Pearls are novel Cajal body-like structures in the Xenopus germinal vesicle that are dependent on RNA pol III transcription
We have identified novel nuclear bodies, which we call pearls, in the giant oocyte nuclei of Xenopus laevis and Xenopus tropicalis. Pearls are attached to the lampbrush chromosomes at specific loci that are transcribed by RNA polymerase III, and they disappear after inhibition of polymerase III activity. Pearls are enriched for small Cajal body-specific RNAs (scaRNAs), which are guide RNAs that modify specific nucleotides on splicing snRNAs. Surprisingly, snRNAs themselves are not present in pearls, suggesting that pearls are not functionally equivalent to Cajal bodies in other systems, which contain both snRNAs and scaRNAs. We suggest that pearls may function in the processing of RNA polymerase III transcripts, such as tRNA, 5S rRNA, and other short non-coding RNAs.
KeywordsCajal bodies histone locus bodies Pearls RNA polymerase III scaRNAs
Cajal body box
Fluorescent in situ hybridization
Fluorescence recovery after photobleaching
Green fluorescent protein
Histone locus body
Oak Ridge 2
Polymerase chain reaction
Small CB-specific RNA
Small nuclear RNA
Because of its large size and wealth of structural detail, the amphibian oocyte nucleus or germinal vesicle (GV) has long been a favorite object for both cytological and molecular studies. In addition to the lampbrush chromosomes (LBCs), the GV contains hundreds to thousands of extrachromosomal bodies that can be classified into a relatively few types based on morphology, molecular composition, and association with specific loci on the LBCs. Three of these—nucleoli, histone locus bodies (HLBs), and speckles—constitute the vast majority of extrachromosomal bodies in Xenopus, and each represents a different relationship between the body and the locus or loci with which they are associated. We will not discuss the speckles here because they have been studied the least (Gall et al. 2004).
HLBs are superficially similar to nucleoli. Like the nucleoli, they occur free in the nucleoplasm and attached to specific loci on the chromosomes, in this case the histone gene loci (Gall et al. 1981; Callan et al. 1991). However, HLBs do not contain the genes coding for histones nor the transcripts derived from them, and in this respect they differ fundamentally from nucleoli (Stephenson et al. 1981; Gall et al. 1983; Fig. 1b). There has been much confusion about the terminology of HLBs, which over the years have been called spheres (Callan and Lloyd 1960), coiled bodies (Gall et al. 1995), Cajal bodies (Gall et al. 1999), and finally HLBs (Nizami et al. 2010). Our lab must take major blame for this confusion, the excuse being that increased knowledge has forced us to distinguish what were thought at first to be the same or similar bodies. The culprit is the protein coilin. Coilin is a protein of unknown function, originally described from the nuclear coiled bodies of mammalian cells (Andrade et al. 1991). Coiled bodies were later renamed Cajal bodies (CBs), and coilin became accepted as a specific marker for CBs. A problem arose when we identified Drosophila coilin and showed that it occurred in two distinctly different bodies (Liu et al. 2009). One of these bodies, the Drosophila CB, shares molecular markers found in CBs of other organisms, particularly snRNAs and the guide RNAs that modify them. These guide RNAs were themselves named small Cajal body-specific (sca) RNAs because of their co-localization with coilin in the CBs of mammalian cells (Darzacq et al. 2002; Richard et al. 2003). The second coilin-positive body in Drosophila cells is the HLB, which we named from its invariant association with the histone gene locus. In addition to coilin, the Drosophila HLB contains the U7 snRNP and other factors involved in histone pre-mRNA processing (Liu et al. 2006; Godfrey et al. 2009; White et al. 2011).
From our studies on Drosophila, it became clear that the coilin-positive bodies we had called CBs in the amphibian GV were, in fact, HLBs (Nizami et al. 2010). Indeed, their association with the histone loci had been recognized decades earlier (Gall et al. 1981; Callan et al. 1991). With the major coilin-positive bodies now recognized as HLBs, the question arises whether there are CBs at all in the amphibian GV. Here, we describe novel coilin-positive bodies from the GV of X. laevis and X. tropicalis, which we call pearls. In addition to coilin, pearls contain scaRNAs and WDR79, a protein known to associate with scaRNAs in other cell types (Tycowski et al. 2009; Venteicher et al. 2009), at first suggesting that pearls are the “real” CBs in the GV. However, unlike CBs in other cell types, pearls do not contain splicing snRNAs. Moreover, they are specifically associated with RNA polymerase (pol) III loci on the chromosomes (Fig. 1c), a feature not shared with CBs in other organisms. For these reasons, we refer to pearls as “CB-like” organelles. Here, we describe the molecular composition of pearls, their association with the LBCs, their dependence on polymerase III (pol III) transcription, and their dynamic behavior.
Materials and methods
Oocytes, oocyte injections, and GV spreads
Pieces of ovary were surgically removed from X. tropicalis and X. laevis adult females and kept in OR2 medium (Wallace et al. 1973) at room temperature (~22 °C). Oocytes remain in usable condition for up to 1 week. For injection of RNA and DNA constructs, precise volumes were injected into oocytes using the Nanoject semi-micro injector (Drummond). GV spread preparations were made from mature oocytes (~0.8 mm diameter in X. tropicalis and ~1.2 mm in X. laevis) as described previously (Gall and Wu 2010). Spreads were fixed in 2–4 % paraformaldehyde for 20 min–1 h before being immunostained for 1 h each in primary and secondary antibodies.
Whole-mount immunostaining and FISH
Immunostaining and fluorescent in situ hybridization (FISH) for Xenopus whole-mount tissues were performed as described (Liu et al. 2009). Briefly, a whole ovary was removed from 3–5 cm X. tropicalis or X. laevis froglets and fixed in 4 % paraformaldehyde in OR2 for 10 min. Tissue was stored long-term in phosphate-buffered saline (PBS) at 4 °C. Antibody staining was performed overnight at room temperature in 10 % horse serum. FISH was carried out in in situ mix at 42 °C for 4–18 h. In situ mix consists of 50 % formamide, 5X SSC, 50 μg/mL heparin, 500 μg/mL yeast tRNA, 9 mM citric acid (pH 6), and 0.1 % Tween-20.
Primary antibodies were as follows: rabbit polyclonal serum C236 against X. laevis coilin (from Zheng’an Wu) used at 1:500–1:1,000 on GV spreads; mouse mAb H1 against X. laevis coilin (Tuma et al. 1993) used at 1:200 on small pieces of whole ovary; mouse mAb against human symplekin (BD Transduction Laboratories) used at 1:1,000; affinity-purified rabbit polyclonal serum against Drosophila melanogaster FLASH used at 1:1,000 (Yang et al. 2009), a gift from Z. Dominski; mouse mAb 72B9 against fibrillarin (Reimer et al. 1987) used at 1:5–1:10; rat mAb 3F10 against the hemagglutinin (HA) tag (Roche) used at 1 μg/mL; rabbit polyclonal serum against RPB6 used at 1:5,000–1:10,000, a gift from Robert Roeder; mouse mAb Y12 against sDMA used at 1:25; and mouse mAb K121 against the trimethylguanosine (TMG) cap on snRNAs (Oncogene Science) used 1 μg/mL. Secondary antibodies were goat or donkey anti-rabbit, anti-mouse, or anti-rat labeled with Alexa 488 or Alexa 594 (Invitrogen).
Clones and in vitro-transcribed RNA
Clones used for making antisense in situ hybridization probes were X. laevis U3 snoRNA in pBluescript (from Rocco Savino) and X. laevis U85 scaRNA in pCRII (cloned by Christine Murphy from genomic DNA). Clones for making sense transcripts for injection were X. laevis U92 scaRNA (pugU2-34/44) in pGEM3Z (Zhao et al. 2002); human mgU2-25/61 scaRNA (Tycowski et al. 2004); GFP X. laevis coilin in pCS2 (Handwerger et al. 2002); X. laevis U7 in pBS (Wu et al. 1996); D. melanogaster mgU2-28 scaRNA wild type; and ΔCAB mutant in pGEM-T (Svetlana Deryusheva). DNA encoding X. laevis telomerase RNA was cloned from genomic DNA into the pGEM-T Easy vector following a protocol modified from Chen et al. (2000) using oligos ZN27 (AAT CAG CGT TTA AAG CTC AAT GTG G, which contains T3 RNA polymerase promoter) and ZN28 (ACA TGT CGG GGA CTG GCT GA). The cloned sequence was validated against the NCBI entry for X. laevis telomerase RNA, NR 003556. X. tropicalis WDR79-HA was cloned from Xenopus ovary RNA into the pGEM-T Easy vector. The oligo pair used for cloning included a Kozak sequence in the forward oligo (ZN51: GCC GCC ACC ATG TTG GGC AGA GAG GAG GAA GA) and an HA tag sequence in the reverse oligo (ZN52: TTT ATT TTT CTA AGC GTA ATC TGG CAC ATC ATA TGG GTA CTC ACG TGC CGC CCC CCC). In vitro-transcribed RNA was generated using standard protocols (Gall et al. 1999). In brief, ~1 μg plasmid DNA or 100 ng PCR product was used as the template in a 20-μL reaction (1× Stratagene transcription buffer, 1–2 mM NTPs, 40 U of RNase inhibitor, 50 U of Strategene T3/T7/SP6 RNA polymerase). For mRNA, 2.5 mM 7-methyl guanosine cap was included in the reaction (NEB). Non-coding RNAs (scaRNAs, snRNAs, and snoRNAs) were directly labeled by including 250 μM fluorescein-UTP (Boehringer Mannheim), Cy3-CTP, Cy5-UTP, or Cy5-CTP (GE Healthcare). Template DNA was subsequently digested using DNase I (Roche), which was then heat-inactivated and chelated with 25 mM EDTA. RNA was purified using Illustra G-50 microspin purification columns (GE Healthcare). RNAs used for injection/targeting experiments were transcribed from templates that included a T3 or T7 promoter immediately upstream of the 5′ end of the molecule, which minimized the presence of non-genomic sequence elements.
Molecular beacons (MBs) for X. laevis U85 scaRNA were generously provided by Salvatore Marras and Sanjay Tyagi (Mhlanga and Tyagi 2006). The probe sequences were as follows: MB Xenopus large 5′ TMR-CCGU CCCUGCCUGGUUUUCCAAU ACGG-BHQ-2 3′; MB Xenopus small no. 1 5′ TMR-CAGC CCUGCCUGGUUUUC GCUG-BHQ-2 3′; MB Xenopus small no. 2: 5′ TMR-CGAA CCCUGCCUGGUU UUCG-BHQ-2 3′; MB scrambled control M8.2 5′ TMR-CTTC GTCCACAAACACAACTCCT GAAG–BHQ-2 3′. The underlined sequences are the complementary arm sequences of the MB probes; the non-underlined regions hybridize to target sequences leading to the unquenching of the fluorophore (TMR is tetramethylrhodamine and BHQ-2 is Black Hole Quencher 2). MBs were injected into mature X. laevis oocytes either into the cytoplasm (23 nL at 50 ng/μL) or into the GV (4.6 nL at 5 ng/μL). Cytoplasmic injection worked as efficiently as GV injection and was used for most experiments.
Extracts of GV proteins were prepared by first isolating intact GVs in cold isolation medium (83 mM KCl, 17 mM NaCl, 6.5 mM Na2HPO4, 3.5 mM KH2PO4, 1 mM MgCl2, 1 mM dithiothreitol). GVs were then dissolved in standard protein sample buffer (0.35 M Tris, pH 6.8, 30 % glycerol, 10 % sodium dodecyl sulfate, 0.6 M dithiothreitol, 0.012 % bromophenol blue). Samples were boiled before loading onto a 12 % polyacrylamide resolving gel. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane and HA-tagged proteins were detected with rat mAb 3F10 against the HA tag (Roche) used at 50 ng/mL, followed by horseradish peroxidase-labeled antibody against rat IgG (Jackson) used at 1:40,000 for 1 h. Chemiluminescence was generated by an ECL reagent (Pierce SuperSignal ECL) and the blot exposed to film for 20 s–5 min. “Rainbow” marker (GE Healthcare RPN 800E) was used for molecular weight calibration.
Fluorescence recovery after photobleaching
Mature X. tropicalis oocytes were injected with 9.2 nL of GFP-labeled X.l. coilin mRNA (~50 pg). Two days later, individual GVs were isolated in oil just prior to each fluorescence recovery after photobleaching (FRAP) experiment. FRAP was performed on a single pearl, followed by FRAP on an HLB from the same GV. FRAP was conducted on a Leica SP5 laser scanning confocal microscope using the FRAP Wizard program. The protocol was as follows. Scanning was conducted at 400 Hz with a line average of 2 with a ×63 oil objective at optical zoom 4. The argon laser power was set to 30 % and the 488-nm laser intensity to 20 %. The scanning order was: pre-bleach; bleach (the region of interest (ROI) was a 10-μm circle around the body, scanned five times at higher laser intensity); post-bleach 1 (two scans at the fastest possible setting, 2.62 s/frame); post-bleach 2 (four scans at 15 s/frame); and post-bleach 3 (30 scans at 60 s/frame). Images were exported as TIFF files and processed with ImageJ software (Rasband W.S., http://imagej.nih.gov/ij/). The mean fluorescence intensity of each frame was normalized by comparing two ROIs of the same size, one with a body and one with background only. Graphs were generated in Microsoft Excel.
For inhibition of RNA pol III, oocytes were injected with either Tagetin (Epicentre) or α-amanitin (Sigma). Tagetin was injected into 0.8-mm oocytes to a final concentration of 5–50 μM (23 nL of 60–600 μM Tagetin). This corresponds to the concentration range ~ 3–10 μM that specifically inhibits RNA pol III but not RNA pol II activity on reporter constructs in X. laevis oocytes (Steinberg et al. 1990). We could monitor the appropriate concentration by visual inspection of LBC loop morphology—the vast majority of loops are transcribed by RNA pol II and they contract or disappear entirely when RNA pol II is inhibited. To inhibit both RNA pol II and RNA pol III withα-amanitin, we injected 4.6 ng of drug into each oocyte (23 nL of 200 μg/mL α-amanitin). This is 1,000× more than is needed to inhibit RNA pol II (Steinberg et al. 1990). GVs were isolated from drug-treated oocytes 1–2 days after injection.
Pearls are novel coilin-positive structures in the Xenopus germinal vesicle
Pearls are consistently present in early diplotene stage Xenopus oocytes
Pearls contain U85 scaRNA and U3 snoRNA
The key molecular feature that distinguishes scaRNAs from snoRNAs is the presence of a short motif (CAB box) on scaRNAs (Richard et al. 2003), which is recognized by the CB-targeting protein, WDR79 (Tycowski et al. 2009; Venteicher et al. 2009). It is generally believed that snoRNAs are absent from CBs because they lack the CAB box. Instead, snoRNAs concentrate in nucleoli, with the well-established exception that U3 snoRNA is present in both nucleoli and CBs in mammalian cells (Verheggen et al. 2002). We therefore used a FISH probe for U3 on whole immature oocytes of X. laevis. We observed that U3 is enriched in both nucleoli and pearls (Fig. 4d–f). Both U3 snoRNA and U85 scaRNA occur in the core region of pearls and are excluded from the shell-like surface (Fig. 4).
To exclude the possibility that the nucleolar localization of the injected scaRNAs is an artifact of overloading, we examined the endogenous localization of X. laevis U85 scaRNA by injecting a “molecular beacon” probe into mature X. laevis oocytes. Molecular beacons are labeled hairpin constructs that fluoresce only when they hybridize to their target nucleic acid (Mhlanga and Tyagi 2006). Nucleoli of mature oocytes were labeled by the U85 molecular beacon, confirming the presence of this scaRNA (ESM Fig. 2h), as we had observed for immature oocytes by in situ hybridization (Fig. 4a–c).
The CAB motif is dispensable for scaRNA targeting to pearls and nucleoli
To determine a possible role of the CAB box in targeting scaRNAs to pearls and nucleoli in the amphibian oocyte, we prepared a Drosophila mgU2-28 construct that lacks the CAB box. We then injected labeled RNA transcribed from this construct into X. tropicalis oocytes. We found that there was no change in the targeting pattern for the CAB deletion mutant, which concentrated in pearls (Fig. 5g–i) and nucleoli (not shown) like the original construct. We conclude that scaRNAs in the Xenopus GV occur in both pearls and nucleoli and that their localization is not dependent on the CAB motif.
The CAB-binding protein WDR79 targets to pearls and HLBs
Pearls lack splicing snRNPs
Live dynamics of coilin in pearls
We also observed some fusion events of pearls to pearls and HLBs to HLBs (ESM Fig. 5). Fusion events have been reported previously for several organelles that lack investing membranes, such as CBs, nucleoli, and P granules (Boudonck et al. 1999; Platani et al. 2000; Brangwynne et al. 2009, 2011). Interestingly, fusing pearls could be found in physical contact with an HLB. This indicates that unlike bodies can be physically associated without fusing, but when like bodies touch, a fusion event can follow. We conclude that pearls are dynamic steady-state structures that exchange components with the nucleoplasm and can fuse with other pearls.
Pearls map to specific gene loci on X. tropicalis lampbrush chromosomes
To identify a potential function for pearls based on association with specific gene loci, we mapped the attachment sites of pearls on LBCs of X. tropicalis. We found that pearls are attached to specific loci on LBC 3, LBC 4, and LBC 10 (ESM Fig. 6). We did not observe extrachromosomal pearls. Although pearls occur at specific and reproducible positions on LBCs, they are not invariably present at these loci in mature GVs (ESM Fig. 7a). Furthermore, the frequency of attachment differs among the different LBCs. Pearls are attached almost invariably at a specific locus on LBC 10 (95 % of GVs), but occur less frequently and with greater variability at two prominent loci on LBCs 3 and 4 (~ 60 % of GVs, with a large standard deviation). There are additional unmapped loci in frogs that have pearls on LBCs 3 and 4 (e.g., frogs 5 and 6; ESM Fig. 7b).
Pearls are dependent on RNA polymerase III transcription
To test whether pearl maintenance is dependent on ongoing pol III transcription, we injected mature X. tropicalis oocytes with two commonly used inhibitors of pol III activity: tagetitoxin (commercially available as Tagetin) and α-amanitin. Tagetin specifically inhibits pol III transcription in various eukaroyotes, including X. laevis oocytes (Steinberg et al. 1990). Unlike Tagetin, α-amanitin does not specifically inhibit pol III. Instead, it inhibits pol II at a low concentration (~1 μg/mL) and both pol II and pol III at high concentrations (~100–200 μg/mL). We injected X. tropicalis oocytes with water (mock injection), Tagetin, or α-amanitin. One day later, we isolated individual GVs and scored for the presence or absence of pearls, counting only those GV spreads in which all ten chromosomes could be identified. For these experiments, we used frogs that displayed a high frequency of pearls before injection. Eighty-five percent of mock-injected oocytes had pearls at one or more of the pearl loci, whereas none of the oocytes injected with Tagetin or α-amanitin had pearls (Fig. 9e). The appearance of the LBCs after drug treatment confirmed the different specificities of the two drugs. Tagetin-injected oocytes had normal-sized pol II loops that stained with the pre-mRNA processing component symplekin (Fig. 9c, c′). In contrast, oocytes injected with α-amanitin had contracted LBCs that were stripped of loops, a hallmark of pol II inhibition (Fig. 9d, d′). We conclude that pearls are dependent on ongoing RNA polymerase III transcription.
Pearls are novel nuclear bodies in Xenopus GVs at RNA polymerase III loci
We have identified pearls as a novel class of nuclear bodies in the Xenopus GV. They are attached specifically to loci that are transcribed by pol III, and they disappear after inhibition of pol III activity. We suggest that pearls may function in processing pol III transcripts, which include 5S ribosomal RNA, tRNAs, 7SK RNA, 7SL RNA, and U6 snRNA (Paule and White 2000). Because the X. tropicalis genome has been sequenced and genetically mapped to ten chromosomes (Kochan et al. 2003; Khokha et al. 2009; Hellsten et al. 2010; Wells et al. 2011), it should eventually be possible to use in situ hybridization to identify the genetic loci at which pearls occur.
Pol III activity has been previously linked to the perinucleolar compartment (PNC) in mammalian cell lines (Matera et al. 1995; Wang et al. 2003; Pollock and Huang 2010). It will be instructive to compare pearls and the PNC since much is known about the composition and dynamics of the PNC. There is also an intriguing correlation between PNC prevalence and malignant tumors (Norton et al. 2008). There are, however, differences between the PNC and pearls: unlike the PNC, pearls are not known to contain pol III transcripts. Moreover, they contain scaRNAs and coilin, which are absent from the PNC.
Pearls are CB-like bodies
Comparison of pearls with other nuclear organelles
Component or property
Xenopus GV pearls
Xenopus GV nucleoli
RNA pol III loci
snRNA gene loci
Histone gene locus
scaRNA localization in pearls and nucleoli of the Xenopus GVs
The localization of scaRNAs in the Xenopus GV is unusual. Not only are they found in pearls but they also occur in nucleoli and can be targeted to nucleoli after exogenous injection. These results are inconsistent with the generally accepted model of scaRNA localization, according to which the CAB box of scaRNAs targets them specifically to CBs (Richard et al. 2003; Tycowski et al. 2009). Earlier observations on scaRNA localization in the GV are also puzzling. Telomerase RNA, a non-guide scaRNA, targeted to nucleoli in Xenopus GVs (Narayanan et al. 1999). However, no specific localization was found in Xenopus GVs for a guide scaRNA that modifies U2 snRNA (Zhao et al. 2002). Furthermore, there are data suggesting that splicing snRNAs can be post-transcriptionally modified in the nucleoli of Xenopus GVs (Ganot et al. 1999; Yu et al. 2001).
If the Xenopus GV does not, in fact, contain a functional CB, then the unusual distribution of scaRNAs can be partially explained by the lack of their usual target. Under these circumstances, scaRNAs might localize by default to the nucleoli due to the inherent nucleolar localization motifs present in box C/D and H/ACA RNAs. However, the lack of a canonical CB cannot explain all of the localization data. We suggest that the CAB box and its binding protein WDR79 are not the only factors that determine how scaRNAs reach their ultimate targets in the nucleus. In particular, we find that elimination of the CAB motif from scaRNAs has no effect on their localization in the GV. Furthermore, there is a discrepancy between the localization of scaRNAs in Xenopus GVs (pearls and nucleoli, but not HLBs) and the localization of WDR79, the protein that binds the CAB box (pearls and HLBs, but not nucleoli). Relevant to this issue, a recent study shows that human WDR79 binds to a CAB box on small Alu RNA transcripts. These Alu transcripts are structurally very similar to H/ACA scaRNAs, but they localize to the nucleoplasm, not to CBs (Jady et al. 2012). These results suggest that RNA complexes that contain WDR79 have novel functions unrelated to posttranscriptional modification of splicing snRNAs. In this light, we speculate that the co-localization of scaRNAs and WDR79 in pearls could reflect a role for scaRNAs in regulating pol III transcripts.
Although the molecular composition of pearls suggests a relationship to CBs in other cell types, and hence to snRNP biogenesis, we believe that the similarities between pearls and CBs may be somewhat misleading. Instead, we suggest that the association of pearls with pol III loci is the key to understanding their molecular function(s). In this respect, it is more useful to compare pearls with HLBs, not because of similarities in composition, which are minimal, but because of the relationship of the body to the locus at which it is attached (Fig. 1). HLBs were named from their association with the histone gene locus (Liu et al. 2006). After their initial identification in Drosophila, it became clear that similar bodies occur at the histone gene loci in other organisms. In particular, we realized that the bodies we had called CBs in the amphibian GV were, in fact, HLBs (Nizami et al. 2010). Terminology aside, the two most important features of HLBs in Drosophila and in the amphibian oocyte are the following. First, although attached to the chromosomes at the histone gene loci, they do not contain histone genes or histone transcripts (Stephenson et al. 1981; Gall et al. 1983). Second, they contain specific processing factors that convert the nascent transcripts into mature histone mRNA (Wu and Gall 1993; Bellini and Gall 1998; Abbott et al. 1999). We suggest that pearls may bear the same relationship to pol III loci as HLBs do to the histone gene loci. We have shown that antibodies against pol III stain loops at approximately 90 loci in X. laevis (Murphy et al. 2002) and about half that number in X. tropicalis (unpublished data). Although the pol III loops at a given locus may be intimately associated with a pearl (when present), the body of the pearl does not stain with the pol III antibody. Like HLBs, pearls are associated with specific loci, but probably do not contain transcripts from these loci. If we carry the analogy further, we may suppose that pearls contain the factors required for processing pol III transcripts.
These considerations point the way to further studies on pearls. A high priority will be to identify the specific loci at which they occur. This can be done in principle by bioinformatic analysis, once there is better correlation between the X. tropicalis genetic and mitotic chromosome maps (Kochan et al. 2003; Khokha et al. 2009; Wells et al. 2011), the LBC cytological maps (Penrad-Mobayed et al. 2009), and the annotated genome (Hellsten et al. 2010). Even without information for all pearl sites, some could be identified by in situ hybridization with specific probes for pol III transcripts (Callan et al. 1988). An equally high priority will be to identify additional components of pearls, particularly known pol III processing factors. If pearls do, indeed, contain such factors, then the analogy with HLBs would be complete: they would contain processing factors for transcripts from the gene loci to which they are attached.
We thank Svetlana Deryusheva (Carnegie Institution) for fluorescently labeled wild-type and mutant Drosophila mgU2-28 scaRNAs. The Xenopus U85 scaRNA molecular beacon was generously made for us by Salvatore Marras and Sanjay Tyagi (Public Health Research Institute, New Jersey). The Xenopus U92 scaRNA construct was a gift from Yi-Tao Yu (University of Rochester Medical Center). The human mgU2-25/61 scaRNA construct was a gift from Kazimierz Tycowski (Yale University). Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award no. R01 GM33397. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. JGG is American Cancer Society Professor of Developmental Genetics.
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