Targeting of U4/U6 small nuclear RNP assembly factor SART3/p110 to Cajal bodies
1 Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany]o, http://www.100md.com
2 Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143]o, http://www.100md.com
Abstract]o, http://www.100md.com
The spliceosomal small nuclear RNAs (snRNAs) are distributed throughout the nucleoplasm and concentrated in nuclear inclusions termed Cajal bodies (CBs). A role for CBs in the metabolism of snRNPs has been proposed but is not well understood. The SART3/p110 protein interacts transiently with the U6 and U4/U6 snRNPs and promotes the reassembly of U4/U6 snRNPs after splicing in vitro. Here we report that SART3/p110 is enriched in CBs but not in gems or residual CBs lacking coilin. The U6 snRNP Sm-like (LSm) proteins, also involved in U4/U6 snRNP assembly, were localized to CBs as well. The levels of SART3/p110 and LSm proteins in CBs were reduced upon treatment with the transcription inhibitor {alpha} -amanitin, suggesting that CB localization reflects active processes dependent on transcription/splicing. The NH2-terminal HAT domain of SART3/p110 was necessary and sufficient for specific protein targeting to CBs. Overexpression of truncation mutants containing the HAT domain had dominant negative effects on U6 snRNP localization to CBs, indicating that endogenous SART3/p110 plays a role in targeting the U6 snRNP to CBs. We propose that U4 and U6 snRNPs accumulate in CBs for the purpose of assembly into U4/U6 snRNPs by SART3/p110.
Key Words: RNA splicing; coiled (Cajal) body; U4/U6 small nuclear ribonucleoprotein; small nuclear RNA; nuclear proteinsu99, 百拇医药
Introductionu99, 百拇医药
The cell nucleus contains numerous, morphologically distinct domains and bodies. One of these, a 0.5–1.0-µm sphere named the Cajal body (CB), was discovered 100 yr ago by Santiago Ramón y Cajal. The molecular characterization of CBs has been facilitated by the discovery of the protein coilin, an unambiguous marker of CBs in Xenopus, mouse, and human cells . Coilin likely plays an important role in cell metabolism because coilin knockout mice exhibit reduced viability . Many factors involved in transcription, RNA processing, and cell cycle regulation are concentrated in CBs, but the function of CBs, with respect to these diverse processes, remains obscure. Although spliceosomal U1, U2, U4, U5, and U6 small nuclear RNAs (snRNAs) are enriched in CBs , CBs are not likely sites of splicing because they are not transcriptionally active and lack many essential non-snRNP splicing factors . snRNP association with CBs is transcription dependent, indicating that CBs play an active role in snRNP metabolism and may be the sites of snRNP assembly rather than storage of inactive snRNPs . What essential function accounts for the concentration of spliceosomal snRNPs in CBs?
The U1, U2, U4, and U5 snRNAs are synthesized by RNA polymerase II, capped at their 5' ends, and transported to the cytoplasm where they are bound by snRNP-specific Sm proteins (for reviews see ). These snRNAs are hypermethylated at their 5' ends, producing their characteristic 2,2,7 trimethylguanosine (TMG) caps and providing an important signal for snRNP import into the nucleus . Because snRNP-specific proteins appear to concentrate in CBs before accumulating in the nucleoplasm, a role for CBs in the maturation of snRNPs has been proposed . The recent identification of RNAs that guide base modification of snRNAs and localization of these guide RNAs to CBs suggest that snRNA base modification may take place in CBs . However, these results do not explain why the U6 snRNA, which is synthesized by RNA polymerase III, is not capped or exported to the cytoplasm, and undergoes base modification in the nucleolus , is present in CBs. The U6 snRNA is present in at least three distinct snRNPs, the U6 snRNP, the U4/U6 snRNP, and the U4/U6U5 tri-snRNP. Formation of all of these snRNP species must occur in the nucleus, but little is known about the subnuclear location of these processes.
After assembly, the U1, U2, and U4/U6U5 snRNPs perform essential functions in spliceosome formation and catalysis. During a process termed the spliceosomal cycle, each snRNP is thought to participate in subsequent rounds of splicing, which then requires the regeneration of snRNPs that have undergone rearrangement during splicing . In particular, the U4/U6 snRNP, which contains two snRNAs base paired with each other, unwinds during splicing as U6 establishes new base-pairing interactions with U2 and the pre-mRNA. Therefore, U4 and U6 must reanneal after splicing to regenerate functional U4/U6 snRNPs. In yeast, the essential protein Prp24 catalyzes this reaction . In the absence of Prp24p, splicing extracts are depleted of the U4/U6 snRNP, demonstrating the importance of snRNP recycling for continuing rounds of pre-mRNA splicing/, 百拇医药
Recently, the tumor rejection antigen SART3/p110 was identified as the human homologue of Prp24p and was shown to be required for U4/U6 snRNP recycling in vitro . SART3/p110 binds specifically and directly to the U6 snRNA and is detectable in the U6 and U4/U6 snRNPs . Unlike other U4, U5, and U6 snRNP–specific proteins, SART3/p110 is not detectable in the U4/U6U5 tri-snRNP , indicating that SART3/p110 dissociates from its U4 and U6 snRNP substrates once they are annealed. In addition to SART3/p110, the Sm-like (LSm) proteins LSm2–8, which assemble on the 3' end of the U6 snRNA as a stable heteromer and persist in the U4/U6 and U4/U6U5 snRNPs , have been implicated in U4/U6 snRNP assembly. The LSm proteins bind directly to Prp24 in yeast , providing a second mode of interaction with the U6 snRNP. In vitro, Prp24p anneals U4 and U6 snRNAs more efficiently in the context of snRNPs , making it likely that the combination of SART3/p110/Prp24p and LSm proteins enables efficient assembly of the U4/U6 snRNP in vivo. Because the role of SART3/p110 in U4/U6 recycling is uniquely transient, the subnuclear distribution of SART3/p110 has the potential to reveal the sites of U4/U6 snRNP assembly.
In this study, we report that SART3/p110 and the LSm proteins, LSm4 and LSm8, are localized in the cell nucleus and concentrated in CBs. We studied the association of SART3/p110 and snRNPs with CBs after transcription inhibition and run-on treatment and in the absence of the CB component coilin. SnRNP and SART3/p110 localizations in CBs were correlated in each of these experimental conditions. Mutant analysis revealed that the HAT domain of SART3/p110 represents the major determinant for specific targeting of SART3/p110 to CBs. Overexpression of mutants lacking the snRNP-binding COOH-terminal domains reduced the concentration of both endogenous SART3/p110 and LSm4 in CBs, suggesting that SART3/p110 is required for U6 snRNP targeting to CBs.70, 百拇医药
Results70, 百拇医药
By immunofluorescent labeling of HeLa cells, SART3/p110 was detected exclusively in the cell nucleus, where it was distributed throughout the nucleoplasm and specifically enriched in large bright dots. Double labeling of cells for SART3/p110 and the TMG cap ( A), a modification found at the 5' end of U1, U2, U4, and U5 snRNAs, revealed complete overlap between SART3/p110 and snRNPs in the large bright structures. These bodies were identified as CBs by double detection of SART3/p110 and coilin, a marker of CB. SART3 was detected in all observed CBs (n = 51 cells). The same results were obtained in cells transiently expressing SART3/p110 conjugated to EGFP (see below and ). Measurement of endogenous SART3/p110 fluorescent intensities in CBs revealed that the concentration of SART3/p110 in CBs is approximately three times higher then in the nucleoplasm (see below, ). We confirmed the SART3/p110 localization patterns in WI-38 primary human fibroblasts . Although the fibroblasts contain fewer CBs, SART3/p110 was concentrated in every CB observed. In contrast to the striking overlap between SART3/p110 and snRNPs in CBs, only partial colocalization of SART3/p110 and snRNPs was observed in the nucleoplasm .
fig.ommtted9#)%.}, http://www.100md.com
SART3/p110 is concentrated in CBs. (A and B) Immunolocalization of SART3/p110 in HeLa cells together with (A) TMG cap, a marker of snRNPs, or (B) with coilin, a marker of CBs. (C) SART3/p110 and coilin were immunolocalized in human primary fibroblasts WI-38. CBs are marked by arrowheads. Bar, 5 µm.9#)%.}, http://www.100md.com
fig.ommtted9#)%.}, http://www.100md.com
Effect of SART3/p110 mutant expression on concentration of the endogenous SART3/p110 and LSm4 in CBs9#)%.}, http://www.100md.com
The survival of motor neuron protein (SMN) is involved in the assembly of snRNPs in the cytoplasm. In addition, SMN and associated gemin proteins are detected in the cell nucleus in structures termed gems . Gems often overlap or are associated with CBs, depending on the cell type . The function of nuclear SMN is currently unknown, although roles in splicing and transcription have been hypothesized . Because a role for gems in snRNP recycling has been proposed, we tested whether SART3/p110 specifically localizes to gems in a HeLa cell line in which gems are often separate from CBs. Double staining of HeLa cells with anti-SART3/p110 and anti-SMN antibodies revealed that SART3/CBs were overlapping or adjacent to gems only 58% of the time (n = 55 cells). Like the HeLa cell line, WI-38 primary fibroblasts often contained gems independent from CBs. We did not detect any enrichment of SART3/p110 in gems in primary fibroblasts . Because 100% of CBs contain SART3/p110 (see above), we conclude from this result that gems, per se, are not sites of SART3/p110 accumulation.
fig.ommtted;3}v/, http://www.100md.com
SART3/p110 is not specifically localized in gems. SART3/p110 and SMN, enriched in gems, were immunolocalized in (A) HeLa cells and (B) human primary fibroblasts WI-38. Examples of CBs are marked by arrowheads, gems by arrows. Inserts are magnified two times, and green (left), red (middle), and merged (right) signals are shown. Bar, 10 µm.;3}v/, http://www.100md.com
To investigate the targeting of SART3/p110 to CBs, we analyzed the role of coilin in SART3/p110 recruitment to CBs. We compared the distribution of SART3/p110 in embryonic fibroblasts derived from a wild-type mouse versus a mouse in which both coilin alleles were disrupted. Wild-type mouse embryonic fibroblasts have snRNPs, SMN, fibrillarin, and Nopp140 localized in CBs . In embryonic fibroblasts derived from the coilin knockout mouse, fibrillarin and Nopp140 are accumulated in so-called "residual" CBs, which fail to recruit snRNPs and SMN; snRNPs are distributed throughout the nucleoplasm, and SMN is concentrated in separate gems . Similar to our previous results, we found that SART3/p110 was detected in the nucleoplasm and in CBs of coilin+/+ cells, labeled either by anti–TMG cap, anti-fibrillarin, or anti-SMN antibodies . In coilin-/- cells, SART3/p110 was distributed throughout the nucleoplasm with no detectable concentration in residual CBs or gems . Double staining of coilin-/- cells with anti–TMG cap and SART3/p110 antibodies revealed the distribution of snRNPs and SART3/p110 throughout the nucleoplasm and never in the highly concentrated sites detectable in wild-type cells (, compare E with F). Thus, coilin expression is required for the recruitment of both snRNPs and SART3/p110 to CBs.
fig.ommtted\#fppnt, http://www.100md.com
SART3/p110 recruitment to CBs requires coilin. Mouse embryonic fibroblasts derived from control (coilin+/+) or coilin knockout (coilin-/-) mice were stained for SART3/p110 together with (A and B) fibrillarin (Fib), (C and D) SMN, and (E and F) TMG cap. (A, C, and E) In control coilin+/+ cells, SART3/p110 concentrates in CBs labeled by fibrillarin, SMN, and TMG cap . (B, D, and F) In cells lacking functional coilin, SART3/p110 is distributed throughout the nucleoplasm and is not recruited to residual CBs labeled by fibrillarin (B) or gems labeled by SMN (D). No CBs were detected by TMG cap (F). Inserts are magnified two times, and red (left), green (middle), and merged (right) signals are shown. Bar, 5 µm.\#fppnt, http://www.100md.com
Splicing largely occurs at transcription sites (for review see ). To test the possibility that SART3/p110 is cotranscriptionally recruited to snRNPs, we labeled nascent RNA with BrUTP and visualized labeled RNA together with SART3/p110 . Nucleoplasmic SART3/p110 did not appreciably overlap with transcription sites. During the course of these experiments, we noticed that cells permeabilized under run-on conditions (see Material and methods) exhibited a dramatic loss of SART3/p110 from CBs (compare B with B). We have further investigated this phenomenon and found that snRNPs were also largely extracted from CBs during run-on transcription as revealed by TMG cap labeling. In contrast, run-on treatment did not affect the detection of coilin, fibrillarin, or SMN in CBs and/or gems . This suggests that snRNPs and SART3/p110 are only weakly associated with CBs, compared with the apparently more stable components, coilin, SMN, and fibrillarin.
fig.ommtted+2, http://www.100md.com
SART3/p110 is depleted from CBs after nuclear run-on. (A) Cells were permeabilized using run-on conditions (see Materials and methods), and newly synthesized RNA was labeled by BrUTP and detected together with SART3/p110. Little colocalization of SART3/p110 with labeled RNA was detected. (B) Cells after run-on treatment were labeled for SART3/p110 and coilin. (B, inset) Representative CB was magnified 3.5 times, and green (left), red (middle), and merged (right) signals are shown. (C) Control fixed cells (control) and cells after run-on (run-on) labeled with coilin together with TMG cap, fibrillarin (Fib), and SMN. For representative CBs, green (left), red (middle), and merged (right) signals are shown in the insets. Bars: (A and C) 5 µm; (B) 10 µm.+2, http://www.100md.com
The presence of U4 and U6 snRNAs and SART3/p110 in CBs indicates that CBs contain the substrates and at least one factor required for U4/U6 snRNP assembly. To determine whether the U6 snRNA–associated LSm proteins that promote U4/U6 annealing in vitro are also present in CBs, we analyzed the distribution of two members of the LSm complex. By immunofluorescence, LSm4 was detected both in the cytoplasm and the nucleus . The cytoplasmic localization may represent LSm complexes involved in mRNA degradation . In the nucleus, LSm4 was detected throughout the nucleoplasm and concentrated in CBs . Double staining of HeLa cells with anti-LSm4 and anti–TMG cap antibodies revealed complete overlap of both antigens in CBs but only partial overlap in the nucleoplasm . Similarly, LSm8 tagged with EYFP was mainly detected in the nucleoplasm and in CBs where it colocalized with LSm4 . Although LSm proteins participate in several distinct complexes, including the Xenopus laevis U8 small nucleolar RNP and a cytoplasmic mRNA degradation complex , it is likely that localization of LSm4 and LSm8 reflects the presence of the mature U6 snRNP in CBs.
fig.ommttedri-, http://www.100md.com
. U6 snRNP proteins LSm4 and LSm8 are enriched in CBs. HeLa cells stained for LSm4 and (A) coilin or (B) TMG cap. LSm4 is concentrated in CBs, where it colocalizes with TMG cap, whereas there is only partial overlap of LSm4 with TMG cap in the nucleoplasm. HeLa cells expressing LSm8–EYFP stained for (C) coilin or (D) Lsm4. LSm8 is diffusely distributed in the cell nucleus and concentrated in CBs. Examples of CBs are marked by arrowheads. For representative CBs, green (left), red (middle), and merged (right) signals are shown in the insets. Bar, 5 µm.ri-, http://www.100md.com
The observations that SART3/p110, snRNPs, and LSm proteins are concentrated in CBs and that snRNP and SART3/p110 association with CBs is correlated combine to suggest that SART3/p110 may accumulate in CBs for the purpose of U4/U6 snRNP assembly. However, an alternative hypothesis is that SART3/p110 is inactive and perhaps stored in CBs. To distinguish between these possibilities, we tested whether SART3/p110 concentration in CBs was affected by transcription activity in the cell. It was previously shown that depleting the cell of pre-mRNA by blocking RNA polymerase II activity leads to a reduction in overall snRNP concentration in CBs . If SART3/p110 is stored in the CB, one would expect SART3/p110 levels to remain constant or even increase upon transcription inhibition. If, in contrast, SART3/p110 in CBs is active in U4/U6 snRNP assembly, then SART3/p110 levels in CBs should decrease when transcription is inhibited.
To determine whether SART3/p110 concentration in CBs is dependent on RNA synthesis, cells were treated with the transcription inhibitor -amanitin. After treatment, all cells had rounded splicing factor compartments (SFCs) labeled with SC-35 , an indication of RNA polymerase II inhibition . Neither SART3/p110 nor LSm4 was enriched in SFCs. The latter result was unexpected, because other snRNPs (U1, U2, U4, and U5) have been shown to concentrate in SFCs after -amanitin treatment After -amanitin treatment, two types of coilin-positive CBs were observed: normal-looking CBs (indistinguishable from CBs in nontreated cells in size and morphology) and enlarged "ring-shaped" CBs, as reported previously . This change in CB morphology is not likely to reflect cell death because 95% of the cells were viable, according to a standard viability test (see Material and methods). In contrast to control untreated cells , we did not detect any specific enrichment of SART3/p110 or LSm4 in any of the coilin-labeled CBs . Quantitation of the fluorescence intensities within normal-looking CBs relative to the nucleoplasmic signal revealed that SART3/p110 levels in CBs decreased threefold (P < 0.0001) and LSm4 levels in CBs decreased twofold (P < 0.0001) after {alpha} -amanitin treatment. These results indicate that SART3/p110 and LSm4 association with CBs is transcription/splicing dependent and not likely due to the storage of inactive molecules.
fig.ommttedte, 百拇医药
SART3/p110 and LSm4 are depleted from CBs after transcription inhibition. (A and B) HeLa cells were treated with -amanitin and stained for a marker of splicing factor compartments (SC-35) and (A) SART3/p110 or (B) LSm4. Enlarged, rounded-up splicing factor compartments indicate that inhibition of RNA polymerase II transcription was effective. (C and D) HeLa cells after -amanitin treatment stained with anti-coilin antibody together with (C) anti-SART3/p110 or (D) anti-LSm4 antibodies. Two types of coilin-labeled structures were observed: normal-looking CBs (arrowheads) and enlarged ring-shaped CBs (arrows). Depicted areas containing CBs were enlarged two times (insets), and green (left), red (middle), and merged (right) signals are shown. Bars, 5 µm.te, 百拇医药
To determine how SART3/p110 is specifically localized to CBs, we investigated the potential of isolated SART3/p110 protein domains fused with EGFP to target CBs. Human SART3/p110 is composed of three major protein domains, a repeat of seven HAT (half a TPR domain) motifs followed by a nuclear localization signal, two RNA recognition motifs (RRMs), and the highly conserved COOH-terminal domain CT10 . The HAT domains and their related TPR domains are present in a number of other RNA processing factors and have been hypothesized to function in protein–protein interactions . Recently, the SART3/p110 HAT domain was shown to interact with the HIV-1 Tat protein . The RRMs have been implicated in U6 snRNA binding , and the CT10 domain likely mediates binding to the U6 snRNP via interaction with LSm proteins, as it does in yeast . In two-hybrid assays, full-length SART3/p110 interacted specifically with LSm7 but not with LSm1, which is not present in the U6 snRNP. Deletion of the CT10 domain reduced the two-hybrid interaction twofold, suggesting that the CT10 domain contributes to SART3/p110 binding to the U6 snRNP via LSm proteins (unpublished data). We created several EGFP-tagged proteins composed of specific SART3/p110 domains . Note that each fusion protein diagrammed (except N-TERM) was also produced with the EGFP tag at the NH2 terminus, and the results were identical to those obtained with the COOH-terminal–tagged constructs (unpublished data), indicating that the protein localization results described below are not due to the addition of the tags at the COOH termini.
fig.ommtted?q4p8e*, http://www.100md.com
The NH2-terminal domain containing the HAT motifs targets SART3/p110 to CBs. (A) Wild-type (WT) SART3/p110 was conjugated to EGFP. Deletion mutants containing amino acids 1–950 (CT10), 1–702 (CT10RRM), 581–963 (HAT), and 119–702 (HAT) were conjugated to EGFP. The expression construct (N-TERM) encodes the NH2-terminal amino acids 1–127 fused to EGFP and a heterologous NLS signal. (B) All constructs including empty vector containing heterologous NLS (EGFP–NLS) were transiently expressed in HeLa cells and stained for coilin. Full-length protein as well as mutants containing the HAT domain accumulated in CBs (arrowheads). The HAT protein and EGFP–NLS accumulated in nucleoli (arrows) and only weakly in CBs. The N-TERM mutant was detected throughout the nucleoplasm and slightly accumulated in nucleoli and CBs. The identification of nucleoli was verified by phase contrast microscopy (not depicted). Bar, 5 µm.?q4p8e*, http://www.100md.com
The full-length SART3/p110–EGFP (WT) revealed the same distribution as endogenous SART3/p110 ( B, WT; compare with ); it localized to the nucleoplasm and was concentrated in CBs, as judged by coilin staining. Deletion of the highly conserved COOH-terminal domain CT-10 (CT10) or both CT-10 and the two RNA recognition motifs (CT10RRM) resulted in similar localization patterns as full-length SART3/p110. In contrast, truncation of the NH2-terminal portion of the protein containing seven HAT repeats (HAT) had strong effects on protein localization. The HAT–EGFP protein was found in the nucleoplasm and concentrated in nucleoli, but was only weakly detectable in CBs. Because EGFP conjugated to an NLS (EGFP–NLS) was also concentrated in nucleoli and faintly detectable in CBs, the simplest interpretation of the data is that the concentration of HAT–EGFP in nucleoli and weak detectability in CBs is nonspecific. To further investigate the role of the NH2 terminus, we created a mutant containing only the HAT domain and the endogenous NLS (HAT). This construct was specifically targeted to CBs. In contrast, the extreme NH2-terminal region of SART3/p110 (N-TERM), lacking the HAT domain and conjugated to EGFP plus a heterologous NLS, was not specifically localized to CBs (compare N-TERM with EGFP–NLS localizations). Interestingly, the N-TERM mutant was not highly concentrated in nucleoli, in contrast to EGFP–NLS alone. Although the function of the extreme NH2-terminal region of SART3/p110 is currently unknown, these results suggest that it may interact with a nucleoplasmic component. We conclude from this analysis that the HAT domain of SART3/p110 is necessary and sufficient for the specific targeting of SART3/p110 to CBs.
The highly conserved CT10 domain was previously shown to interact with LSm proteins . To determine whether overexpression of SART3/p110 mutants lacking the COOH-terminal region affects the localization of endogenous SART3/p110 and/or LSm4, we transfected HeLa cells with WT–, HAT–, CT10–, and CT10RRM–EGFP mutants and determined the localization patterns of endogenous SART3/p110 and LSm4 by immunofluorescence (, WT–EGFP and CT10RRM–EGFP constructs shown only). Quantitation of fluorescence intensities within CBs relative to the nucleoplasm revealed that the localization of endogenous SART3/p110 was significantly reduced in CT10- and CT10RRM-expressing cells, compared with untransfected controls (. This indicates that both mutant proteins effectively compete for SART3/p110 binding sites within CBs. Moreover, if SART3/p110 plays a role in U6 snRNP localization to CBs, then expression of CT10 and/or CT10RRM mutants may have dominant negative effects on LSm4 localization in CBs. Indeed, LSm4 concentration in CBs was significantly reduced upon overexpression of CT10 or CT10RRM mutants by 30% . The expression of WT–EGFP also influenced the concentration of LSm4 in CBs, but the effect was less pronounced than in the case of CT10 or CT10RRM mutant We noticed that CBs were disrupted in some cells expressing high levels of CT10 or CT10RRM mutant (unpublished data). The HAT–EGFP mutant, which is aberrantly localized to nucleoli, did not show any effects on a localization of LSm4 (unpublished data). These data suggest that SART3/p110 plays a role in U6 snRNP targeting to CBs, largely through the CT10 domain of SART3/p110.
fig.ommtted*}hy[(, 百拇医药
Expression of CT10RRM–EGFP mutant reduces the concentration of endogenous SART3/p110 and LSm4 in CBs. HeLa cells were transiently transfected with (A) WT–EGFP or (B and C) CT10RRM–EGFP constructs and, after fixation, immunostained with (A and C) anti-LSm4 or (B) anti-SART3/p110 antibodies. Note that anti-SART3/p110 antibodies were raised against the COOH terminus of SART3/p110 and thus do not react with fusion proteins lacking the COOH-terminal part. Projections of five optical sections are shown. CBs in transfected cells are marked by arrowheads, in nontransfected cells by arrows. Examples of cytoplasmic accumulations of LSm4 are marked by double arrowheads. Bar, 10 µm.*}hy[(, 百拇医药
Discussion*}hy[(, 百拇医药
SART3/p110 is concentrated in CBs*}hy[(, 百拇医药
The CB is a nuclear structure identified in many organisms and cell lines, but little is known about its formation and function In this study, we show for the first time that SART3/p110, a protein transiently associated with U4/U6 snRNP and with a defined function in the U4/U6 snRNP assembly , is concentrated in CBs. In HeLa cells, primary human fibroblasts, and mouse embryonic fibroblasts, we detected SART3/p110 by immunofluorescence throughout the nucleoplasm and enriched in CBs, where snRNPs were also highly concentrated. The localization of SART3/p110 in CBs was confirmed by expression of EGFP-tagged SART3/p110. Previous studies demonstrated the nuclear localization of SART3/p110 , but these experiments did not specifically address whether SART3/p110 was detectable in CBs. We did not find any significant overlap of nucleoplasmic SART3/p110 with sites of RNA synthesis, consistent with SART3/p110's absence from the U4/U6U5 tri-snRNP and spliceosomesand further suggesting that SART3/p110 is not recruited to snRNPs cotranscriptionally.
To address how SART3/p110 is specifically targeted to CBs, we constructed and expressed EGFP fusion proteins containing distinct domains of SART3/p110. We found that the SART3/p110 HAT domain with NLS is necessary and sufficient to specifically localize EGFP-tagged constructs to CBs. CstF77, a protein containing 10 HAT repeats, was not concentrated in CBs (unpublished data), indicating that the SART3/p110 HAT domain, and not all HAT domains in general, can target proteins to CBs. Although all constructs containing the HAT domain were specifically targeted to CBs, mutants lacking the COOH-terminal region (CT10, CT10RRM, and HAT) showed less intense signal than the full-length protein. Thus, although the COOH-terminal half of SART3/p110 was not specifically localized in CBs, the RRMs and/or CT-10 domain may enhance SART3/p110 retention in CBs by promoting binding to snRNPs\;, 百拇医药
Because SART3/p110 acts during snRNP assembly, we wanted to test the hypothesis that gems, a nuclear structure often associating with CBs, are the sites of snRNP regeneration after splicing, as previously suggested . In HeLa cells and primary human fibroblasts, where gems are often separated from CBs, we found that SART3/p110 was not concentrated in gems, as judged by double immunofluorescence with anti-SMN . Moreover, mouse embryonic fibroblasts, which lack the CB-specific protein coilin, also contain gems separate from residual CBs , and these gems lacked SART3/p110 as well . Therefore, our data suggest that steps in U4/U6 snRNP assembly or regeneration involving SART3/p110 are unlikely to occur in gems.
Correlation of SART3/p110 and snRNP accumulation in CBs8{x*(&|, http://www.100md.com
The spliceosomal snRNPs are concentrated in CBs even though CBs are not thought to be sites of pre-mRNA splicing . Because newly synthesized snRNPs transit CBs en route to the nucleoplasm, and because snRNPs are depleted from CBs upon inhibition of transcription, a role for CBs in snRNP assembly and regeneration has been proposed . If this is true, then assembly factors like SART3/p110 might interact with snRNPs in CBs. In this study, three independent lines of evidence demonstrate a correlation between SART3/p110 and snRNP accumulation in CBs, suggesting that SART3/p110 associates with CBs in a complex with snRNPs.8{x*(&|, http://www.100md.com
First, we show that SART3/p110 accumulation in CBs is dependent on the expression of the CB-specific protein coilin. In an embryonic fibroblast cell line established from a coilin-/- mouse, the CB components fibrillarin and Nopp140 remain concentrated in so-called residual CBs, which fail to recruit snRNPs . We found that SART3/p110 was detectable in the nucleoplasm of these coilin-/- cells but, like snRNPs, was absent from residual CBs Thus, coilin is required for snRNP assembly into CBs along with factors involved in their metabolism, such as SART3/p110.
Second, we show that SART3/p110 and snRNPs are depleted from CBs by nuclear run-on treatment, in which the activity of RNA polymerase II is preserved. In contrast, fibrillarin, coilin, and SMN remained associated with CBs after run-on treatment. The loss of SART3/p110 and snRNPs under these conditions suggests that SART3/p110 and snRNPs associate weakly with CBs and are not stable structural components of CBs. In light of recent efforts to characterize the proteomic environment of a variety of subnuclear structures , these results indicate that at least some important nuclear body components may be removed during purification and therefore subsequently escape detection. In this regard, it is noteworthy that a number of known nucleolar proteins were indeed absent in the proteomic analysis of isolated nucleoli5lk$, 百拇医药
Third, we show that SART3/p110 and LSm4, which are normally concentrated in CBs and 5), are not specifically concentrated in CBs after -amanitin treatment, indicating that SART3/p110 and LSm4 association with CBs is transcription/splicing dependent. These results coincide with previous findings , which describe the depletion of snRNPs from CBs after -amanitin treatment. However, in contrast to other snRNPs, the U6 snRNP component LSm4 and SART3/p110 were not concentrated in SFCs after RNA polymerase II inhibition. The dependence of SART3/p110 and snRNP accumulation in CBs on transcription and splicing is consistent with the hypothesis that CBs play an active role in snRNP metabolism and do not represent storage sites for snRNPs or SART3/p110.
CBs: sites of snRNP assembly?5nf{z, 百拇医药
The observations described here suggest that specific steps in snRNP biogenesis, namely the binding of SART3/p110 to its U4 and U6 snRNP substrates and possibly U4/U6 snRNA annealing itself, occur in CBs. We propose a model in which CBs are the sites of U4/U6 snRNP assembly . This may occur after snRNP nuclear import and/or after each round of splicing, although direct evidence that snRNPs cycle repeatedly through CBs is currently lacking. This working hypothesis is consistent with the detection of U4 and U6 snRNAs as well as TMG cap, Sm, and LSm proteins in CBs , the latter indicating that mature snRNPs are present . Importantly, newly synthesized snRNPs imported from the cytoplasm first concentrate in CBs. The model is further supported by our data, which correlate snRNP and SART3/p110 association with CBs under the conditions of coilin knockout, run-on treatment, and transcription inhibition (see above). The fact that SART3/p110 associates with U6 and U4/U6 snRNPs only transiently and is not detectable in the U4/U6U5 tri-snRNP is a key point in the proposal that the SART3/p110 concentration in CBs reflects its function there.
fig.ommtted%':w+, http://www.100md.com
A model for U4/U6 snRNP assembly in CBs. To form a functional U4/U6 snRNP, U4 and U6 snRNAs have to be annealed. The annealing occurs after their synthesis (and reimport from the cytoplasm in the case of U4 snRNP) as well as after each round of splicing. The U4/U6 snRNP subsequently interacts with U5 snRNP (yellow ball) to form U4/U6U5 tri-snRNP. We propose that U4/U6 snRNP assembly occurs in CBs. After splicing, the U6 snRNP is translocated to CBs as a single particle or in a complex with SART3/p110, which promotes the U4 and U6 annealing. The assembly of the U4/U6 snRNP takes place in CBs, and SART3/p110 leaves the complex as U4/U6U5 tri-snRNP is formed. U4/U6 snRNP assembly may also occur in the nucleoplasm (not depicted), which is likely the case in cells lacking morphologically defined CBs.%':w+, http://www.100md.com
The expression of mutant SART3/p110 lacking the CT10 domain or both CT10 and RRM domains reduced the levels of endogenous SART3/p110 and LSm4 in CBs . These data indicate that endogenous SART3/p110 can be depleted from CBs by overexpression of mutant proteins lacking the COOH-terminal region. These mutants also have a dominant negative effect on LSm4 localization to CBs, most likely due to the absence of the CT10 domain. This observation is in agreement with the interaction observed between the CT10 domain of Prp24, the yeast homologue of SART3/p110, and LSm proteins and further support the model that SART3/p110 is in a complex with the U6 snRNP in CBs. This observation satisfies one requirement of the proposal that U4/U6 snRNP assembly occurs in CBs . Moreover, these results suggest that SART3/p110 plays a role in the targeting of the U6 snRNP to CBs. In future studies, it will be important to determine how the HAT domain specifies SART3/p110 and U6 snRNP localization to CBs.
The presence of SART3/p110 in the nucleoplasm indicates that SART3/p110 binding to its substrates and/or U4/U6 annealing may also occur outside CBs. In cells lacking morphologically defined CBs, U4/U6 assembly and recycling likely takes place in the nucleoplasm, although it is currently unknown whether other CB components, such as nucleoplasmic coilin, also participate. The other possibility is that besides snRNP regeneration, nucleoplasmic SART3/p110 may be involved in other nuclear processes, as suggested by others[d)7(c, 百拇医药
Splicing involves not only unwinding of U4 and U6 snRNAs but also rearrangements within other snRNPs . We speculate that CBs could be involved in the assembly and/or recycling of snRNPs other than U4/U6, for example the U4atac/U6atac snRNP, the U4/U6U5 tri-snRNP, and/or the U2 snRNP. This proposal is supported by the recent finding that a 61-kD protein involved in formation of the U4/U6U5 and the U4atac/U6atacU5 tri-snRNPs is also present in CBs . Interestingly, the U4atac/U6atac snRNP also contains LSm proteins , suggesting that SART3/p110 may also promote annealing of the U4atac/U6atac snRNP. The concentration of these processes in CBs might represent an efficient pathway for the assembly and recycling of transcription and splicing factors in highly active cells with elevated levels of transcription and splicing
Materials and methods)#]%, http://www.100md.com
Cell lines and antibodies)#]%, http://www.100md.com
HeLa cells, WI-38 primary fibroblasts (passages 16–18), and mouse embryonic fibroblasts 42 (coilin-/-) and 26 (coilin+/+) were cultured in DME supplemented with 10% fetal calf serum, penicillin, and streptomycin . Rabbit anti-SART3/p110 antibodies were raised against the COOH-terminal 16 amino acids (PKMSNADFAKLFLRKC) and affinity purified. The following additional antibodies were used: mAb anti-coilin (5P10) (; provided by M. Carmo-Fonseca, Universidade de Lisboa, Lisboa, Portugal), mAb anti-SMN (2B1) (provided by G. Dreyfuss, University of Pennsylvania School of Medicine, Philadelphia, PA; ), mAb anti-TMG (Oncogene Research Products), mAb anti-fibrillarin (17C12) (; provided by K. Koberna and I. Raka, Institute of Experimental Medicine, Prague, Czech Republic), mAb SC-35 , rat mAb anti-BrdU (Harlan Sera Lab), and a rabbit antibody against LSm4 (provided by T. Achsel and R. Luhrmann, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; ).
SART3/p110 cloning and protein tagging6, http://www.100md.com
The SART3/p110 gene was amplified from 293 cell RNA by RT-PCR in two pieces, using DNA oligonucleotides A (GAATTCGCCACCATGGCGACTGCGGCCGAAACCTCGGC) and B (GCTATCCCAGAGTTCCCGGGCTTTCTGC) for the 5' portion (nt 1–1474) and C (GCAGAAAGCCCGGGAACTCTGGGATAGC) and D (GGAGATCTGACTTTCTCAGAAACA-GCTTGGCAAAATCGGCATTGG) for the 3' portion (nt 1466–2889). Oligonucleotide A contained an EcoRI restriction site as well as a Kozak sequence, oligos B and C contained an XmaI site, and D contained a BglII site. Reverse transcription was performed using MMLV reverse transcriptase, and PCR was performed using Pfu polymerase according to the manufacturer's instructions. The two fragments were sequentially ligated into the BglII, XmaI, and EcoRI sites of plasmid pTYB4 , and the entire EcoRI–BglII fragment was subcloned into the EcoRI–BamHI sites of pBOS-H2BGFP Three independent bacterial transformants were sequenced (at the University of California San Francisco Biological Resource Center), revealing two silent mutations (A127G and C2815T) as well as a G507C mutation that results in a GlyAla change relative to the sequence in GenBank/EMBL/DDBJ (accession no. ). Full-length SART3/p110 as well as deletion mutants (CT10 aa 1–950; CT10RRM aa 1–702, HAT aa 581–963, and HAT aa 119–702) were amplified by Expand long template PCR system (Roche) and cloned into EGFP-N1 and -C3 vectors using BglII and EcoRI sites. The N-TERM (aa 1–127) fusion construct was cloned using HindIII and KpnI sites into EGFP-N2 vector containing at the COOH terminus three tandem repeats of the NLS from simian virus large T-antigen (gift of W. Haubensak, Max Planck Institute of Molecular Cell Biology and Genetics). The NLS signal was cloned from EYFP-Nuc vector using BsrGI and AflII sites.
The mouse LSm8 cDNA was obtained from R. Luhrmann and T. Achsel. The mouse LSm8 protein has the same amino acid sequence as its human homologue . The full-length cDNA was amplified by Expand long template PCR and cloned into EYFP-N1 vector using BglII and KpnI restriction sites.;13e^, 百拇医药
All fusion constructs were confirmed by sequencing. Fugene 6 (Roche) was used for transfection of cells with the SART3/p110–EGFP and LSm8–EYFP constructs.;13e^, 百拇医药
Indirect immunofluorescence;13e^, 百拇医药
Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized for 5 min with 0.2% Triton X-100, and incubated with the indicated antibodies. Secondary anti–mouse antibodies conjugated with TRITC or FITC, anti–rat antibody conjugated with FITC, and anti–rabbit antibodies conjugated with TRITC, FITC, or Cy5 were used. Immunodetection of SART3/p110 or LSm4 in cells expressing EGFP constructs was done 24–48 h after transfection. Images were collected using the microscope system (Applied Precision) coupled with IX70 microscope. Stacks of 25 z-sections with 200-nm z-step were collected per sample and subjected to mathematical deconvolution (SoftWorx; Applied Precision). If not indicated otherwise, the images shown here are single sections of the resulting three-dimensional reconstructions.
Run-on transcription assay/{, http://www.100md.com
Cells were permeabilized and RNA was labeled by BrUTP as previously described . In brief, cells were incubated in glycerol buffer (20 mM Tris-Cl, pH 7.4, 5 mM MgCl2, 25% glycerol, 0.5% PMSF, 0.5% EGTA) for 2 min at 37°C, overlaid with BTB buffer (100 mM KCl, 50 mM Tris-Cl, pH 7.4, 5 mM MgCl2, 0.5 mM EGTA, 25% glycerol, 2.5% PVA, and 0.1% Triton X-100) containing 0.5 mM ATP, GTP, and CTP and 0.2 mM BrUTP, incubated for 10 min at 37°C, fixed with 4% paraformaldehyde, and processed for immunofluorescence./{, http://www.100md.com
Transcription inhibition/{, http://www.100md.com
-Amanitin treatment was performed as previously described . HeLa cells were placed in fresh medium, and -amanitin (Sigma) was added to a final concentration of 50 µg/ml. Cells were incubated for 5 h and prepared for immunofluorescence as described above. To test cell viability after -amanitin treatment, the dye FM 4–64 (Molecular Probes) was added to culture medium (final concentration 16 nM). The dye incorporation into living cells was observed after 10 min and compared with untreated control cells.
Measurement of fluorescence intensities;-, 百拇医药
Fluorescence intensities were quantified with MetaVue software using deconvolved images (see above). The optical sections were merged, and the intensities in random regions of the nucleoplasm divided by the region area were taken as the values to which the intensities within the CBs were compared. CB area was defined by SART3/p110–EGFP constructs or by coilin labeling; intensities of SART3/p110 or LSm4 were measured within the CB and divided by the CB area. Data were collected from 20–50 normal-looking CBs after {alpha} -amanitin treatment and CBs in control cells, and 100–160 CBs in cells expressing different SART3/p110–EGFP mutants or control untransfected cells.;-, 百拇医药
Acknowledgments;-, 百拇医药
The authors are grateful to C. Guthrie for support and helpful discussions, A.G. Matera (Case Western Reserve University, Cleveland, OH) for mouse embryonic fibroblasts 42 (coilin-/-) and 26 (coilin+/+), M. Carmo-Fonseca for the gift of anti-coilin antibody, G. Dreyfuss for the anti-SMN antibody, K. Koberna and I. Raska for the anti-fibrillarin antibody, and T. Achsel and R. Luhrmann for the polyclonal antibody specific for LSm4 and for the LSm8 cDNA. We thank A. Bindereif for communicating unpublished results and T. Misteli, K. Kotovic, J. Geiger, and J. Görnemann for discussion and comments on the manuscript.
This work was supported by a Research Project grant (RPG-00-110-01-MGO) from the American Cancer Society, the Max Planck Gesellschaft, and National Institutes of Health grant GM21119. S.D. Rader was supported by a National Institutes of Health postdoctoral fellowship (grant 5 F32 GM18312) and by an American Heart Association postdoctoral fellowship.y&\8%$, 百拇医药
Note added in proof. The cytoplasmic distribution of LSm proteins 1–7 and their colocalization with mRNA-degrading enzymes has been recently published (Ingelfinger, D., D.J. Arndt-Jovin, R. Lührmann, and T. Achsel. 2002. RNA. 8:1489–1501).y&\8%$, 百拇医药
Submitted: 15 October 2002y&\8%$, 百拇医药
Revised: 13 January 2003y&\8%$, 百拇医药
Accepted: 13 January 2003y&\8%$, 百拇医药
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2 Department of Biochemistry and Biophysics, University of California, San Francisco, CA, 94143]o, http://www.100md.com
Abstract]o, http://www.100md.com
The spliceosomal small nuclear RNAs (snRNAs) are distributed throughout the nucleoplasm and concentrated in nuclear inclusions termed Cajal bodies (CBs). A role for CBs in the metabolism of snRNPs has been proposed but is not well understood. The SART3/p110 protein interacts transiently with the U6 and U4/U6 snRNPs and promotes the reassembly of U4/U6 snRNPs after splicing in vitro. Here we report that SART3/p110 is enriched in CBs but not in gems or residual CBs lacking coilin. The U6 snRNP Sm-like (LSm) proteins, also involved in U4/U6 snRNP assembly, were localized to CBs as well. The levels of SART3/p110 and LSm proteins in CBs were reduced upon treatment with the transcription inhibitor {alpha} -amanitin, suggesting that CB localization reflects active processes dependent on transcription/splicing. The NH2-terminal HAT domain of SART3/p110 was necessary and sufficient for specific protein targeting to CBs. Overexpression of truncation mutants containing the HAT domain had dominant negative effects on U6 snRNP localization to CBs, indicating that endogenous SART3/p110 plays a role in targeting the U6 snRNP to CBs. We propose that U4 and U6 snRNPs accumulate in CBs for the purpose of assembly into U4/U6 snRNPs by SART3/p110.
Key Words: RNA splicing; coiled (Cajal) body; U4/U6 small nuclear ribonucleoprotein; small nuclear RNA; nuclear proteinsu99, 百拇医药
Introductionu99, 百拇医药
The cell nucleus contains numerous, morphologically distinct domains and bodies. One of these, a 0.5–1.0-µm sphere named the Cajal body (CB), was discovered 100 yr ago by Santiago Ramón y Cajal. The molecular characterization of CBs has been facilitated by the discovery of the protein coilin, an unambiguous marker of CBs in Xenopus, mouse, and human cells . Coilin likely plays an important role in cell metabolism because coilin knockout mice exhibit reduced viability . Many factors involved in transcription, RNA processing, and cell cycle regulation are concentrated in CBs, but the function of CBs, with respect to these diverse processes, remains obscure. Although spliceosomal U1, U2, U4, U5, and U6 small nuclear RNAs (snRNAs) are enriched in CBs , CBs are not likely sites of splicing because they are not transcriptionally active and lack many essential non-snRNP splicing factors . snRNP association with CBs is transcription dependent, indicating that CBs play an active role in snRNP metabolism and may be the sites of snRNP assembly rather than storage of inactive snRNPs . What essential function accounts for the concentration of spliceosomal snRNPs in CBs?
The U1, U2, U4, and U5 snRNAs are synthesized by RNA polymerase II, capped at their 5' ends, and transported to the cytoplasm where they are bound by snRNP-specific Sm proteins (for reviews see ). These snRNAs are hypermethylated at their 5' ends, producing their characteristic 2,2,7 trimethylguanosine (TMG) caps and providing an important signal for snRNP import into the nucleus . Because snRNP-specific proteins appear to concentrate in CBs before accumulating in the nucleoplasm, a role for CBs in the maturation of snRNPs has been proposed . The recent identification of RNAs that guide base modification of snRNAs and localization of these guide RNAs to CBs suggest that snRNA base modification may take place in CBs . However, these results do not explain why the U6 snRNA, which is synthesized by RNA polymerase III, is not capped or exported to the cytoplasm, and undergoes base modification in the nucleolus , is present in CBs. The U6 snRNA is present in at least three distinct snRNPs, the U6 snRNP, the U4/U6 snRNP, and the U4/U6U5 tri-snRNP. Formation of all of these snRNP species must occur in the nucleus, but little is known about the subnuclear location of these processes.
After assembly, the U1, U2, and U4/U6U5 snRNPs perform essential functions in spliceosome formation and catalysis. During a process termed the spliceosomal cycle, each snRNP is thought to participate in subsequent rounds of splicing, which then requires the regeneration of snRNPs that have undergone rearrangement during splicing . In particular, the U4/U6 snRNP, which contains two snRNAs base paired with each other, unwinds during splicing as U6 establishes new base-pairing interactions with U2 and the pre-mRNA. Therefore, U4 and U6 must reanneal after splicing to regenerate functional U4/U6 snRNPs. In yeast, the essential protein Prp24 catalyzes this reaction . In the absence of Prp24p, splicing extracts are depleted of the U4/U6 snRNP, demonstrating the importance of snRNP recycling for continuing rounds of pre-mRNA splicing/, 百拇医药
Recently, the tumor rejection antigen SART3/p110 was identified as the human homologue of Prp24p and was shown to be required for U4/U6 snRNP recycling in vitro . SART3/p110 binds specifically and directly to the U6 snRNA and is detectable in the U6 and U4/U6 snRNPs . Unlike other U4, U5, and U6 snRNP–specific proteins, SART3/p110 is not detectable in the U4/U6U5 tri-snRNP , indicating that SART3/p110 dissociates from its U4 and U6 snRNP substrates once they are annealed. In addition to SART3/p110, the Sm-like (LSm) proteins LSm2–8, which assemble on the 3' end of the U6 snRNA as a stable heteromer and persist in the U4/U6 and U4/U6U5 snRNPs , have been implicated in U4/U6 snRNP assembly. The LSm proteins bind directly to Prp24 in yeast , providing a second mode of interaction with the U6 snRNP. In vitro, Prp24p anneals U4 and U6 snRNAs more efficiently in the context of snRNPs , making it likely that the combination of SART3/p110/Prp24p and LSm proteins enables efficient assembly of the U4/U6 snRNP in vivo. Because the role of SART3/p110 in U4/U6 recycling is uniquely transient, the subnuclear distribution of SART3/p110 has the potential to reveal the sites of U4/U6 snRNP assembly.
In this study, we report that SART3/p110 and the LSm proteins, LSm4 and LSm8, are localized in the cell nucleus and concentrated in CBs. We studied the association of SART3/p110 and snRNPs with CBs after transcription inhibition and run-on treatment and in the absence of the CB component coilin. SnRNP and SART3/p110 localizations in CBs were correlated in each of these experimental conditions. Mutant analysis revealed that the HAT domain of SART3/p110 represents the major determinant for specific targeting of SART3/p110 to CBs. Overexpression of mutants lacking the snRNP-binding COOH-terminal domains reduced the concentration of both endogenous SART3/p110 and LSm4 in CBs, suggesting that SART3/p110 is required for U6 snRNP targeting to CBs.70, 百拇医药
Results70, 百拇医药
By immunofluorescent labeling of HeLa cells, SART3/p110 was detected exclusively in the cell nucleus, where it was distributed throughout the nucleoplasm and specifically enriched in large bright dots. Double labeling of cells for SART3/p110 and the TMG cap ( A), a modification found at the 5' end of U1, U2, U4, and U5 snRNAs, revealed complete overlap between SART3/p110 and snRNPs in the large bright structures. These bodies were identified as CBs by double detection of SART3/p110 and coilin, a marker of CB. SART3 was detected in all observed CBs (n = 51 cells). The same results were obtained in cells transiently expressing SART3/p110 conjugated to EGFP (see below and ). Measurement of endogenous SART3/p110 fluorescent intensities in CBs revealed that the concentration of SART3/p110 in CBs is approximately three times higher then in the nucleoplasm (see below, ). We confirmed the SART3/p110 localization patterns in WI-38 primary human fibroblasts . Although the fibroblasts contain fewer CBs, SART3/p110 was concentrated in every CB observed. In contrast to the striking overlap between SART3/p110 and snRNPs in CBs, only partial colocalization of SART3/p110 and snRNPs was observed in the nucleoplasm .
fig.ommtted9#)%.}, http://www.100md.com
SART3/p110 is concentrated in CBs. (A and B) Immunolocalization of SART3/p110 in HeLa cells together with (A) TMG cap, a marker of snRNPs, or (B) with coilin, a marker of CBs. (C) SART3/p110 and coilin were immunolocalized in human primary fibroblasts WI-38. CBs are marked by arrowheads. Bar, 5 µm.9#)%.}, http://www.100md.com
fig.ommtted9#)%.}, http://www.100md.com
Effect of SART3/p110 mutant expression on concentration of the endogenous SART3/p110 and LSm4 in CBs9#)%.}, http://www.100md.com
The survival of motor neuron protein (SMN) is involved in the assembly of snRNPs in the cytoplasm. In addition, SMN and associated gemin proteins are detected in the cell nucleus in structures termed gems . Gems often overlap or are associated with CBs, depending on the cell type . The function of nuclear SMN is currently unknown, although roles in splicing and transcription have been hypothesized . Because a role for gems in snRNP recycling has been proposed, we tested whether SART3/p110 specifically localizes to gems in a HeLa cell line in which gems are often separate from CBs. Double staining of HeLa cells with anti-SART3/p110 and anti-SMN antibodies revealed that SART3/CBs were overlapping or adjacent to gems only 58% of the time (n = 55 cells). Like the HeLa cell line, WI-38 primary fibroblasts often contained gems independent from CBs. We did not detect any enrichment of SART3/p110 in gems in primary fibroblasts . Because 100% of CBs contain SART3/p110 (see above), we conclude from this result that gems, per se, are not sites of SART3/p110 accumulation.
fig.ommtted;3}v/, http://www.100md.com
SART3/p110 is not specifically localized in gems. SART3/p110 and SMN, enriched in gems, were immunolocalized in (A) HeLa cells and (B) human primary fibroblasts WI-38. Examples of CBs are marked by arrowheads, gems by arrows. Inserts are magnified two times, and green (left), red (middle), and merged (right) signals are shown. Bar, 10 µm.;3}v/, http://www.100md.com
To investigate the targeting of SART3/p110 to CBs, we analyzed the role of coilin in SART3/p110 recruitment to CBs. We compared the distribution of SART3/p110 in embryonic fibroblasts derived from a wild-type mouse versus a mouse in which both coilin alleles were disrupted. Wild-type mouse embryonic fibroblasts have snRNPs, SMN, fibrillarin, and Nopp140 localized in CBs . In embryonic fibroblasts derived from the coilin knockout mouse, fibrillarin and Nopp140 are accumulated in so-called "residual" CBs, which fail to recruit snRNPs and SMN; snRNPs are distributed throughout the nucleoplasm, and SMN is concentrated in separate gems . Similar to our previous results, we found that SART3/p110 was detected in the nucleoplasm and in CBs of coilin+/+ cells, labeled either by anti–TMG cap, anti-fibrillarin, or anti-SMN antibodies . In coilin-/- cells, SART3/p110 was distributed throughout the nucleoplasm with no detectable concentration in residual CBs or gems . Double staining of coilin-/- cells with anti–TMG cap and SART3/p110 antibodies revealed the distribution of snRNPs and SART3/p110 throughout the nucleoplasm and never in the highly concentrated sites detectable in wild-type cells (, compare E with F). Thus, coilin expression is required for the recruitment of both snRNPs and SART3/p110 to CBs.
fig.ommtted\#fppnt, http://www.100md.com
SART3/p110 recruitment to CBs requires coilin. Mouse embryonic fibroblasts derived from control (coilin+/+) or coilin knockout (coilin-/-) mice were stained for SART3/p110 together with (A and B) fibrillarin (Fib), (C and D) SMN, and (E and F) TMG cap. (A, C, and E) In control coilin+/+ cells, SART3/p110 concentrates in CBs labeled by fibrillarin, SMN, and TMG cap . (B, D, and F) In cells lacking functional coilin, SART3/p110 is distributed throughout the nucleoplasm and is not recruited to residual CBs labeled by fibrillarin (B) or gems labeled by SMN (D). No CBs were detected by TMG cap (F). Inserts are magnified two times, and red (left), green (middle), and merged (right) signals are shown. Bar, 5 µm.\#fppnt, http://www.100md.com
Splicing largely occurs at transcription sites (for review see ). To test the possibility that SART3/p110 is cotranscriptionally recruited to snRNPs, we labeled nascent RNA with BrUTP and visualized labeled RNA together with SART3/p110 . Nucleoplasmic SART3/p110 did not appreciably overlap with transcription sites. During the course of these experiments, we noticed that cells permeabilized under run-on conditions (see Material and methods) exhibited a dramatic loss of SART3/p110 from CBs (compare B with B). We have further investigated this phenomenon and found that snRNPs were also largely extracted from CBs during run-on transcription as revealed by TMG cap labeling. In contrast, run-on treatment did not affect the detection of coilin, fibrillarin, or SMN in CBs and/or gems . This suggests that snRNPs and SART3/p110 are only weakly associated with CBs, compared with the apparently more stable components, coilin, SMN, and fibrillarin.
fig.ommtted+2, http://www.100md.com
SART3/p110 is depleted from CBs after nuclear run-on. (A) Cells were permeabilized using run-on conditions (see Materials and methods), and newly synthesized RNA was labeled by BrUTP and detected together with SART3/p110. Little colocalization of SART3/p110 with labeled RNA was detected. (B) Cells after run-on treatment were labeled for SART3/p110 and coilin. (B, inset) Representative CB was magnified 3.5 times, and green (left), red (middle), and merged (right) signals are shown. (C) Control fixed cells (control) and cells after run-on (run-on) labeled with coilin together with TMG cap, fibrillarin (Fib), and SMN. For representative CBs, green (left), red (middle), and merged (right) signals are shown in the insets. Bars: (A and C) 5 µm; (B) 10 µm.+2, http://www.100md.com
The presence of U4 and U6 snRNAs and SART3/p110 in CBs indicates that CBs contain the substrates and at least one factor required for U4/U6 snRNP assembly. To determine whether the U6 snRNA–associated LSm proteins that promote U4/U6 annealing in vitro are also present in CBs, we analyzed the distribution of two members of the LSm complex. By immunofluorescence, LSm4 was detected both in the cytoplasm and the nucleus . The cytoplasmic localization may represent LSm complexes involved in mRNA degradation . In the nucleus, LSm4 was detected throughout the nucleoplasm and concentrated in CBs . Double staining of HeLa cells with anti-LSm4 and anti–TMG cap antibodies revealed complete overlap of both antigens in CBs but only partial overlap in the nucleoplasm . Similarly, LSm8 tagged with EYFP was mainly detected in the nucleoplasm and in CBs where it colocalized with LSm4 . Although LSm proteins participate in several distinct complexes, including the Xenopus laevis U8 small nucleolar RNP and a cytoplasmic mRNA degradation complex , it is likely that localization of LSm4 and LSm8 reflects the presence of the mature U6 snRNP in CBs.
fig.ommttedri-, http://www.100md.com
. U6 snRNP proteins LSm4 and LSm8 are enriched in CBs. HeLa cells stained for LSm4 and (A) coilin or (B) TMG cap. LSm4 is concentrated in CBs, where it colocalizes with TMG cap, whereas there is only partial overlap of LSm4 with TMG cap in the nucleoplasm. HeLa cells expressing LSm8–EYFP stained for (C) coilin or (D) Lsm4. LSm8 is diffusely distributed in the cell nucleus and concentrated in CBs. Examples of CBs are marked by arrowheads. For representative CBs, green (left), red (middle), and merged (right) signals are shown in the insets. Bar, 5 µm.ri-, http://www.100md.com
The observations that SART3/p110, snRNPs, and LSm proteins are concentrated in CBs and that snRNP and SART3/p110 association with CBs is correlated combine to suggest that SART3/p110 may accumulate in CBs for the purpose of U4/U6 snRNP assembly. However, an alternative hypothesis is that SART3/p110 is inactive and perhaps stored in CBs. To distinguish between these possibilities, we tested whether SART3/p110 concentration in CBs was affected by transcription activity in the cell. It was previously shown that depleting the cell of pre-mRNA by blocking RNA polymerase II activity leads to a reduction in overall snRNP concentration in CBs . If SART3/p110 is stored in the CB, one would expect SART3/p110 levels to remain constant or even increase upon transcription inhibition. If, in contrast, SART3/p110 in CBs is active in U4/U6 snRNP assembly, then SART3/p110 levels in CBs should decrease when transcription is inhibited.
To determine whether SART3/p110 concentration in CBs is dependent on RNA synthesis, cells were treated with the transcription inhibitor -amanitin. After treatment, all cells had rounded splicing factor compartments (SFCs) labeled with SC-35 , an indication of RNA polymerase II inhibition . Neither SART3/p110 nor LSm4 was enriched in SFCs. The latter result was unexpected, because other snRNPs (U1, U2, U4, and U5) have been shown to concentrate in SFCs after -amanitin treatment After -amanitin treatment, two types of coilin-positive CBs were observed: normal-looking CBs (indistinguishable from CBs in nontreated cells in size and morphology) and enlarged "ring-shaped" CBs, as reported previously . This change in CB morphology is not likely to reflect cell death because 95% of the cells were viable, according to a standard viability test (see Material and methods). In contrast to control untreated cells , we did not detect any specific enrichment of SART3/p110 or LSm4 in any of the coilin-labeled CBs . Quantitation of the fluorescence intensities within normal-looking CBs relative to the nucleoplasmic signal revealed that SART3/p110 levels in CBs decreased threefold (P < 0.0001) and LSm4 levels in CBs decreased twofold (P < 0.0001) after {alpha} -amanitin treatment. These results indicate that SART3/p110 and LSm4 association with CBs is transcription/splicing dependent and not likely due to the storage of inactive molecules.
fig.ommttedte, 百拇医药
SART3/p110 and LSm4 are depleted from CBs after transcription inhibition. (A and B) HeLa cells were treated with -amanitin and stained for a marker of splicing factor compartments (SC-35) and (A) SART3/p110 or (B) LSm4. Enlarged, rounded-up splicing factor compartments indicate that inhibition of RNA polymerase II transcription was effective. (C and D) HeLa cells after -amanitin treatment stained with anti-coilin antibody together with (C) anti-SART3/p110 or (D) anti-LSm4 antibodies. Two types of coilin-labeled structures were observed: normal-looking CBs (arrowheads) and enlarged ring-shaped CBs (arrows). Depicted areas containing CBs were enlarged two times (insets), and green (left), red (middle), and merged (right) signals are shown. Bars, 5 µm.te, 百拇医药
To determine how SART3/p110 is specifically localized to CBs, we investigated the potential of isolated SART3/p110 protein domains fused with EGFP to target CBs. Human SART3/p110 is composed of three major protein domains, a repeat of seven HAT (half a TPR domain) motifs followed by a nuclear localization signal, two RNA recognition motifs (RRMs), and the highly conserved COOH-terminal domain CT10 . The HAT domains and their related TPR domains are present in a number of other RNA processing factors and have been hypothesized to function in protein–protein interactions . Recently, the SART3/p110 HAT domain was shown to interact with the HIV-1 Tat protein . The RRMs have been implicated in U6 snRNA binding , and the CT10 domain likely mediates binding to the U6 snRNP via interaction with LSm proteins, as it does in yeast . In two-hybrid assays, full-length SART3/p110 interacted specifically with LSm7 but not with LSm1, which is not present in the U6 snRNP. Deletion of the CT10 domain reduced the two-hybrid interaction twofold, suggesting that the CT10 domain contributes to SART3/p110 binding to the U6 snRNP via LSm proteins (unpublished data). We created several EGFP-tagged proteins composed of specific SART3/p110 domains . Note that each fusion protein diagrammed (except N-TERM) was also produced with the EGFP tag at the NH2 terminus, and the results were identical to those obtained with the COOH-terminal–tagged constructs (unpublished data), indicating that the protein localization results described below are not due to the addition of the tags at the COOH termini.
fig.ommtted?q4p8e*, http://www.100md.com
The NH2-terminal domain containing the HAT motifs targets SART3/p110 to CBs. (A) Wild-type (WT) SART3/p110 was conjugated to EGFP. Deletion mutants containing amino acids 1–950 (CT10), 1–702 (CT10RRM), 581–963 (HAT), and 119–702 (HAT) were conjugated to EGFP. The expression construct (N-TERM) encodes the NH2-terminal amino acids 1–127 fused to EGFP and a heterologous NLS signal. (B) All constructs including empty vector containing heterologous NLS (EGFP–NLS) were transiently expressed in HeLa cells and stained for coilin. Full-length protein as well as mutants containing the HAT domain accumulated in CBs (arrowheads). The HAT protein and EGFP–NLS accumulated in nucleoli (arrows) and only weakly in CBs. The N-TERM mutant was detected throughout the nucleoplasm and slightly accumulated in nucleoli and CBs. The identification of nucleoli was verified by phase contrast microscopy (not depicted). Bar, 5 µm.?q4p8e*, http://www.100md.com
The full-length SART3/p110–EGFP (WT) revealed the same distribution as endogenous SART3/p110 ( B, WT; compare with ); it localized to the nucleoplasm and was concentrated in CBs, as judged by coilin staining. Deletion of the highly conserved COOH-terminal domain CT-10 (CT10) or both CT-10 and the two RNA recognition motifs (CT10RRM) resulted in similar localization patterns as full-length SART3/p110. In contrast, truncation of the NH2-terminal portion of the protein containing seven HAT repeats (HAT) had strong effects on protein localization. The HAT–EGFP protein was found in the nucleoplasm and concentrated in nucleoli, but was only weakly detectable in CBs. Because EGFP conjugated to an NLS (EGFP–NLS) was also concentrated in nucleoli and faintly detectable in CBs, the simplest interpretation of the data is that the concentration of HAT–EGFP in nucleoli and weak detectability in CBs is nonspecific. To further investigate the role of the NH2 terminus, we created a mutant containing only the HAT domain and the endogenous NLS (HAT). This construct was specifically targeted to CBs. In contrast, the extreme NH2-terminal region of SART3/p110 (N-TERM), lacking the HAT domain and conjugated to EGFP plus a heterologous NLS, was not specifically localized to CBs (compare N-TERM with EGFP–NLS localizations). Interestingly, the N-TERM mutant was not highly concentrated in nucleoli, in contrast to EGFP–NLS alone. Although the function of the extreme NH2-terminal region of SART3/p110 is currently unknown, these results suggest that it may interact with a nucleoplasmic component. We conclude from this analysis that the HAT domain of SART3/p110 is necessary and sufficient for the specific targeting of SART3/p110 to CBs.
The highly conserved CT10 domain was previously shown to interact with LSm proteins . To determine whether overexpression of SART3/p110 mutants lacking the COOH-terminal region affects the localization of endogenous SART3/p110 and/or LSm4, we transfected HeLa cells with WT–, HAT–, CT10–, and CT10RRM–EGFP mutants and determined the localization patterns of endogenous SART3/p110 and LSm4 by immunofluorescence (, WT–EGFP and CT10RRM–EGFP constructs shown only). Quantitation of fluorescence intensities within CBs relative to the nucleoplasm revealed that the localization of endogenous SART3/p110 was significantly reduced in CT10- and CT10RRM-expressing cells, compared with untransfected controls (. This indicates that both mutant proteins effectively compete for SART3/p110 binding sites within CBs. Moreover, if SART3/p110 plays a role in U6 snRNP localization to CBs, then expression of CT10 and/or CT10RRM mutants may have dominant negative effects on LSm4 localization in CBs. Indeed, LSm4 concentration in CBs was significantly reduced upon overexpression of CT10 or CT10RRM mutants by 30% . The expression of WT–EGFP also influenced the concentration of LSm4 in CBs, but the effect was less pronounced than in the case of CT10 or CT10RRM mutant We noticed that CBs were disrupted in some cells expressing high levels of CT10 or CT10RRM mutant (unpublished data). The HAT–EGFP mutant, which is aberrantly localized to nucleoli, did not show any effects on a localization of LSm4 (unpublished data). These data suggest that SART3/p110 plays a role in U6 snRNP targeting to CBs, largely through the CT10 domain of SART3/p110.
fig.ommtted*}hy[(, 百拇医药
Expression of CT10RRM–EGFP mutant reduces the concentration of endogenous SART3/p110 and LSm4 in CBs. HeLa cells were transiently transfected with (A) WT–EGFP or (B and C) CT10RRM–EGFP constructs and, after fixation, immunostained with (A and C) anti-LSm4 or (B) anti-SART3/p110 antibodies. Note that anti-SART3/p110 antibodies were raised against the COOH terminus of SART3/p110 and thus do not react with fusion proteins lacking the COOH-terminal part. Projections of five optical sections are shown. CBs in transfected cells are marked by arrowheads, in nontransfected cells by arrows. Examples of cytoplasmic accumulations of LSm4 are marked by double arrowheads. Bar, 10 µm.*}hy[(, 百拇医药
Discussion*}hy[(, 百拇医药
SART3/p110 is concentrated in CBs*}hy[(, 百拇医药
The CB is a nuclear structure identified in many organisms and cell lines, but little is known about its formation and function In this study, we show for the first time that SART3/p110, a protein transiently associated with U4/U6 snRNP and with a defined function in the U4/U6 snRNP assembly , is concentrated in CBs. In HeLa cells, primary human fibroblasts, and mouse embryonic fibroblasts, we detected SART3/p110 by immunofluorescence throughout the nucleoplasm and enriched in CBs, where snRNPs were also highly concentrated. The localization of SART3/p110 in CBs was confirmed by expression of EGFP-tagged SART3/p110. Previous studies demonstrated the nuclear localization of SART3/p110 , but these experiments did not specifically address whether SART3/p110 was detectable in CBs. We did not find any significant overlap of nucleoplasmic SART3/p110 with sites of RNA synthesis, consistent with SART3/p110's absence from the U4/U6U5 tri-snRNP and spliceosomesand further suggesting that SART3/p110 is not recruited to snRNPs cotranscriptionally.
To address how SART3/p110 is specifically targeted to CBs, we constructed and expressed EGFP fusion proteins containing distinct domains of SART3/p110. We found that the SART3/p110 HAT domain with NLS is necessary and sufficient to specifically localize EGFP-tagged constructs to CBs. CstF77, a protein containing 10 HAT repeats, was not concentrated in CBs (unpublished data), indicating that the SART3/p110 HAT domain, and not all HAT domains in general, can target proteins to CBs. Although all constructs containing the HAT domain were specifically targeted to CBs, mutants lacking the COOH-terminal region (CT10, CT10RRM, and HAT) showed less intense signal than the full-length protein. Thus, although the COOH-terminal half of SART3/p110 was not specifically localized in CBs, the RRMs and/or CT-10 domain may enhance SART3/p110 retention in CBs by promoting binding to snRNPs\;, 百拇医药
Because SART3/p110 acts during snRNP assembly, we wanted to test the hypothesis that gems, a nuclear structure often associating with CBs, are the sites of snRNP regeneration after splicing, as previously suggested . In HeLa cells and primary human fibroblasts, where gems are often separated from CBs, we found that SART3/p110 was not concentrated in gems, as judged by double immunofluorescence with anti-SMN . Moreover, mouse embryonic fibroblasts, which lack the CB-specific protein coilin, also contain gems separate from residual CBs , and these gems lacked SART3/p110 as well . Therefore, our data suggest that steps in U4/U6 snRNP assembly or regeneration involving SART3/p110 are unlikely to occur in gems.
Correlation of SART3/p110 and snRNP accumulation in CBs8{x*(&|, http://www.100md.com
The spliceosomal snRNPs are concentrated in CBs even though CBs are not thought to be sites of pre-mRNA splicing . Because newly synthesized snRNPs transit CBs en route to the nucleoplasm, and because snRNPs are depleted from CBs upon inhibition of transcription, a role for CBs in snRNP assembly and regeneration has been proposed . If this is true, then assembly factors like SART3/p110 might interact with snRNPs in CBs. In this study, three independent lines of evidence demonstrate a correlation between SART3/p110 and snRNP accumulation in CBs, suggesting that SART3/p110 associates with CBs in a complex with snRNPs.8{x*(&|, http://www.100md.com
First, we show that SART3/p110 accumulation in CBs is dependent on the expression of the CB-specific protein coilin. In an embryonic fibroblast cell line established from a coilin-/- mouse, the CB components fibrillarin and Nopp140 remain concentrated in so-called residual CBs, which fail to recruit snRNPs . We found that SART3/p110 was detectable in the nucleoplasm of these coilin-/- cells but, like snRNPs, was absent from residual CBs Thus, coilin is required for snRNP assembly into CBs along with factors involved in their metabolism, such as SART3/p110.
Second, we show that SART3/p110 and snRNPs are depleted from CBs by nuclear run-on treatment, in which the activity of RNA polymerase II is preserved. In contrast, fibrillarin, coilin, and SMN remained associated with CBs after run-on treatment. The loss of SART3/p110 and snRNPs under these conditions suggests that SART3/p110 and snRNPs associate weakly with CBs and are not stable structural components of CBs. In light of recent efforts to characterize the proteomic environment of a variety of subnuclear structures , these results indicate that at least some important nuclear body components may be removed during purification and therefore subsequently escape detection. In this regard, it is noteworthy that a number of known nucleolar proteins were indeed absent in the proteomic analysis of isolated nucleoli5lk$, 百拇医药
Third, we show that SART3/p110 and LSm4, which are normally concentrated in CBs and 5), are not specifically concentrated in CBs after -amanitin treatment, indicating that SART3/p110 and LSm4 association with CBs is transcription/splicing dependent. These results coincide with previous findings , which describe the depletion of snRNPs from CBs after -amanitin treatment. However, in contrast to other snRNPs, the U6 snRNP component LSm4 and SART3/p110 were not concentrated in SFCs after RNA polymerase II inhibition. The dependence of SART3/p110 and snRNP accumulation in CBs on transcription and splicing is consistent with the hypothesis that CBs play an active role in snRNP metabolism and do not represent storage sites for snRNPs or SART3/p110.
CBs: sites of snRNP assembly?5nf{z, 百拇医药
The observations described here suggest that specific steps in snRNP biogenesis, namely the binding of SART3/p110 to its U4 and U6 snRNP substrates and possibly U4/U6 snRNA annealing itself, occur in CBs. We propose a model in which CBs are the sites of U4/U6 snRNP assembly . This may occur after snRNP nuclear import and/or after each round of splicing, although direct evidence that snRNPs cycle repeatedly through CBs is currently lacking. This working hypothesis is consistent with the detection of U4 and U6 snRNAs as well as TMG cap, Sm, and LSm proteins in CBs , the latter indicating that mature snRNPs are present . Importantly, newly synthesized snRNPs imported from the cytoplasm first concentrate in CBs. The model is further supported by our data, which correlate snRNP and SART3/p110 association with CBs under the conditions of coilin knockout, run-on treatment, and transcription inhibition (see above). The fact that SART3/p110 associates with U6 and U4/U6 snRNPs only transiently and is not detectable in the U4/U6U5 tri-snRNP is a key point in the proposal that the SART3/p110 concentration in CBs reflects its function there.
fig.ommtted%':w+, http://www.100md.com
A model for U4/U6 snRNP assembly in CBs. To form a functional U4/U6 snRNP, U4 and U6 snRNAs have to be annealed. The annealing occurs after their synthesis (and reimport from the cytoplasm in the case of U4 snRNP) as well as after each round of splicing. The U4/U6 snRNP subsequently interacts with U5 snRNP (yellow ball) to form U4/U6U5 tri-snRNP. We propose that U4/U6 snRNP assembly occurs in CBs. After splicing, the U6 snRNP is translocated to CBs as a single particle or in a complex with SART3/p110, which promotes the U4 and U6 annealing. The assembly of the U4/U6 snRNP takes place in CBs, and SART3/p110 leaves the complex as U4/U6U5 tri-snRNP is formed. U4/U6 snRNP assembly may also occur in the nucleoplasm (not depicted), which is likely the case in cells lacking morphologically defined CBs.%':w+, http://www.100md.com
The expression of mutant SART3/p110 lacking the CT10 domain or both CT10 and RRM domains reduced the levels of endogenous SART3/p110 and LSm4 in CBs . These data indicate that endogenous SART3/p110 can be depleted from CBs by overexpression of mutant proteins lacking the COOH-terminal region. These mutants also have a dominant negative effect on LSm4 localization to CBs, most likely due to the absence of the CT10 domain. This observation is in agreement with the interaction observed between the CT10 domain of Prp24, the yeast homologue of SART3/p110, and LSm proteins and further support the model that SART3/p110 is in a complex with the U6 snRNP in CBs. This observation satisfies one requirement of the proposal that U4/U6 snRNP assembly occurs in CBs . Moreover, these results suggest that SART3/p110 plays a role in the targeting of the U6 snRNP to CBs. In future studies, it will be important to determine how the HAT domain specifies SART3/p110 and U6 snRNP localization to CBs.
The presence of SART3/p110 in the nucleoplasm indicates that SART3/p110 binding to its substrates and/or U4/U6 annealing may also occur outside CBs. In cells lacking morphologically defined CBs, U4/U6 assembly and recycling likely takes place in the nucleoplasm, although it is currently unknown whether other CB components, such as nucleoplasmic coilin, also participate. The other possibility is that besides snRNP regeneration, nucleoplasmic SART3/p110 may be involved in other nuclear processes, as suggested by others[d)7(c, 百拇医药
Splicing involves not only unwinding of U4 and U6 snRNAs but also rearrangements within other snRNPs . We speculate that CBs could be involved in the assembly and/or recycling of snRNPs other than U4/U6, for example the U4atac/U6atac snRNP, the U4/U6U5 tri-snRNP, and/or the U2 snRNP. This proposal is supported by the recent finding that a 61-kD protein involved in formation of the U4/U6U5 and the U4atac/U6atacU5 tri-snRNPs is also present in CBs . Interestingly, the U4atac/U6atac snRNP also contains LSm proteins , suggesting that SART3/p110 may also promote annealing of the U4atac/U6atac snRNP. The concentration of these processes in CBs might represent an efficient pathway for the assembly and recycling of transcription and splicing factors in highly active cells with elevated levels of transcription and splicing
Materials and methods)#]%, http://www.100md.com
Cell lines and antibodies)#]%, http://www.100md.com
HeLa cells, WI-38 primary fibroblasts (passages 16–18), and mouse embryonic fibroblasts 42 (coilin-/-) and 26 (coilin+/+) were cultured in DME supplemented with 10% fetal calf serum, penicillin, and streptomycin . Rabbit anti-SART3/p110 antibodies were raised against the COOH-terminal 16 amino acids (PKMSNADFAKLFLRKC) and affinity purified. The following additional antibodies were used: mAb anti-coilin (5P10) (; provided by M. Carmo-Fonseca, Universidade de Lisboa, Lisboa, Portugal), mAb anti-SMN (2B1) (provided by G. Dreyfuss, University of Pennsylvania School of Medicine, Philadelphia, PA; ), mAb anti-TMG (Oncogene Research Products), mAb anti-fibrillarin (17C12) (; provided by K. Koberna and I. Raka, Institute of Experimental Medicine, Prague, Czech Republic), mAb SC-35 , rat mAb anti-BrdU (Harlan Sera Lab), and a rabbit antibody against LSm4 (provided by T. Achsel and R. Luhrmann, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany; ).
SART3/p110 cloning and protein tagging6, http://www.100md.com
The SART3/p110 gene was amplified from 293 cell RNA by RT-PCR in two pieces, using DNA oligonucleotides A (GAATTCGCCACCATGGCGACTGCGGCCGAAACCTCGGC) and B (GCTATCCCAGAGTTCCCGGGCTTTCTGC) for the 5' portion (nt 1–1474) and C (GCAGAAAGCCCGGGAACTCTGGGATAGC) and D (GGAGATCTGACTTTCTCAGAAACA-GCTTGGCAAAATCGGCATTGG) for the 3' portion (nt 1466–2889). Oligonucleotide A contained an EcoRI restriction site as well as a Kozak sequence, oligos B and C contained an XmaI site, and D contained a BglII site. Reverse transcription was performed using MMLV reverse transcriptase, and PCR was performed using Pfu polymerase according to the manufacturer's instructions. The two fragments were sequentially ligated into the BglII, XmaI, and EcoRI sites of plasmid pTYB4 , and the entire EcoRI–BglII fragment was subcloned into the EcoRI–BamHI sites of pBOS-H2BGFP Three independent bacterial transformants were sequenced (at the University of California San Francisco Biological Resource Center), revealing two silent mutations (A127G and C2815T) as well as a G507C mutation that results in a GlyAla change relative to the sequence in GenBank/EMBL/DDBJ (accession no. ). Full-length SART3/p110 as well as deletion mutants (CT10 aa 1–950; CT10RRM aa 1–702, HAT aa 581–963, and HAT aa 119–702) were amplified by Expand long template PCR system (Roche) and cloned into EGFP-N1 and -C3 vectors using BglII and EcoRI sites. The N-TERM (aa 1–127) fusion construct was cloned using HindIII and KpnI sites into EGFP-N2 vector containing at the COOH terminus three tandem repeats of the NLS from simian virus large T-antigen (gift of W. Haubensak, Max Planck Institute of Molecular Cell Biology and Genetics). The NLS signal was cloned from EYFP-Nuc vector using BsrGI and AflII sites.
The mouse LSm8 cDNA was obtained from R. Luhrmann and T. Achsel. The mouse LSm8 protein has the same amino acid sequence as its human homologue . The full-length cDNA was amplified by Expand long template PCR and cloned into EYFP-N1 vector using BglII and KpnI restriction sites.;13e^, 百拇医药
All fusion constructs were confirmed by sequencing. Fugene 6 (Roche) was used for transfection of cells with the SART3/p110–EGFP and LSm8–EYFP constructs.;13e^, 百拇医药
Indirect immunofluorescence;13e^, 百拇医药
Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized for 5 min with 0.2% Triton X-100, and incubated with the indicated antibodies. Secondary anti–mouse antibodies conjugated with TRITC or FITC, anti–rat antibody conjugated with FITC, and anti–rabbit antibodies conjugated with TRITC, FITC, or Cy5 were used. Immunodetection of SART3/p110 or LSm4 in cells expressing EGFP constructs was done 24–48 h after transfection. Images were collected using the microscope system (Applied Precision) coupled with IX70 microscope. Stacks of 25 z-sections with 200-nm z-step were collected per sample and subjected to mathematical deconvolution (SoftWorx; Applied Precision). If not indicated otherwise, the images shown here are single sections of the resulting three-dimensional reconstructions.
Run-on transcription assay/{, http://www.100md.com
Cells were permeabilized and RNA was labeled by BrUTP as previously described . In brief, cells were incubated in glycerol buffer (20 mM Tris-Cl, pH 7.4, 5 mM MgCl2, 25% glycerol, 0.5% PMSF, 0.5% EGTA) for 2 min at 37°C, overlaid with BTB buffer (100 mM KCl, 50 mM Tris-Cl, pH 7.4, 5 mM MgCl2, 0.5 mM EGTA, 25% glycerol, 2.5% PVA, and 0.1% Triton X-100) containing 0.5 mM ATP, GTP, and CTP and 0.2 mM BrUTP, incubated for 10 min at 37°C, fixed with 4% paraformaldehyde, and processed for immunofluorescence./{, http://www.100md.com
Transcription inhibition/{, http://www.100md.com
-Amanitin treatment was performed as previously described . HeLa cells were placed in fresh medium, and -amanitin (Sigma) was added to a final concentration of 50 µg/ml. Cells were incubated for 5 h and prepared for immunofluorescence as described above. To test cell viability after -amanitin treatment, the dye FM 4–64 (Molecular Probes) was added to culture medium (final concentration 16 nM). The dye incorporation into living cells was observed after 10 min and compared with untreated control cells.
Measurement of fluorescence intensities;-, 百拇医药
Fluorescence intensities were quantified with MetaVue software using deconvolved images (see above). The optical sections were merged, and the intensities in random regions of the nucleoplasm divided by the region area were taken as the values to which the intensities within the CBs were compared. CB area was defined by SART3/p110–EGFP constructs or by coilin labeling; intensities of SART3/p110 or LSm4 were measured within the CB and divided by the CB area. Data were collected from 20–50 normal-looking CBs after {alpha} -amanitin treatment and CBs in control cells, and 100–160 CBs in cells expressing different SART3/p110–EGFP mutants or control untransfected cells.;-, 百拇医药
Acknowledgments;-, 百拇医药
The authors are grateful to C. Guthrie for support and helpful discussions, A.G. Matera (Case Western Reserve University, Cleveland, OH) for mouse embryonic fibroblasts 42 (coilin-/-) and 26 (coilin+/+), M. Carmo-Fonseca for the gift of anti-coilin antibody, G. Dreyfuss for the anti-SMN antibody, K. Koberna and I. Raska for the anti-fibrillarin antibody, and T. Achsel and R. Luhrmann for the polyclonal antibody specific for LSm4 and for the LSm8 cDNA. We thank A. Bindereif for communicating unpublished results and T. Misteli, K. Kotovic, J. Geiger, and J. Görnemann for discussion and comments on the manuscript.
This work was supported by a Research Project grant (RPG-00-110-01-MGO) from the American Cancer Society, the Max Planck Gesellschaft, and National Institutes of Health grant GM21119. S.D. Rader was supported by a National Institutes of Health postdoctoral fellowship (grant 5 F32 GM18312) and by an American Heart Association postdoctoral fellowship.y&\8%$, 百拇医药
Note added in proof. The cytoplasmic distribution of LSm proteins 1–7 and their colocalization with mRNA-degrading enzymes has been recently published (Ingelfinger, D., D.J. Arndt-Jovin, R. Lührmann, and T. Achsel. 2002. RNA. 8:1489–1501).y&\8%$, 百拇医药
Submitted: 15 October 2002y&\8%$, 百拇医药
Revised: 13 January 2003y&\8%$, 百拇医药
Accepted: 13 January 2003y&\8%$, 百拇医药
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