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. Author manuscript; available in PMC: 2013 Nov 30.
Published in final edited form as: Mol Cell. 2012 Oct 18;48(4):509–520. doi: 10.1016/j.molcel.2012.09.010

A Tetrahymena Piwi bound to mature tRNA 3' fragments activates the exonuclease Xrn2 for RNA processing in the nucleus

Mary T Couvillion 1, Gergana Bounova 2, Elizabeth Purdom 2, Terence P Speed 2, Kathleen Collins 1,*
PMCID: PMC3513674  NIHMSID: NIHMS409009  PMID: 23084833

SUMMARY

Emerging evidence suggests that Argonaute (Ago)/Piwi proteins have diverse functions in the nucleus as well as the cytoplasm, but the molecular mechanisms employed in the nucleus remain poorly defined. The Tetrahymena thermophila Ago/Piwi protein Twi12 is essential for growth and functions in the nucleus. Twi12-bound sRNAs are 3' tRNA fragments that contain modified bases and thus are attenuated for base-pairing to targets. We show that Twi12 assembles an unexpected complex with the nuclear exonuclease Xrn2. Twi12 functions to stabilize and localize Xrn2, as well as to stimulate its exonuclease activity. Twi12 function depends on small RNA (sRNA) binding, which is required for its nuclear import. Depletion of Twi12 or Xrn2 induces a cellular ribosomal RNA (rRNA) processing defect known to result from limiting Xrn2 activity in other organisms. Our findings suggest a role for an Ago/Piwi protein and 3' tRNA fragments in nuclear RNA metabolism.

INTRODUCTION

Ago/Piwi-protein complexes are central players in RNA silencing pathways, with bound sRNAs directing the sequence-specific recognition of target nucleic acids. Cytoplasmic Ago/Piwi RNPs typically induce mRNA decay and/or translational repression (Ghildiyal and Zamore, 2009). Nuclear Ago/Piwi RNPs typically guide heterochromatin formation for genome maintenance (Grewal, 2010). Studies in many organisms have revealed that these and other Ago/Piwi cellular functions are carried out by diverse mechanisms. In some cases Ago/Piwi proteins with slicer activity cleave a target transcript directly (Tolia and Joshua-Tor, 2007). In other cases the mechanisms of Ago/Piwi function depend on partner protein associations. Ago proteins loaded with microRNAs interact with a GW-domain protein to promote translational repression and deadenylation (Eulalio et al., 2009). Piwi proteins loaded with animal germline Piwi-interacting RNAs associate with Tudor domain-containing proteins to mediate transposon silencing by RNA degradation and DNA methylation (Juliano et al., 2011). S. pombe Ago1 recruits a histone methyltransferase complex to direct heterochromatin formation at centromeres, telomeres, and mating type loci (Bayne et al., 2010; Buhler and Moazed, 2007). S. pombe Ago1 and associated factors also have roles in promoting RNAP II termination and DNA release coordinated with the cell cycle (Gullerova and Proudfoot, 2008; Zaratiegui et al., 2011) and in co-transcriptional mRNA degradation at stress-inducible genes (Woolcock et al., 2012). In animals, nuclear Ago/Piwi proteins affect RNAP II transcription at protein-coding genes through mechanisms that are not yet well defined (Burkhart et al., 2011; Cernilogar et al., 2011; Guang et al., 2010; Moshkovich et al., 2011).

Exonucleases are important factors in many RNA silencing pathways. They contribute to sRNA biogenesis (Kawaoka et al., 2011), sRNA turnover (Chatterjee and Grosshans, 2009; Ramachandran and Chen, 2008), Ago/Piwi cleavage product degradation (Orban and Izaurralde, 2005; Souret et al., 2004), and decay of translationally repressed mRNAs (Rehwinkel et al., 2005). The 5' to 3' exonuclease activity in these cytoplasmic processes is mediated by members of the XRN nuclease family. However, the nuclear-localized XRN, Xrn2/Rat1, has not been implicated in RNA silencing. Studies in yeast and mammalian cells have demonstrated that Xrn2/Rat1 functions in pre-ribosomal RNA (pre-rRNA) processing and in the degradation of truncated or improperly processed rRNA precursors (Geerlings et al., 2000; Henry et al., 1994; Wang and Pestov, 2011). In addition, Xrn2/Rat1 has roles in RNAP I and RNAP II termination (El Hage et al., 2008; Kaneko et al., 2007; Kawauchi et al., 2008; Kim et al., 2004; Luo et al., 2006; West et al., 2004) and the regulation of productive mRNA synthesis (Brannan et al., 2012; Davidson et al., 2012; Jimeno-Gonzalez et al., 2010).

The ciliate Tetrahymena thermophila encodes a large family of Piwi proteins with distinct expression, localization, and associated sRNAs (Couvillion et al., 2009; Chalker and Yao, 2011). During vegetative culture growth by cell fissions, the Tetrahymena germline micronucleus is packaged into heterochromatin while the somatic macronucleus that lacks heterochromatin is expressed. In this stage of the life cycle, several Tetrahymena Piwi (Twi) proteins bind 23–24 nt sRNA products of the constitutively expressed and genetically essential Dicer 2, but curiously the only Tetrahymena Piwi individually essential for growth is the most divergent family member Twi12 (Couvillion et al., 2009). Twi12-associated sRNAs are not produced by either Tetrahymena Dicer, and Twi12 itself does not conserve slicer catalytic residues (Couvillion et al., 2009; Couvillion et al., 2010). We previously characterized Twi12 association with RNA fragments derived predominantly from the 3’ end of mature tRNAs (Couvillion et al., 2010). These Twi12-bound tRNA fragments are one example of the newly recognized diversity of tRNA processing and tRNA-fragment biology across eukaryotes (Haussecker et al., 2010; Thompson and Parker, 2009). Twi12-bound 18–22 nt tRNA fragment termini are uniform, with the predominant 5' end in the TΨC loop and the 3' end at the mature tRNA 3' terminus. A second, larger size range of Twi12-enriched sRNAs corresponds to tRNA 5' fragments that are not tightly Twi-bound and are thought to be passenger strands from the RNA loading/RNP maturation process (Couvillion et al., 2010).

To elucidate the biological role of Twi12 and its bound tRNA fragments, we have investigated the protein interaction partners of Twi12. We find that Twi12 RNP functions in a multisubunit complex that includes the evolutionarily conserved nuclear 5' to 3' exonuclease, Xrn2/Rat1. We demonstrate that Tetrahymena Xrn2 functions only in the context of this complex; it is destabilized in vivo and inactive in vitro without sRNA-loaded Twi12. Cellular depletion of Tetrahymena Twi12 or Xrn2 inhibits Xrn2-dependent pre-rRNA processing. Our findings uncover a new biological role and mechanism of function for Ago/Piwi RNPs.

RESULTS

Full-length Twi12 binds tRNA-derived fragments from the 3' ends of mature tRNAs

In a previous study we reported that overexpressed, tagged Twi12 is bound to tRNA fragments (Couvillion et al., 2010). While creating a Tetrahymena strain with Twi12 tagged at its endogenous locus, we discovered an in-frame ATG upstream of the start codon predicted by genome annotation. The corresponding upstream AUG codon is included in the transcript (data not shown). Therefore, we created strains expressing full-length Twi12 for comparison to the original Twi12, now called Twi12 short (Twi12S) (Figure 1A). We resolved RNAs co-purified with each Twi12 by denaturing polyacrylamide gel electrophoresis (PAGE) (Figure 1B). While Twi12S co-purified 18–22 nt and also 25–30 nt RNA populations, Twi12 preferentially co-purified the 18–22 nt RNAs, previously shown to be the more tightly bound sRNAs that can be crosslinked to Twi12S in vivo (Couvillion et al., 2010). We deep sequenced 18–22 nt RNAs co-purified with Twi12 N-terminally tagged with two Protein A domains (ZZ), a Tobacco Etch Virus (TEV) protease cleavage site, and three FLAG peptide (F) sequences (ZZF-Twi12) expressed at the endogenous TWI12 promoter. We found that the majority of reads mapped to tRNAs without any evident biases across different tRNA molecules (Figure 1C and data not shown). Thus, like Twi12S, Twi12 selectively co-purifies specific bound fragments of tRNAs.

Figure 1. Full-length Twi12 binds tRNA fragments.

Figure 1

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(A) Schematic of Twi12 compared to Twi12S, which is truncated by 17 amino acids at the N-terminus. See Figure S1 for further description of strains.

(B) RNA co-purified with ZZF-Twi12 or ZZ-Twi12S, resolved by urea-PAGE and stained by SYBR Gold. Total RNA is from the same cells, which express ZZF-Twi12 or ZZ-Twi12S from the uninduced MTT1 promoter and do not express endogenous Twi12.

(C) Library composition of sequenced 18–22 nt sRNAs co-purified with ZZF-Twi12 expressed from the endogenous TWI12 promoter, mapped allowing for one internal mismatch and a 3' overhang of C, CC, or CCA.

Twi12 is essential for growth; therefore the TWI12 locus cannot be replaced by a drug-resistance cassette in a wild-type background (Couvillion et al., 2009). However, in the presence of a transgene expressing ZZF-Twi12, the endogenous TWI12 locus could be fully replaced (Figure S1A), indicating that tagged Twi12 is functional. ZZ-tagged Twi12S (ZZ-Twi12S) transgene expression can also substitute for endogenous Twi12 (Couvillion et al., 2010), but unlike expression of tagged full-length Twi12, cellular expression of only tagged Twi12S slowed culture growth in rich medium (Figure S1B). Accordingly, we detected endogenous untagged Twi12 migrating with Twi12 rather than Twi12S (Figure S1C).

Because Twi12S could have reduced function, additional characterization of associated sRNAs was done for tagged full-length Twi12. Eukaryotic tRNAs are post-transcriptionally modified by 3'-terminal untemplated CCA addition. Therefore, in mapping reads we allowed for 3' terminal mismatches of C, CC, or CCA. About 75% of tRNA-derived sRNA reads contain these untemplated nucleotides, compared to less than 10% of the reads in all other categories (Figure 2A), which result from a mixture of reads mapping to unannotated tRNAs and misannotated reads. Reads were first mapped allowing for no internal mismatch, then unmapped reads were mapped allowing for one mismatch. For reads mapping to annotated tRNA, mismatches were only allowed at positions that we annotated as putative base modification sites (see Supplemental Experimental Procedures). Analysis of the position of reads across tRNA lengths confirmed that the majority are 3' fragments starting at the pseudouridine (Ψ) in the TΨC loop and ending at the mature tRNA 3' end (Figures 2B and 2C, and data not shown).

Figure 2. Twi12-bound sRNAs are derived from the 3' ends of mature tRNAs.

Figure 2

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(A) Annotated reads in each category analyzed for the fraction that map perfectly (black), with one mismatch (gray), or with a 3' overhang of C, CC, or CCA (blue and light blue, see key). Reads annotated as mRNA and rRNA are most likely misannotated.

(B) Plot showing number of reads with 5' ends at each tRNA location: 5' end, D stem-loop, anticodon stem-loop (A), variable stem-loop (V), or TΨC stem-loop (T).

(C) Two-dimensional thin layer chromatographic analysis of 32P post-labeled 5' monophosphate nucleosides. The diagram at right shows the positions of the conserved tRNA base modifications that are indicated in the chromatographs. The solid black line indicates the Twi12-bound fragment. Asterisks indicate modified base positions in the Twi12-bound fragment that are pseudouridine (Ψ) or 1-methyl adenosine (m1A).

Mature tRNA base modifications are important for folding and/or activity in translation (Phizicky and Alfonzo, 2010). To determine whether Twi12-bound tRNA fragments contain modified bases, we used two-dimensional thin layer chromatography to compare labeled nucleosides from the Twi12-bound sRNAs to those of full-length mature tRNAs and the bulk 23–24 nt sRNAs, which are mostly bound to Twi2 (Figure 2C). Consistent with their processing from functional mature tRNAs, Twi12-bound 18–22 nt RNAs contained Ψ and 1-methyladenosine (m1A), the two conserved modifications found in the T loop of tRNAs (Figure 2C, middle panel and schematic at right). These sRNAs did not contain thymidine (T) or dihydrouridine (D), which are clearly identifiable in nucleosides from full-length tRNA (Figure 2C, right panel). The conserved m1A modification at tRNA position 58 (Twi12-bound sRNA position 4) is also evident in sequence reads, because this base is often misread by reverse transcriptase during library preparation, resulting in a relatively high frequency of mismatch at this position (see Supplemental Experimental Procedures). Together, presence of the 3' untemplated CCA and the base modifications provide strong support for the conclusion that Twi12-bound sRNAs derive from mature tRNAs.

Twi12 forms a nuclear complex with Xrn2 and Tan1

To gain insight into the function of Twi12, we next investigated interacting proteins. SDS-PAGE and silver staining after a two-step immunoprecipitation (IP) of ZZF-Twi12 expressed from the endogenous TWI12 promoter revealed several co-enriched polypeptides with apparent masses between 85 and 120 kDa (Figure 3A). Mass spectrometry identified two proteins in addition to Twi12, each with significant peptide coverage and each not found in the mock-purification control: a 12.3 kDa protein with no homologs that we named Tan1 (Twi-associated novel 1) and a 126 kDa protein containing a 5' to 3' monophosphate-dependent exonuclease domain (Figure 3B). The Tetrahymena genome encodes three putative proteins with a 5' to 3' exoribonuclease domain. Two are most similar to Xrn1 from yeast and human, and consistent with Xrn1-like function they have cytoplasmic localization (Douglas Chalker, personal communication), whereas the one identified in association with Twi12 is more similar to human XRN2 and yeast Rat1 and thus we named it Xrn2.

Figure 3. Twi12 interacts with Xrn2 and Tan1.

Figure 3

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(A) SDS-PAGE and silver staining after two-step IP of ZZF-Twi12 expressed from the endogenous locus. The first step was IgG IP and TEV protease elution, and the second step was anti-FLAG IP and urea elution.

(B) Table of ZZF-Twi12-associated proteins identified by mass spectrometry. See also Figures S2A and S2B.

(C) SDS-PAGE and silver staining after one-step IP of the proteins identified by mass spectrometry. Filled circles indicate the tagged protein in each lane, which runs as two bands (ZZF-tagged and F-tagged) because of proteolytic clipping between the tag segments. Labels at right indicate the migration positions of untagged proteins, and small Xs mark Xrn2 N-terminal proteolysis fragments. Note that tagged Tan1 does not silver stain strongly. See also Figures S2C and S2D.

(D) Illustrated model of TXT with bound sRNA. Asterisks on the line representing sRNA indicate base modifications. Direct interaction of Tan1 with Twi12 is not established.

To verify the interaction of Twi12 with Tan1 and Xrn2, we created cell lines expressing Tan1 or Xrn2 tagged at their endogenous loci. IP of Tan1-FZZ and Xrn2-FZZ confirmed their interaction with Twi12 and each other (Figure 3C). We refer to the Twi12/Xrn2/Tan1 complex as TXT (Figure 3D; see below for analysis of sRNA in TXT). No other proteins associated with any of the three subunits were detectable by silver staining. The ladder of silver-stained bands between 85 and 120 kDa visible with IP of Twi12 was also evident with IP of Tan1 but not C-terminally tagged Xrn2 (Figure 3C). We therefore suspect that the ladder derives from proteolysis of Xrn2 near its C-terminus.

To test whether Tan1 and Xrn2, like Twi12, are essential for Tetrahymena growth, we attempted to replace their endogenous loci with a drug-resistance cassette (neo2) on all chromosomes in the somatic macronucleus by phenotypic assortment. TAN1 could be fully replaced by neo2 (Figure S2A), and therefore it is not essential. XRN2 could be only partially replaced (Figure S2B), suggesting that it is essential. We used Tan1 knockout (KO) cell lines to investigate the function of Tan1 in TXT. IP of Xrn2 in the Tan1 KO background still recovered Twi12, indicating that Tan1 is not required for Twi12/Xrn2 association (Figure S2C). Additionally, IP of Twi12 in the Tan1 KO background still recovered 18–22 nt RNA, indicating that Tan1 is not required for sRNA binding (Figure S2D).

Xrn2/Rat1 homologs are generally nuclear, so the interaction of Twi12 with Xrn2 was surprising because overexpressed ZZ-Twi12S is predominantly cytoplasmic (Couvillion et al., 2009). However, indirect immunofluoresence (IF) to detect ZZF-Twi12, Tan1-FZZ, and Xrn2-FZZ expressed from their endogenous promoters revealed each of them to be predominantly nuclear. Each protein was enriched in the expressed macronucleus (Figure 4A), consistent with the assembly of these three subunits as a TXT complex. IF analysis of ZZF-Twi12 expressed from the cadmium-inducible MTT1 promoter revealed mostly nuclear localization when only slightly overexpressed (without promoter induction) but predominantly cytoplasmic localization when highly overexpressed (data not shown), accounting for the previously reported localization of ZZ-Twi12S.

Figure 4. TXT is a nuclear complex with 5' monophosphate-dependent exonuclease activity.

Figure 4

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(A) Indirect IF for each subunit in TXT.

(B) Nuclease activity assay on TXT purified by each subunit. Top panel: silver stained proteins after one-step IP. Middle panel: RNA after in vitro incubation with TXT. Note that N-terminally tagged Xrn2 is catalytically inactive. Bottom panel: sRNAs associated with each population of TXT in vivo. See Figure S3 for analysis of a potential Tan1 activity.

Xrn2 has 5' monophosphate-dependent exonuclease activity

Using purified TXT, we assayed for the expected 5' monophosphate-dependent single-stranded RNA exonuclease activity of XRN-family enzymes (Jinek et al., 2011). TXT purified by ZZF-Twi12, Tan1-FZZ, or Xrn2-FZZ did degrade 5' monophosphorylated RNA, e.g. 5.8S rRNA, but not 5' triphosphorylated RNA, e.g. 5S rRNA, from a mixture of purified, low molecular weight Tetrahymena cellular RNA (Figure 4B). We conclude that Xrn2 has 5' monophosphate-dependent activity in vitro in the context of TXT.

The Xrn2 homolog in yeast, Rat1, is stabilized for catalytic activity in vitro by the small associated protein Rai1 (Xue et al., 2000), which has a pyrophosphohydrolase activity that converts triphosphorylated RNAs to monophosphorylated Rat1 substrates (Xiang et al., 2009). Although we could not identify any sequence or structural similarity between Tan1 and Rai1, we tested for Tan1 pyrophosphohydrolase activity. None could be detected, either for recombinant Tan1 purified from E. coli or for Tan1 in the context of TXT (Figure S3A). Furthermore, Tan1 was not required for Xrn2 exonuclease activity in vitro (Figure S3B).

One potential function of Xrn2 in TXT is to trim anticodon loop-cleaved tRNAs to produce the 5' ends of the Twi12-bound tRNA fragments. To test this hypothesis, we used N-terminally tagged Xrn2 (ZZF-Xrn2), which cannot functionally substitute for untagged Xrn2 in vivo (data not shown) and which has reduced catalytic activity in vitro (Figure 4B). TXT complexes containing ZZF-Xrn2 purified the identical size range and amount of 18–22 nt sRNAs as the TXT complexes purified by ZZF-Twi12, Tan1-FZZ, or Xrn2-FZZ, each of which contained active Xrn2 (Figure 4B, bottom panel), suggesting that Xrn2 catalytic activity is not required for Twi12-bound sRNA maturation within its TXT complex. Furthermore, the similar amounts of 18–22 nt sRNAs enriched by TXT purification using each separately tagged subunit indicate that sRNA is an integral component of the TXT RNP.

RNA binding is required for Twi12 nuclear import

Twi12 is a divergent Ago/Piwi protein family member (Couvillion et al., 2009; Seto et al., 2007), leaving open the possibility that sRNA binding is a vestigial characteristic not essential to Twi12 function. To address the significance of Twi12 sRNA binding, we made cell lines expressing tagged Twi12 sequence variants. In Twi12Y524E, a single amino acid substitution replaces a conserved tyrosine (Y) with a negatively charged glutamate (E) (Figure S4A) in the binding pocket for the sRNA 5' phosphate (Ma et al., 2005). Similar phosphate mimicry was shown to abrogate sRNA binding by human AGO2 (Rudel et al., 2011). Another conserved residue in the 5' phosphate binding pocket is a glutamine (Q) that Twi12 lacks; Twi12 instead has a serine (S) at the analogous position (Figure S4A). The structure of the Archaeoglobus fulgidas Piwi with bound sRNA (Ma et al., 2005; Figure S4B) shows this Q, Q137, at a position where it could sterically hinder loading of a 5'-extended precursor (Figure S4B) required as a biogenesis step in our model for Twi12 loading with full-length or anticodon-nicked tRNA (Couvillion et al., 2010). Therefore, we also made cell lines expressing tagged Twi12S540Q, which has a single amino acid substitution restoring the conserved Q.

Two different cell lines were made to express each Twi12 sequence variant described above: one for overexpression, in which the tagged Twi12 is expressed from the MTT1 cadmium-inducible promoter at the ectopic BTU1 locus, and one in which the tagged Twi12 is expressed from the endogenous promoter at the endogenous locus. Consistent with disruption of critical recognition of the sRNA 5' monophosphate, ZZF-Twi12Y524E did not associate with sRNA whether slightly overexpressed from the uninduced MTT1 promoter (Figure 5A) or expressed at or below endogenous level from the endogenous TWI12 promoter (Figure S4C). We note that unloaded Twi12 may be more susceptible to proteolysis than Twi12 RNP in vivo and/or in cell lysate, as observed for Drosophila Piwi (Olivieri et al., 2010), because less ZZF-Twi12Y524E was purified than ZZF-Twi12 or ZZF-Twi12S540Q from an equivalent amount of cell extract (Figures 5A and S4C). Unlike ZZF-Twi12Y524E, ZZF-Twi12S540Q retained sRNA binding (Figures 5A and S4C). IF analysis revealed that wild-type ZZF-Twi12 and ZZF-Twi12S540Q accumulated in the macronucleus, while ZZF-Twi12Y524E and ZZF-Twi12S (with an altered sRNA size profile; Figure 5A and see also ZZ-Twi12S sRNAs in Figure 1B) accumulated to higher levels in the cytoplasm, whether slightly overexpressed from the uninduced MTT1 promoter (Figure 5B) or expressed at or below endogenous level from the endogenous TWI12 promoter (Figure S4D). Consistent with a loss of function, ZZF-Twi12Y524E expression did not allow for KO of endogenous TWI12; in contrast, ZZF-Twi12S540Q expression did (data not shown). We conclude that sRNA binding is a prerequisite for Twi12 nuclear localization and physiological function.

Figure 5. Twi12 nuclear localization is dependent on sRNA binding.

Figure 5

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(A) SDS-PAGE and silver staining after one-step IP of ZZF-Twi12 wild-type and variants expressed from the uninduced MTT1 promoter (top), with SYBR Gold-staining of associated sRNA (bottom).

(B) Indirect IF for ZZF-Twi12 wild-type and variants expressed from the uninduced MTT1 promoter. See also Figure S4.

Depletion of Twi12 or Xrn2 induces a pre-rRNA processing defect

To test whether Tetrahymena Xrn2 and Twi12 cellular functions are interrelated, as suggested by their stable physical association, we made inducible knockdown (iKD) cell lines for conditional expression of each protein. First, F-tagged transgenes encoding each protein were separately integrated at the MTT3 locus under the control of the cadmium-inducible MTT3 promoter (Figures 6A and S5). This locus was chosen because of its low basal (uninduced) expression (Miao et al., 2009). Subsequently, replacement of the endogenous TWI12 or XRN2 locus with a drug-resistance cassette could be driven to complete assortment when cultures were grown in the presence of cadmium to induce transgene expression, as assessed by Southern blot hybridization (Figure S5). Cells were maintained in a cadmium concentration that supported near endogenous levels of protein expression, as judged by western blotting of ZZF-Twi12 expressed from the endogenous promoter (data not shown). Within 6 to 7 population doublings after removal of cadmium, culture growth dramatically slowed and protein accumulation level diminished to less than 20% for F-Twi12 or less than 50% for Xrn2-F of the protein level in cells grown in cadmium, relative to a tubulin loading control (Figure 6A).

Figure 6. Depletion of Twi12 or Xrn2 induces a pre-rRNA processing defect.

Figure 6

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(A) Twi12 or Xrn2 depletion was achieved by cadmium removal from cells with the genotypes schematized at top (see also Figure S5). Bottom: western blot probed with anti-FLAG and anti-tubulin, with loading normalized for cell equivalents of whole-cell extract.

(B) Top: schematic of the Tetrahymena rRNA RNAP I transcript. Gray lines show positions of probes, and the 26S rRNA intron is also shown. Bottom: Northern blot analysis of total RNA samples normalized by cell equivalents. Blots were made from two gels loaded with the same RNA samples; both blots were probed for RPL21 mRNA as a control for equivalent loading. The asterisk indicates full-length or near full-length pre-rRNA transcript. Numbers below each panel indicate signal intensity normalized to that of wild-type cells.

(C) SYTO RNA select staining of Twi12 iKD and Xrn2 iKD cells cultured with and without cadmium. Note that both RNA and DNA are stained by SYTO RNA select in these paraformaldehyde-fixed cells.

A well-established role for Xrn2/Rat1 in yeast and human cells is in the 5’-3’ trimming required to remove internal transcribed spacer regions of the primary rRNA transcript following its initial endonucleolytic cleavage (Geerlings et al., 2000; Henry et al., 1994; Wang and Pestov, 2011). To investigate the impact of Twi12 or Xrn2 depletion on Tetrahymena pre-rRNA processing, we probed total RNA from wild-type, Twi12 iKD, and Xrn2 iKD cells using an oligonucleotide complementary to the internal transcribed spacer 5’ of mature 5.8S rRNA (Figure 6B, top), which is a substrate for Xrn2 processing in other organisms (Henry et al., 1994). Strikingly, pre-rRNA processing intermediates increased in accumulation in Twi12 iKD and Xrn2 iKD cells upon depletion of either protein (Figure 6B). In parallel mature 5.8S rRNA accumulation was reduced, a change that was particularly evident for the more effective TXT depletion by shut off of Twi12 (Figures 6A and 6B). In contrast there was no change in the accumulation of mature 17S rRNA (Figure 6B), which is not known to be processed by Xrn2. No pre-rRNA processing phenotypes were observed in wild-type cells across a range of growth rate (data not shown), indicating that the phenotype is specific to depletion of TXT.

Total RNA from wild-type, Twi12 iKD, and Xrn2 iKD cells was also probed to detect RPL21 mRNA, an abundant ribosomal protein mRNA typically used as a Tetrahymena RNA loading control. We were surprised to detect increased accumulation of this mRNA in Twi12 iKD and Xrn2 iKD cells depleted for TXT (Figure 6B). Unlike RPL21 mRNA, the mitochondrially encoded YMF66 mRNA was unchanged in accumulation upon Twi12 or Xrn2 depletion (Figure 6B). There was also no impact of Twi12 or Xrn2 depletion on the accumulation of tRNAs processed from primary transcripts of RNAP III (Figure 6B). However, a full-length or near full-length rRNA precursor transcript of RNAP I was increased upon Twi12 or Xrn2 depletion (Figure 6B, asterisk). The combination of these results raises the possibility that Tetrahymena TXT has role(s) similar to human and yeast Xrn2/Rat1 in the co-transcriptional regulation of RNAP I and RNAP II (Brannan et al., 2012; Davidson et al., 2012; El Hage et al., 2008; Jimeno-Gonzalez et al., 2010; Kaneko et al., 2007; Kawauchi et al., 2008; Kim et al., 2004; Luo et al., 2006; West et al., 2004). However, future experiments will be necessary to investigate any co-transcriptional regulatory function of TXT.

To investigate TXT-dependent cellular RNA accumulation using another approach, we stained whole cells with the fluorescent dye SYTO RNA select. Using cells fixed with paraformaldehyde before staining, we found that the dye stains DNA as well as RNA, as evidenced by staining of transcriptionally silent micronuclei (Figure 6C). Twi12- or Xrn2-depleted cells had notably increased staining at the macronuclear periphery, which is evident as an enlargement of the stained macronuclear volume compared to that stained by DAPI (Figure 6C). This increased staining mirrors the localization of Tetrahymena nucleoli (Gorovsky, 1973), which are positioned just beneath the macronuclear envelope around the DAPI-staining central area of bulk chromatin. We suggest that the change in pattern of SYTO RNA select staining upon depletion of Twi12 or Xrn2 (Figure 6C) reflects the greatly increased abundance of rRNA processing intermediates detected at the molecular level (Figure 6B).

Twi12 is necessary for Xrn2 accumulation, localization, and activity

We next investigated how Twi12 influences Xrn2 activity at a biochemical level. To detect and purify Xrn2 before and after conditional depletion of Twi12, we introduced a transgene encoding ZZ-tagged Xrn2 (Xrn2-ZZ) in the Twi12 iKD line (Figure 7A). Xrn2-ZZ was expressed under the control of the copper-inducible MTT2 promoter (Diaz et al., 2007). Xrn2-ZZ level was greatly increased upon addition of copper to the growth medium (Figure 7B). This allowed us to independently vary the cellular expression of Xrn2 and Twi12 by adding copper and/or removing cadmium from the growth medium.

Figure 7. Xrn2 requires Twi12 for accumulation, nuclear localization, and activity.

Figure 7

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(A) Genetic strategy for generation of the cell line in which Xrn2 is tagged in the Twi12 iKD background.

(B) Western blot probed with IgG or anti-FLAG, with loading normalized for cell equivalents of whole-cell extract.

(C) SDS-PAGE and silver staining after one-step IgG IP of Xrn2-ZZ from cell extracts.

(D) Indirect IF for Xrn2-ZZ in cells with Twi12 expressed (+Cd2+) or depleted (−Cd2+). Note that there is some cell-to-cell variability in transgene expression level.

(E) Nuclease activity. Top panel: silver stained samples after purification and depletion as indicated. Bottom panel: RNA after in vitro incubation with each purified sample.

Even if Xrn2-ZZ was overexpressed by copper addition simultaneously with Twi12 depletion by cadmium removal, we were unable to IP an excess of Xrn2-ZZ over F-Twi12 from whole cell extract (Figure 7C). These results suggest that Xrn2 cellular stability depends on its association with Twi12. Consistent with this conclusion, Twi12 depletion reduced the amount of functional, nuclear Xrn2-ZZ monitored by IF (Figure 7D) and also total Xrn2-ZZ monitored by western blot (data not shown). Based on the stabilizing influence of the proteasome inhibitor MG132 (see below), we suggest that Xrn2 that is not associated with Twi12 is degraded by protein turnover in the cytoplasm. We propose that Twi12, once loaded with a tRNA fragment, binds Xrn2 to facilitate Xrn2 cellular accumulation and nuclear import through formation of TXT.

Finally, we tested whether Twi12 is required for Xrn2 catalytic activity. To obtain Xrn2 without other TXT subunits, we exploited cells overexpressing Xrn2-ZZ in the Twi12 iKD background (Figures 7A and 7B) with Xrn2-ZZ expression induced in the presence of the proteasome inhibitor MG132. ZZ-Xrn2 IP was performed by binding to IgG agarose, using extract from cells overexpressing Xrn2-ZZ or cells lacking tagged protein (mock). Subsequently, IgG-purified Xrn2 was depleted of F-Twi12 using FLAG antibody resin (Figure 7E, lane 3). In parallel, as a control, IgG-purified mock and Xrn2 samples were mock-depleted at the FLAG antibody resin step to control for incubation time as well as nonspecifically enriched activities and/or inhibitors (Figure 7E, lanes 1 and 2). All of these depleted samples, the eluate from the FLAG antibody resin containing enriched TXT complex (Figure 7E, lane 4), and F-Twi12 from ZZF-Twi12-overexpressing cells (Figure 7E, lane 5) were tested for exonuclease activity. Strikingly, Xrn2 lacking associated Twi12 had no detectable exonuclease activity (Figure 7E, lane 3). TXT on the other hand selectively degraded RNA with a 5’ monophosphate (Figure 7E, lane 4) and as expected, F-Twi12 without Xrn2 had no nuclease activity (Figure 7E, lane 5). These findings strongly suggest that Tetrahymena Xrn2 functions only in association with Twi12 RNP.

DISCUSSION

TXT assembly and nuclear import

Analogous to the RNA loading of many Ago/Piwi proteins, we suggest that Twi12 initially binds a double-stranded RNA structure formed by the mature tRNA acceptor and TΨC stems (Couvillion et al., 2010). RNP maturation would then involve tRNA nicking and passenger-strand degradation and/or unwinding activities. The nuclease(s) responsible for trimming Twi12-bound tRNAs could be broad rather than tRNA-specific in their substrate cleavage specificity, as shown for the Tetrahymena RNase T2 enzymes involved in starvation-induced tRNA cleavages (Andersen and Collins, 2012). An N-terminally truncated form of Twi12 (Twi12S) shows increased retention of tRNA 5' fragments under native purification conditions (Couvillion et al., 2010; Figure 1B). These 5' fragments likely represent bona fide loading intermediates, because the Argonaute N-terminal domain has been established to be important for unwinding the sRNA duplex during loading (Kwak and Tomari, 2012). Twi12-bound sRNAs are scarce compared to mature tRNAs, so it seems likely that only a small fraction of the mature tRNA pool is degraded by a mechanism involving Twi12. The evolutionary specialization of Twi12 loading with tRNAs could in part reflect the expedience of tRNA as an available base-paired cytoplasmic RNA, rather than a biologically selected role for Twi12 in tRNA regulation. Or, if tRNA availability for Twi12 loading changes with cellular conditions, Twi12 specialization for tRNA binding could be part of a physiological regulation of ribosome biogenesis.

Two lines of evidence support the hypothesis that Twi12 nuclear import depends on sRNA binding and maturation. First, Twi12 defective for sRNA binding is not imported (Figures 5B and S4C). Second, Twi12S, which is impaired in duplex unwinding, remains predominantly cytoplasmic (Figures 5B and S4C). Nuclear import dependent on RNA binding has been reported for the Ago/Piwi proteins Tetrahymena Twi1, C. elegans NRDE-3, mouse Miwi2, Drosophila Piwi, and Arabidopsis AGO4 (Ishizu et al., 2011; Ye et al., 2012). Also, for Twi1 and AGO4, unwinding of the bound sRNA duplex is an established prerequisite for nuclear import (Noto et al., 2010; Ye et al., 2012). It seems likely to be a general principle that sRNA loading and RNP maturation provide a checkpoint for import of nuclear-localized Ago/Piwi proteins.

The TXT complex with Twi12 and Xrn2 also contains Tan1 (Figure 3). Tan1 is not necessary for Twi12 nuclear accumulation (data not shown), suggesting that Tan1 functions downstream of sRNA loading. Although Tan1 is part of the functional TXT RNP (Figure 4B), unlike Twi12 and Xrn2 it is not a genetically essential subunit (Figure S2A). Furthermore, Tan1 KO does not impose an obvious slow-growth phenotype (data not shown). Tan1 was not critical for Xrn2 catalytic activity in vitro (Figure S3), but it is possible that Tan1 stimulates Xrn2 degradation of a subset of TXT substrates whose altered accumulation does not confer a cellular disadvantage under typical laboratory growth conditions.

TXT function

Our results suggest that Twi12 functions as an essential activator of Xrn2. All of the depletion phenotypes of Twi12 and Xrn2 are consistent with known roles for Xrn2 in pre-rRNA processing or co-transcriptional RNA degradation. Although the role of an Ago/Piwi protein in Xrn2 activation was unanticipated, it could be general. Indeed, Ago/Piwi proteins have been implicated in pre-rRNA processing in human cells (Liang and Crooke, 2011) and in transcriptional or co-transcriptional silencing of protein-coding genes in C. elegans, S. pombe, and Drosophila (Cernilogar et al., 2011; Guang et al., 2010; Moshkovich et al., 2011; Woolcock et al., 2012). These mechanisms of silencing could depend on direct RNAP II interaction and/or co-transcriptional RNA degradation.

Xrn2 homologs have diverse functions. For this reason we initially expected Tetrahymena Xrn2 to interact with protein partners in addition to Twi12 and Tan1 and for its role in TXT to involve the biogenesis of precisely processed Twi12-bound tRNA fragments. On the contrary, IP of Xrn2 did not co-purify any detectable proteins other than Twi12 and Tan1 (Figures 3C and S2C). Furthermore, Xrn2 catalytic activity was not required for the associated Twi12 RNA loading (Figure 4B), although subunit exchange among assembled TXT complexes would complicate the interpretation of this result. Combined with our inability to IP Xrn2 free of Twi12 without proteasome inhibition and with the Twi12 requirement for Xrn2 catalytic activity (Figures 7C and 7E), we suggest that Tetrahymena Xrn2 may not have functions beyond those mediated by TXT.

Twi12-dependent accumulation, localization, and activity of Xrn2

In budding and fission yeasts, the Xrn2 ortholog Rat1 gains conformational stability and improved activity through direct interaction with its cofactor Rai1 (Xiang et al., 2009; Xue et al., 2000). The amino acid side chains that contribute to the Rai1-Rat1 interface are conserved across fungal species but not all eukaryotes, consistent with a lack of human Xrn2 protein-protein interaction with the proposed human Rai1 ortholog Dom3Z (Xiang et al., 2009). Structural alignment of Tetrahymena Xrn2 with S. pombe Rat1 suggests that Tetrahymena Xrn2 also does not conserve the yeast Rat1 surface of Rai1 interaction (data not shown). Sequence alignment further revealed that the predicted nuclear localization signal of S. pombe Rat1 (TKKTK) (UniProt, 2012) is not conserved in Tetrahymena Xrn2. The lack of a nuclear localization signal could underlie the dependence of Xrn2 nuclear import on assembly with Twi12 RNP as TXT.

A major question is whether the Twi12-bound tRNA fragment can guide TXT to a target RNA substrate, as is the paradigm for Ago/Piwi RNPs. Twi12-bound sRNAs derive almost exclusively from a precise region of the sense strand of mature tRNAs that contains a bulky m1A base modification (Figure 2C), which would disrupt the hydrogen-bonding surface of the seed sequence for target RNA recognition. If this base modification is sufficient to preclude sRNA pairing to any target RNA, sRNA binding by Twi12 may be required only to favor the Twi12 conformation that binds, stabilizes, imports to the nucleus, and catalytically activates Xrn2. Alternatively, in addition to those roles, Twi12 RNP could influence the affinity and/or specificity of cellular RNA selection as a substrate for Xrn2. Weak base-pairing of sRNA and target RNA could be optimal in order to balance target recruitment and its subsequent release to allow complete degradation.

In tRNA tertiary structure, the acceptor and T stems stack end-on-end to form a short duplex with the 3' end of the tRNA as a single-stranded overhang, mimicking the features of a Dicer product. Notably, in S. pombe dcr1Δ cells, tRNA fragment representation in an Ago1-bound sRNA library is increased 5-fold, more so than rRNA (less than 2-fold) or mRNA (2-fold) (Halic and Moazed, 2010). This could be explained by tRNAs becoming the preferred substrate for Ago1 loading in the absence of Dicer products. Interestingly, deep sequencing reads matching tRNA fragments are found in most Ago/Piwi-bound sRNA libraries but are often filtered out before analysis. We suggest that there may be an evolutionarily broad Ago/Piwi-protein binding capacity for sRNAs derived from mature tRNAs. Beyond this potentially general Ago/Piwi loading with tRNA-derived sRNAs, it remains to be explored whether other Ago/Piwi proteins share the newly discovered Twi12 function of stimulating RNA processing in the nucleus.

EXPERIMENTAL PROCEDURES

Purifications

Affinity purifications from cell extract were performed largely as described (Couvillion and Collins, 2012). Briefly, lysate was cleared at 16,000 × g for 15 min. Binding to rabbit IgG agarose (Sigma) or mouse anti-FLAG M2 resin (Sigma) was in 20 mM Tris-HCl pH 7.5, 0.1 M NaCl, 10% glycerol, 0.2% Igepal, 0.1% Triton X-100, 1 mM MgCl2, and protease inhibitors. Washes were in binding buffer with 0.1% Igepal, 0.1% Tween-20, and no Triton X-100. Complexes were eluted with TEV protease or 150 ng/µl triple FLAG peptide. To obtain Xrn2 depleted of Twi12, Xrn2-ZZ expression was induced by the addition of 250 µM CuSO4 for the last 3 hours of culture growth. The proteasome inhibitor MG132 was added to cultures at a final concentration of 13 µM for the last 1 hour of culture growth, and 5 µM MG132 was added to cell lysate. For FLAG antibody depletion of IgG-purified samples, the TEV protease eluate was diluted 2-fold in binding buffer with 0.2 M NaCl and incubated with FLAG antibody resin for 30 min at room temperature. Bound complexes were eluted with triple FLAG peptide.

Activity assays

For most exonuclease assays, complexes purified from Tetrahymena were incubated with ~1 µg Tetrahymena total low molecular weight RNA (Lee and Collins, 2006) in 10 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM MgCl2 at 30°C for 60 min. For the assay in Figure 7E, complexes or proteins in limiting amounts were incubated with 25 ng each gel-purified 5.8S rRNA and 5S rRNA in 20 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM MgCl2, 0.5 mM DTT at 30°C for 10 min. Time courses were also performed to verify enzyme turnover (data not shown).

Supplementary Material

01

HIGHLIGHTS.

  • Twi12, an essential Piwi protein, forms a stable nuclear complex with the exonuclease Xrn2

  • Twi12 is required for Xrn2 cellular accumulation, localization, and activity

  • Twi12 nuclear localization requires tRNA-derived small RNA binding

  • The Twi12-Xrn2 complex mediates ribosomal RNA processing

ACKNOWLEDGMENTS

This work was supported by NIH grant GM54198 (K.C.) and a predoctoral fellowship from the Genentech Foundation (M.T.C.). We thank members of the Collins lab for helpful discussion and Emily Egan for critical reading of the manuscript.

Footnotes

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ACCESSION NUMBERS

GenBank accession numbers and Tetrahymena Genome Database annotations are: TWI12: EF507507, TTHERM_00653810; XRN2: XM_001011167.1, TTHERM_00145270; TAN1: XM_001014010.3, TTHERM_00399370. The sRNA sequencing library is deposited in the Gene Expression Omnibus with the accession number GSE38507, as data set GSM943743.

SUPPLEMENTAL INFORMATION

Supplemental Information includes five Supplemental Figures, Supplemental Experimental Procedures, and Supplemental References.

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