb1p-YFP was observed specifi-cally at the poles of these nuclei, at either one or both endsdepending on the developmental stage. This micronuclear lo-calization pattern is quite distinct from that of Dcl1p, which isfound throughout the nucleoplasm of the crescent micronu-cleus, and suggests that Drb1p may not be a critical Dcl1pprotein partner (42, 49). Point localization of Drb1p-YFP wasseen early in conjugation once the micronucleus began to elon-gate at one end, and later, after the crescent micronucleus fullyelongated, it was seen at both ends of the micronucleus (Fig.2A, top row, and data not shown). Upon anaphase of meiosisI, Drb1p-YFP micronuclear localization is lost. While it islikely that DRB1 and DRB2 arose from an ancient gene dupli-cation, differential localization and expression patterns indi-cate that each DSRM-containing protein has specific cellularroles.

DRB2, but not DRB1, is essential for growth and develop-ment. We created strains lacking each gene to establishwhether and when each protein functions during the T. ther-mophila life cycle. Constructs containing the NEO3 selectablemarker, flanked by up- and downstream homology regions toeither DRB1 or DRB2, were biolistically transformed into cellsduring conjugation to generate heterozygous micronuclear/macronuclear knockout strains. By taking advantage of therandom assortment of alleles during amitotic macronucleardivision, we obtained strains for which all wild-type DRB1 genecopies in the macronucleus were replaced with the knockoutallele, which revealed that Drb1p is not required for vegetativegrowth (Fig. 3A and B and data not shown).

To further verify that DRB1 is not essential, homozygousmicronuclear knockout strains were crossed to produce com-plete DRB1 knockout cell lines. Southern blot analysis ofgenomic DNA isolated from these strains detected only theDRB1 knockout allele (Fig. 3A). RT-PCR of the DRB1 knock-out strains during conjugation confirmed loss of all DRB1expression (Fig. 3B). While these complete DRB1 knockoutstrains showed no growth defects, matings between two DRB1knockout strains generated progeny at a reduced rate relativeto crosses of wild-type strains (Table 2). The DRB1 knockoutcells that were able to complete conjugation arrested with twonew macronuclei and a single micronucleus, as do wild-typeconjugants, until they were returned to growth medium andstarted vegetative growth (Fig. 4). The observation that only afraction of mated DRB1 knockout cells progressed to zygoticdevelopment suggests that Drb1p is important, but not essen-tial, for prezygotic development. The lack of Drb1p during thisstage(s) of early conjugation resulted in substantial prematureabortion of conjugation (data not shown).

Unlike our experience with DRB1, we were unable to iden-tify strains in which all macronuclear copies of DRB2 weredisrupted, which indicates that vegetative DRB2 expression is

essential (Fig. 3C and D). To verify this, we first performedgenomic exclusion crosses between the original heterozygousmicronuclear knockout strains and “star” strains (B*VI andB*VII) to create strains homozygous for the knockout cassettein the micronucleus while maintaining wild-type copies ofDRB2 in the macronucleus to support growth (see Materialsand Methods for details). These homozygous micronuclearknockout strains were then crossed in an attempt to generatestrains homozygous for the knockout cassette in both the mi-cro- and macronucleus, thus eliminating all wild-type DRB2gene copies. Despite each individual DRB2 micronuclearknockout strain being able to produce progeny when comple-mented by crossing to wild-type strains, when these lines werecrossed to each other no viable progeny emerged (Table 3).

Further analysis revealed that DRB2 micronuclear knockoutstrains are unable to reach the terminal stage of conjugationwith 2 macronuclei and 1 micronucleus even 30 h after pairing,but instead arrest with 2 macronuclei and 2 micronuclei (Fig.4). Thus, not only is DRB2 expression necessary for vegetativegrowth, but zygotic DRB2 expression is essential for comple-tion of conjugation as well (Fig. 3C and 4). As observed inother mutants that arrest at the 2-macronuclei, 2-micronucleistage, conjugating DRB2 mic knockouts underamplified theirmacronuclear DNA relative to zygotic macronuclei of wild-type conjugants at their terminal stage prior to refeeding (15,42, 47, 49). Although DRB2 mic knockout strains only lackzygotic expression of DRB2, the majority of conjugants arrestat the 2-macronuclei, 2-micronuclei stage, while the remainderarrest after elimination of one of the remaining micronuclei(Fig. 4, bottom). RT-PCR analysis of DRB2 mic knockoutmatings showed reduced, but not complete loss of, expressionafter 12 h of conjugation relative to wild-type cells, when zy-gotic DRB2 expression normally should predominate (Fig.3D). Unmated cells as well as parentally expressed DRB2mRNA in the DRB2 mic knockout mating population ac-counted for the DRB2 mRNA detected. The residual, paren-tally expressed DRB2 transcripts may enable a fraction of cellsto proceed further into conjugation and eliminate one micro-nucleus.

DRB2 mic knockouts fail to remodel chromosomes late inconjugation. The DRB2 conjugation arrest phenotype is com-monly observed in knockouts of genes necessary for genomerearrangement in T. thermophila, including DCL1, TWI1, andPDD1 (15, 42, 47, 49). To determine whether the DRB2 micknockout arrest is accompanied by failure of RNA-directedDNA elimination or due to some other perturbation duringconjugation, we monitored the rearrangements of several IESs.Genomic DNA was isolated from mated cell populations 30 hafter initiating conjugation, when all genome reorganizationshould be completed in wild-type cells. PCR using primers ableto detect both the unrearranged (micronuclear form of thelocus) and rearranged (macronuclear form) IESs allowed as-sessment of the level of excision. Whereas DNA from wild-typemating populations showed predominantly the rearranged lo-cus for each IES, DRB2 mic knockout or control DCL1 knock-out matings exhibited accumulation of the unrearranged formof both IES B and the M IES (Fig. 4 and data not shown). IESB is a 327-bp IES found within the LIA2 gene, and the M IESis a well-studied intergenic IES that undergoes alternative re-arrangement that removes either 0.6 kb or the complete 0.9-kb



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IES (2, 22a). PCR analysis of IES B clearly showed that the597-bp product indicative of the micronuclear locus was over-represented in the DCL1 and DRB2 mic knockout matingsrelative to wild type (Fig. 5A). It is important to note that thecell populations monitored included some percentage of un-mated cells, whose DNA likely contributed much of the tem-plate for the 270-bp product representing the rearranged formin the mutant cell lines. The PCR analysis of the M IES utilizedthree primers for PCR, which we have found provides a morequantitative assessment of its rearrangement. Two bands at

1,192 bp and 386 bp resulted from amplification of micronu-clear DNA containing the IES, while two other bands at 592 bpand 292 bp were the products of removal of either 0.6 kb or 0.9kb of the M IES locus. As observed for IES B, the unrear-ranged form of the M IES was overrepresented in the DCL1and DRB2 mic knockout mating populations relative to wild-type matings (Fig. 5B). This difference was less apparent inDRB2 mic knockout matings than in the DCL1 mutant, whichmay have been due to persistence of parental Drb2p. Analysisof other IESs further demonstrated that these mutants exhibit

FIG. 3. Generation of DRB1 complete and DRB2 mic knockout strains, based on Southern blot analysis of knockout strain genomic DNA.(A) Genomic DNA isolated from wild-type (WT) CU428, four DRB1 macronuclear/micronuclear knockout strains (DRB1 KO), and fourmicronuclear strains (B*VI and B*VII�D1/�D1) was digested with XmnI prior to gel electrophoresis. (C) Genomic DNA from DRB2 mic knockoutheterozygous strains (DRB2 mic KO het) was digested with ClaI and SacI prior to analysis. The diagram of WT (DRB1/2) and KO (MTT1/NEO3)alleles is shown on the right of each panel. Black arrowhead, band expected for the knockout allele; white arrowhead, band expected for thewild-type fragment. (B and D) RT-PCR expression analysis of DRB KO strain matings. RNA isolated 4 h (parental expression) and 12 h (zygoticexpression) into conjugation was converted to cDNA (RT�), and PCR using gene-specific primers was used to assess loss/reduction of expression.A control reaction with reverse transcriptase omitted (RT-) is also shown. Primers specific to the �-tubulin gene (ATU1) provided a normalizationcontrol between samples. In panel B, the star marks a nonspecific RT-PCR band detected with DRB1 primers. Diagrams of the DRB1/2 and ATU1loci; the relative locations of forward and reverse PCR primers (black arrows) are shown on the right.



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substantial failure of RNA-directed DNA elimination (datanot shown).

Assessment of chromosome breakage near the LIA1 locusalso showed that DRB2 mic knockout progeny fail to properlyfragment chromosomes (Fig. 5C). Before the completion ofconjugation, the chromosomes in the zygotic macronuclei,which contain 5 chromosomes amplified to between 4 and 8copies, are fragmented at approximately 180 chromosomebreakage sites (CBSs) to produce the shortened macronuclearchromosomes. In knockouts of genes essential for genomerearrangement, including DCL1 and TWI1, chromosomebreakage fails, as does IES elimination (42, 47). In a Southernblot assay of wild-type progeny, chromosome breakage at theLIA1 locus resulted in a band of approximately 2.5 kb inthe zygotic macronuclei. The copies of this chromosome fromthe parental macronucleus were visible as a 2.6-kb band, asthey have longer telomeres relative to newly fragmented ends.Unbroken micronuclear chromosomes were detected as a10.5-kb band. The probe also detected a 7.8-kb fragment pres-ent in all nuclei. Due to the increased copy number of the locusin the macronucleus in the progeny of wild-type crosses, the2.5-kb and 2.6-kb fragments are more intense than the larger10.5-kb micronucleus-specific fragment. As in the control mat-ings of DCL1 knockout cells, the postconjugation populationsof DRB2 mic knockout crosses have increased levels of the10.5-kb unrearranged fragment and lack the 2.5-kb fragmentindicative of de novo chromosome breakage (Fig. 4C). A pre-vious report on chromosome breakage in a somatic knockoutof PDD1 showed that chromosome fragmentation was able tooccur (15). Here we report that crosses of homozygous PDD1knockout strains showed failure of chromosome breakage, asobserved with DCL1 and DRB2 mic knockouts, emphasizingthe importance of zygotic expression of PDD1 and DRB2 inchromosome breakage (Fig. 5C).

DRB2 colocalizes with Pdd1p in DNA elimination bodies.Failure of DNA elimination and chromosome breakage inDRB2 mic knockout strain matings indicated that the conju-gation arrest phenotype described earlier was a result of failureto complete RNA-directed DNA elimination. The localizationof Drb2p-CFP into large foci 14 h into conjugation, which iswhen DNA elimination normally occurs, prompted us to as-certain whether Drb2p-CFP was localized into DNA elimina-

tion bodies. These nuclear structures are enriched for the es-sential DNA elimination, chromodomain-containing proteinPdd1p and are the putative sites of IES removal. Strains ex-pressing Drb2p-CFP or Pdd1p-YFP were mated, and localiza-tion of both proteins was monitored at 8 h into conjugation,very early in zygotic macronuclear differentiation, and later at14 h into conjugation, when DNA elimination occurs (Fig. 6).As was previously reported, Pdd1-YFP was diffusely localizedin the zygotic macronuclei at 8 h, and as conjugation pro-ceeded toward DNA elimination around 14 h, Pdd1p-YFPlocalization gradually became unevenly dispersed, forming firstsmall foci and then finally large foci (Fig. 6) (40, 41). Local-ization of Drb2p-CFP in the zygotic macronuclei at 8 h intoconjugation was not markedly different from Pdd1p-YFP lo-calization, with small Drb2p-CFP foci throughout the nucleus(Fig. 5). However, at 14 h into conjugation Drb2p-CFP fociaggregated into larger foci, which colocalized with the Pdd1p-

FIG. 4. Zygotic expression of DRB2 is necessary for completion ofconjugation. (Top) Terminal arrest phenotype of wild-type (WT),�DCL1, �DRB1, and �DRB2 mic cells 30 h into conjugation. WT,�DCL1, �DRB1, and �DRB2 mic cells were mated and harvested after30 h into conjugation. Cells were then DAPI stained, and DIC (left)and DAPI (right) images were obtained. White arrowheads, micronu-clei; white arrows, zygotic macronuclei. (Bottom) Cells with the indi-cated terminal arrest phenotype of WT, �DCL1, �DRB1, and �DRB2mic 30 h into conjugation.

TABLE 2. Progeny production of DRB1 knockouts in wild-type andknockout matings

Cross % pair survival(S/N)a

% progenyproduction

(P/S)b

CU427 � DRB1 KO 5.1.3 97.2 (171/176) 96.5 (165/171)CU427 � DRB1 KO 6.1.6 98.8 (87/88) 98.9 (86/87)CU427 � DRB1 KO 6.1.12.1 96.0 (169/176) 98.2 (166/169)CU427 � DRB1 KO 6.1.12.2 98.9 (174/176) 97.1 (169/174)CU427 � DRB1 KO 7.1 97.7 (129/132) 98.4 (127/129)CU427 � DRB1 KO 7.7.2 99.2 (131/132) 94.7 (124/131)DRB1 KO 5.1.3 � 6.1.12.1 93.5 (247/264) 33.3 (6/18)DRB1 KO 5.1.3 � 6.1.12.2 94.7 (250/264) 51.5 (35/68)

a The pair survival is the percentage of pairs alive (S) of the total pairs (N)isolated.

b Progeny production is the percentage of surviving pairs (S) that successfullycompleted conjugation and made new macronuclei (P).



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YFP-containing DNA elimination bodies, indicating a possibleinteraction with each other in zygotic macronuclei.

Localization of Pdd1-YFP and Drb2p-CFP is not exclusiveto the zygotic macronuclei. Residual localization of both pro-teins was seen in the parental macronucleus as well. At 8 h intoconjugation, both proteins formed strong, distinct foci in theparental macronucleus, with Pdd1p-YFP foci localized to thenuclear periphery and Drb2p-CFP foci found in the nuclearinterior. During DNA elimination at 14 h into conjugation,remaining Pdd1p-YFP was found throughout the parental ma-cronucleus but away from the interior, while Drb2p-CFP wasstill seen only in the interior. The significance of this lateparental macronuclear localization remains to be explored.

Pdd1p fails to form DNA elimination bodies in DRB2 micknockouts. To understand if Pdd1p and Drb2p colocalizationis relevant to the conjugation arrest phenotype and failure ofDNA elimination in DRB2 mic knockouts, we sought to deter-mine how Pdd1p localization was affected in DRB2 mic knock-out strain matings. DRB2 mic knockout strains were trans-formed with an inducible Pdd1p-YFP expression construct,and the resulting transformants were mated and their Pdd1p-YFP localization was examined. At 10 h into conjugation dur-ing zygotic macronuclear differentiation, Pdd1p-YFP localiza-tion in both DRB2 mic knockouts crossed to wild-type strains,which rescues loss of DRB2 from the mating partner, andDRB2 mic knockout matings appeared mottled throughout thedeveloping zygotic macronucleus without obvious defects (Fig.7A). However, late in conjugation (14 h), Pdd1p-YFP failed toform DNA elimination bodies in zygotic macronuclei in DRB2mic knockout matings (Fig. 7B). Thus, Pdd1p-YFP foci fail tomature into DNA elimination bodies without zygotic DRB2expression. These data indicate that DRB2 participates in thematuration of DNA elimination bodies and implicates a pos-sible role for uncharacterized dsRNAs in genome reorganiza-tion.

DISCUSSION

Our analyses of DRB1 and DRB2 have revealed that eachhas unique and important functions. While both are predom-inantly nuclear proteins, they localize into distinct subnuclearfoci. Furthermore, disruption of the each gene showed thatDrb2p has essential functions during both growth and devel-

opment, while Drb1p appears to be important for prezygoticdevelopment. The similarities of these two proteins outsidetheir predicted DSRMs suggest that they may have arisen froman ancestral gene duplication. If that is the case, they havesignificantly diverged in function since the duplication event.

FIG. 5. DNA rearrangements of IESs and chromosome breakageare impaired in DRB2 mic knockouts. (A and B) Rearrangements ofIES B (A) and the M IES (B) were assessed by two- or three-primerPCR, respectively, in genomic DNA isolated from wild-type (WT),�DCL1, and �DRB2 mic cells postconjugation. White arrowheads, theunrearranged/micronuclear form; black arrowheads, the rearranged/macronuclear form; unlabeled bands, nonspecific products. Diagramsof each IES locus are shown below the gel image. IES, white and darkgray boxes; flanking DNA, gray boxes; PCR primers, black and grayarrows. The M IES undergoes alternative rearrangement throughelimination of the 0.6-kb (white box) or the 0.9-kb (white and dark grayboxes) sequence. The expected PCR product size is provide besideeach form. (C) Chromosome breakage fails in DRB2 mic knockouts.(Left) Southern blot hybridization of total genomic DNA isolated fromWT or mutant cells postconjugation. White arrowhead, micronucleus-specific fragment; gray arrowhead, parental macronucleus-specificfragment; black arrowhead, zygotic macronucleus-specific fragment.(Right) Diagram of CBS near the LIA1 locus in the micro- and ma-cronuclei. Southern blot band sizes are listed next to each locus dia-gram. White circle, CBS; white arrow, LIA1 gene; Tel, telomere.

TABLE 3. Progeny production of DRB2 mic knockouts in wild-typeand knockout matings

Cross % pair survival(S/N)a

% progenyproduction

(P/S)b

B*VII427 � B*VI�D2/�D2 1 99.6 (263/264) 98.8 (260/263)B*VII427 � B*VI�D2/�D2 6 99.2 (262/264) 99.2 (260/262)CU427 � B*VII�D2/�D2 1 95.1 (251/264) 99.6 (250/251)CU427 � B*VII�D2/�D2 2 97.3 (257/264) 100 (257/257)B*VI�D2/�D2 1 � B*VII�D2/�D2 1 2.8 (5/176) 0.0 (0/5)B*VI�D2/�D2 1 � B*VII�D2/�D2 2 1.7 (3/176) 0.0 (0/3)B*VI�D2/�D2 6 � B*VII�D2/�D2 1 0.0 (0/176) 0.0 (0/0)B*VI�D2/�D2 6 � B*VII�D2/�D2 2 1.1 (2/176) 0.0 (0/2)

a Pair survival is the percentage of pairs alive (S) of the total pairs (N) isolated.b Progeny production is the percentage of surviving pairs (S) that successfully

completed conjugation and made new macronuclei (P).



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Upon initial recognition that the T. thermophila genomeencodes two DSRM-containing proteins, we looked for evi-dence that would connect them as protein partners for theDicer homologues encoded by DCL1 and DCR2 (42, 49). Tan-dem DSRM-containing partner proteins for Dicer and Droshafamily proteins, including R2D2, Loqs, and Pasha in D. mela-nogaster, RDE-4 in C. elegans, and TRBP2 and DGCR8 in H.sapiens and other mammals, play vital roles in RNAi by ensur-ing proper sRNA delivery and in many cases cleavage of sRNAprecursors (12, 17, 23, 27, 37, 57, 65). Our analyses providedlittle support that Drb1p or Drb2p serve as major Dicer part-ners. Neither protein showed abundant localization in meioticmicronuclei, where Dcl1p acts (Fig. 2A and B, top rows) (42,49). We also did not find defects in scnRNA accumulation incomplete DRB1 knockouts (data not shown). As Drb2p isessential for growth, we were unable to generate full knockoutswith which to examine scnRNA accumulation upon its loss.The T. thermophila Dicer protein, Dcr2p, is also essential forgrowth, but a previously published characterization of Dcr2pcomplexes did not find Drb2p to be an interacting protein(34, 35).

While we did not find evidence that these proteins act withDcl1p, we uncovered a critical role for Drb2p in the RNAi-directed DNA elimination pathway. Loss of zygotic expressionwas sufficient to block DNA rearrangement; thus, Drb2p isneeded well downstream of scnRNA biogenesis by Dcl1p (Fig.5A and B). Colocalization of Drb2p with Pdd1p-containingDNA elimination bodies and loss of these DNA eliminationbodies in DRB2 mic knockouts implicate zygotically expressedDrb2p in promoting development or stabilizing these largenucleoprotein structures (Fig. 6 and 7). This may indicate that

Drb2p/RNA complexes mediate the formation of matureDNA elimination bodies through facilitating protein-RNA orprotein-protein interactions within these structures. Althoughthe exact mechanism of Drb2p action remains to be discov-ered, its importance in late stages of genome reorganizationsuggests an unrecognized role for dsRNA in RNAi-directedDNA elimination.

Drb2p is also required for vegetative growth, as we wereunable to replace all wild-type DRB2 gene copies with thedisrupted allele. We tried extensively to assort DRB2 out of themacronucleus without success (data not shown). Furthermore,when DRB2 partial knockout strains were grown in nonselec-tive medium (without paromomycin), the remaining wild-typeDRB2 copies rapidly replaced the DRB2 knockout allele (datanot shown). As both Drb2p and Dcr2p are essential for growth,it remains possible that they act in the same pathway (34, 35).We cannot rule out the possibility that these proteins tran-siently interact, as do RDE-4 and DCR-1 in C. elegans (65).Further investigation of the function of Drb2p during growthmay provide key insights into the role of this protein duringboth growth and genome reorganization.

While Drb1p is predominantly a macronuclear protein, italso localizes to one or both ends of the crescent micronucleusduring the prophase of meiosis I (Fig. 2 and data not shown).Further investigation of this micronuclear point localizationindicated that colocalization of Drb1p with cenH3, the centro-meric histone H3 (unpublished data) (9, 16, 39). Knockouts ofDRB1 were able to complete conjugation, yet a significantpercentage of pairs aborted mating without forming new ma-cronuclei. Together, the localization of Drb1p near centrom-eres and possibly with telomeres and the reduction in knockout

FIG. 6. Drb2p colocalizes with the essential conjugation chromodomain protein Pdd1p in DNA elimination bodies. Cells expressing Drb2p-CFP were mated with cells expressing Pdd1p-YFP. Both proteins localized to the developing zygotic macronucleus (8 h) and in DNA eliminationbodies (14 h). The bottom panels show a magnified view of one zygotic macronucleus from the 14-h cell above. White arrowheads, micronuclei;black arrowheads, parental macronuclei; white arrows, zygotic macronuclei.



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cells completing prezygotic stages of development are consis-tent with a role for Drb1p in maintaining micronuclear chro-mosome structure (Fig. 2 and data not shown). Thus, theanalysis of both of these DSRM-containing proteins stronglysuggests that they perform critical chromosomal functions.

Although many tandem DSRM-containing proteins havebeen found to interact with Dicer and Drosha family proteins,this is by no means the only job that these proteins containingDSRMs undertake (12, 17, 23, 27, 37, 57, 65; reviewed inreferences 20 and 67). Roles for these proteins include cleav-age of long noncoding RNAs into sRNAs by RNase III familymembers, RNA editing by the ADAR family, translation inhi-bition in response to viruses by PKR family members, anddevelopmental RNA localization by the Staufen family (3, 4,26, 28, 30, 36, 45, 50, 55, 62, 63). Besides the partner proteinsfor the Dicer and Drosha families, at least one other proteinfamily, the NFAT family, also encodes tandem DSRMs. TheNFAT family proteins, which contain a DZF protein domain in

addition to tandem DSRMs, are putative nuclear, nucleotidetransferases that participate in DNA repair and RNA trans-port (32, 59, 60, 73; reviewed in reference 31). Further study ofDRB1 and DRB2 in T. thermophila may reveal new roles fortandem DSRM-containing proteins. The great evolutionarydistance between ciliates and other eukaryotes could also fa-cilitate understanding of how DSRM-containing proteinsevolved within the eukaryotic lineage (54). Much remains to begleaned about the roles of DSRM-containing proteins in eu-karyotes, and we expect further investigation of Drb1p andDrb2p functions will provide greater understanding of RNAi-directed DNA elimination and roles for dsRNA in regulatingchromosome structure.

ACKNOWLEDGMENT

This research was supported primarily by a grant from the NationalScience Foundation (NSF MCB-0642162) to D.L.C.

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FIG. 7. Failure of DNA elimination bodies to form in DRB2 micknockouts late in conjugation. (A) Normal zygotic macronuclear lo-calization of Pdd1p in DRB2 mic knockout matings midway throughconjugation. DRB2 mic knockouts ectopically expressing Pdd1p-YFPwere mated with wild-type or with DRB2 mic knockouts. Pdd1p-YFPlocalized to the developing zygotic macronucleus in both matings.White arrowheads, micronuclei; black arrowheads, parental macronu-clei; white arrows, zygotic macronuclei. (B) Ectopically expressedPdd1p fails to form DNA elimination bodies in DRB2 mic knockouts.DRB2 mic knockouts ectopically expressing Pdd1p-YFP were mated asdescribed for panel A. When DRB2 mic knockouts were mated toDRB2 mic knockouts, Pdd1p-YFP failed to form DNA eliminationbodies in the developing zygotic macronucleus. White arrowheads,micronuclei; black arrowheads, parental macronuclei; white arrows,zygotic macronuclei.



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Zygotic Expression of the Double-Stranded RNA Binding ... · Similarly, the entire PDD1 coding sequence was ampliﬁed from genomic DNA (primers are listed in Table 1) and cloned