The recruitment of the U5 snRNP to nascent transcripts requires internal loop 1 of U5 snRNA
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In this study, we take advantage of the high spatial resolution offered by the nucleus and lampbrush chromosomes of the amphibian oocyte to investigate the mechanisms that regulate the intranuclear trafficking of the U5 snRNP and its recruitment to nascent transcripts. We monitor the fate of newly assembled fluorescent U5 snRNP in Xenopus oocytes depleted of U4 and/or U6 snRNAs and demonstrate that the U4/U6.U5 tri-snRNP is not required for the association of U5 snRNP with Cajal bodies, splicing speckles, and nascent transcripts. In addition, using a mutational analysis, we show that a non-functional U5 snRNP can associate with nascent transcripts, and we further characterize internal loop structure 1 of U5 snRNA as a critical element for licensing U5 snRNP to target both nascent transcripts and splicing speckles. Collectively, our data support the model where the recruitment of snRNPs onto pre-mRNAs is independent of spliceosome assembly and suggest that U5 snRNP may promote the association of the U4/U6.U5 tri-snRNP with nascent transcripts.
KeywordsCajal bodies Lampbrush chromosomes RNA splicing spliceosome snRNAs
Interchromatin granule cluster
Polymerase chain reaction
RNA polymerase II
Small nuclear ribonucleoprotein particle
Small nuclear RNA
One of the most prominent features of the lampbrush chromosomes (LBCs) is their very high level of RNA polymerase II (RNAPII) activity (reviewed in Callan 1986; Morgan 2002; see Macgregor ibid). This property is structurally showcased by numerous transcription units in the form of loops that are projected laterally, away from the relatively condensed chromosomal axes. While individual loops are easily distinguished using a light microscope, it is important to note that their chromatin axes are only revealed by labeling with antibodies against DNA (see Morgan, ibid) or proteins associated with DNA, such as histones (Austin et al. 2009) and active RNAPII (Gall et al. 1999, see Gall ibid). In essence, the lateral loops correspond to nascent RNAPII transcripts that are associated with numerous RNA processing and export factors. Together, they create a ribonucleoprotein matrix, which is dense enough to be seen by phase contrast or DIC, surrounding a highly decondensed chromatin axis. LBC loops, therefore, represent a unique cellular system to investigate the mechanisms controlling the major steps in RNA transcription and processing, particularly the recruitment of the various machineries involved in these processes. Indeed, it has become well accepted that pre-mRNA transcription and maturation, including splicing, are two interrelated processes in all eukaryotes (Bentley 2005; de Almeida and Carmo-Fonseca 2008; Martins et al. 2011).
A critical step in pre-mRNA maturation is the removal of introns and ligation of exons by the spliceosome, a multi-subunit and dynamic enzyme (Zhou et al. 2002; Jurica and Moore 2003; Nilsen 2003; Patel and Steitz 2003). At the heart of the spliceosome are its catalytic components, the five major small nuclear ribonucleoproteins U1, U2, U4, U5, and U6 snRNPs. Each spliceosomal snRNP consists of a small nuclear RNA (snRNA) associated with a specific set of proteins. The biogenesis of most snRNPs follows a complex maturation pathway that involves both a cytoplasmic and a nuclear phase (Patel and Bellini 2008). Within the nucleus, snRNPs are often detected in association with several discrete domains such as Cajal bodies (CBs), nucleoli, and interchromatin granule clusters (IGCs). While the functional implications of these associations are not immediately clear, the current view is that these domains are involved in the maturation and/or storage of snRNPs and other processing factors prior to their recruitment to nascent transcripts. Previously, we demonstrated that the recruitment of the splicing snRNP to nascent transcripts is independent of the spliceosomal assembly. Additionally, we showed that discrete elements of the U1 and U2 snRNPs control their intranuclear trafficking and recruitment to nascent transcripts (Patel et al. 2007; Paschedag et al., in revision). Here, we extend our analysis to the U5 snRNP, which integrates into the spliceosome as part of the U4/U6–U5 tri-snRNP complex. Interestingly, we find that the U5 snRNP still targets nascent transcripts in the absence of the U4 and/or U6 snRNPs. In addition, we define the small internal loop 1 (IL1) of U5 snRNA as a critical element for the association of the U5 snRNP with both IGCs and nascent transcripts.
Materials and methods
In vitro transcription and labeling
The cDNA clone coding for the full-length Xenopus laevis U5 snRNA was generously provided by Dr. Joseph Gall and was used to generate all templates for in vitro transcription of the wild-type and mutant U5 snRNAs. DNA templates were obtained by PCR, and in all cases, the T3 promoter was introduced immediately upstream of the sequence to be transcribed. DNA primers (Integrated DNA Technologies) used were as follows (T3 promoter is underlined):
U5 WT: 5′CGGAATTCAATTAACCCTCACTAAAGGG and 3′ATACCTGGTGTGAACCAGGCTTC
U5∆5′47: 5′CGAATTAACCCTCACTAAAGGAAAGATTTCCGTGG and 3′ATACCTGGTGTGAACCAGGCTTC
U5∆5′68: CGAATTAACCCTCACTAAAGGCGACCATGAGTTTCG and 3′ATACCTGGTGTGAACCAGGCTTC
U5∆3′96: 5′CGGAATTCAATTAACCCTCACTAAAGGG and 3′TTCAAAAAATTGAACGAAACTCATGGTCG
ΔSL1: Step 1: 5′CCTCTGGTTTCTCTTCAAATTCGAATAAATCTTTTTCGAAAGATTTCCG and 3′ATACCTGGTGTGAACCAGGCTTC;
Step 2: 5′AATTAACCCTCACTAAAGGATACTCTGGTTTCTCTTCAAATTCG and 3′-ATACCTGGTGTGAACCAGGCTTC
ΔIL1: Step 1: 5′AATTAACCCTCACTAAAGGATACTTTTCTCTTCAAATTCGAAT and 3′TGAACCAGGCTTCAAAAAATTGAACGAAACTTTCCTCTCCACGG
Step2: 5′AATTAACCCTCACTAAAGGATACTTTTCTCTTCAAATTCGAAT and 3′ATACCTGGTGTGAACCAGGCTTC
ΔIL2: Step 1: 5′CGCCTTTTACTAAAGATGAGAGGAACGACCAT and 3′ATACCTGGTGTGAACCAGGCTTC;
Step 2: 5′AATTAACCCTCACTAAAGGATACTCTGGTTTCTCTTATCTTTCGCCTTTTAC and 3′ATACCTGGTGTGAACCAGGCTTC
Amplified DNA templates were gel-purified using 0.45 mm cellulose acetate spin-X filters (Corning Inc.), phenol extracted, and ethanol precipitated before transcription. Fluorescently labeled U5 snRNA and mutants were synthesized using T3 polymerase (Stratagene) in the presence of 25 μM fluorescein-12-UTP (Roche), 625 μM ATP and CTP, 312.5 μM UTP, 250 μM GTP, and 1.25 mM m7G(5′)ppp(5′)G cap analog (New England Biolabs, Inc.). Reactions were carried out for 3 h at 37 °C, treated with RQ1 RNase-free DNase (Fisher Scientific) for 10 min at 37 °C, and newly made transcripts were purified on NucAway Spin columns (Ambion).
Oocytes and microinjections
Fragments of ovary were surgically removed from female adult frogs that were anesthetized in 0.15 % tricaine methane sulfonate (MS222; Sigma-Aldrich). Oocytes were defolliculated for 2 h at room temperature in saline buffer OR2 (Wallace et al. 1973) containing 0.2 % collagenase (type II; Sigma-Aldrich). OR2 buffer is 82.5 mM NaCl, 2.5 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 1.0 mM Na2HPO4, and 5.0 mM HEPES. Stage IV–V oocytes were subsequently isolated and maintained at 18 °C in OR2. All injections were performed directly into the cytoplasm of oocytes using ~10–20 fmol of RNA, with volumes no greater than 30 nL. For the U4 and U6 depletion experiments, 50 ng of U4d and U6f oligonucleotides (Integrated DNA Technologies) was injected per oocyte 4 h prior to the injection of the fluorescent U5 WT snRNA. The U4d sequence, TATTGGGAAAAGTTT, is complementary to nucleotides 66–80 of U4 snRNA. The U6f sequence, TCGTTCCAATTTTAG, is complementary to nucleotides 25–39 of U6 snRNA. Glass needles were prepared using a horizontal pipette puller (P-97; Sutter Instruments Co.). Injections were performed under a dissecting microscope (Leica) using the nanojetII micro-injector from Drummond.
Nuclear spreads and immunofluorescence
Nuclear spreads were prepared as described in (Patel et al. 2007). Preparations were fixed in phosphate-buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.46 mM KH2PO4, pH 7.2) containing 2 % paraformaldehyde and 1 mM MgCl2, for 1 h at room temperature, and blocked in PBS with 0.5 % BSA (Sigma-Aldrich) + 0.5 % gelatin (from cold water fish) for 10 min. The fluorescent signal emitted by fluorescein-labeled RNAs was amplified as follows: Spread preparations were incubated with primary antibody Alexa Fluor 488 anti-fluorescein (Invitrogen) diluted at 2.5 μg/ml, washed with PBS, and then incubated with secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) at 2.5 μg/ml. Spreads were washed again with PBS and mounted in 50 % glycerol in PBS + 1 mg/ml phenylenediamine and 10 pg/ml DAPI. Microscopy was performed using a fluorescent microscope (DMR; Leica) and a Fluotar 100× NA 1.30 oil objective (Leica). Images were captured with a Retiga EXI monochrome CCD camera (QImaging) and In Vivo software (version 3.2.0, Media Cybernetics), processed with Photoshop CS version 8.0 (Adobe), and assembled with InDesign CS version 3.0 (Adobe).
Two sets of ten oocytes were injected with the U4d and U6f oligonucleotides, and ten uninjected oocytes were used as a control. After 4 h of incubation at 18 °C, the nuclei were collected and homogenized in 10 mM Tri-HCl, pH 8.0, 1 mM EDTA, and 0.2 % sodium dodecyl sulfate. Total RNAs were phenol-extracted and ethanol-precipitated. Northern blots were performed as described in Patel et al. (2007) with following modifications to the protocol: The membranes were cross-linked at 120 mJ/cm2, and antisense U4, U5, and U6 snRNAs were used as probes.
Newly assembled U5 snRNPs associate with CBs, IGCs, and LBC loops
The recruitment of U5 snRNP to nascent transcripts does not require U4 or U6 snRNPs
Distinct elements of the U5 snRNP regulate its subnuclear distribution
Localization of wild-type and mutant U5 snRNPs as defined on nuclear spreads
While the biogenesis of the spliceosomal snRNPs is well documented (reviewed in Patel and Bellini 2008), the mechanisms that regulate their targeting to nascent transcripts still remain unclear. The current view is that snRNPs are recruited in a stepwise manner for the formation of the spliceosome on target pre-mRNAs. Recent findings, however, indicate that the recruitment of some snRNPs to nascent transcripts can occur in absence of spliceosomal assembly (Patel et al. 2007; Spiluttini et al. 2010; Paschedag et al., in revision). This conclusion stems from two main observations. First, the recruitment of snRNPs is unaffected by the absence of the U2 snRNP, which is critical for proper spliceosomal assembly and activity. Second, a non-functional snRNP that cannot comprise part of the spliceosome still associates with nascent transcripts. These data strongly suggest that distinct populations of snRNPs could co-exist on nascent transcripts. One possibility is that snRNPs are initially recruited to nascent transcripts, perhaps as a staging event to increase their local concentrations and allow efficient spliceosome formation upon the emergence during transcription of the necessary cis-acting RNA elements. Another possibility is that snRNPs may function in roles other than splicing. Interestingly, the U1 snRNP was recently shown to protect nascent transcripts from premature cleavage and polyadenylation independently of splicing (Kaida et al. 2010; Berg et al. 2012).
While IGCs and LBC loops are sites targeted by fully assembled snRNPs, CBs and nucleoli are often described as nuclear domains involved in the maturation and assembly of snRNPs (Gall et al. 1999; Yu et al. 2001; Gall 2003; Gerbi et al. 2003a; Matera 2003). In particular, CBs were directly implicated in the internal modification of the snRNAs by pseudouridylation and 2′-O-methylation (Darzacq et al. 2002; Jady et al. 2003). Here, we find that CBs accumulate newly formed wild-type and mutant U5 snRNPs at high concentrations. Interestingly, Gall et al. (1999) used in situ hybridizations on nuclear spread preparations more than a decade ago to reveal that endogenous U5 snRNPs, as well as all the other spliceosomal snRNPs, are present at a very low concentration in the oocyte CBs. We recently showed that the newly formed fluorescent U2 snRNPs that accumulate in CBs are incompletely assembled and lack the proteins that are required for targeting nascent transcripts (Paschedag et al., in revision). Similarly, the endogenous U1 snRNP rapidly accumulates in CBs upon truncation of the first stem-loop structure (U1SL1) of the U1 snRNA (Gall et al. 1999), which was subsequently shown to be both necessary and sufficient for targeting U1 snRNP to IGCs and LBC loops (Patel et al. 2007). It is likely, then, that the newly formed U5 snRNPs accumulated in CBs correspond primarily to incomplete RNP complexes. In addition, since the U5 snRNP comprises part of the tri-snRNP, a stoichiometric imbalance resulting from injecting fluorescent U5 snRNAs into oocytes may also contribute to the accumulation of fluorescent U5 snRNPs in CBs. Indeed, CBs are nuclear bodies where the formation of the U4/U6.U5 tri-snRNP is enhanced (Stanek et al. 2003; Schaffert et al. 2004; Stanek and Neugebauer 2004), and one could speculate that monomeric U5 snRNP remains dynamically associated with CBs until assembly of the U4/U6.U5 tri-snRNP occurs.
We previously demonstrated that and U4 and U5 snRNPs target LBC loops in absence of spliceosomal assembly (Patel et al. 2007). Here, we present several pieces of data that further indicate a definite separation between the snRNP elements controlling intranuclear trafficking and those involved in catalyzing pre-mRNA splicing. First, the deletion of SL1 has no effect on the intranuclear distribution of U5 snRNPs, at least qualitatively. In fact, U5∆SL1 snRNP targets nascent transcripts and IGCs as efficiently as the wild-type U5 snRNP despite SL1 being essential for the splicing activity of the U5 snRNP, as shown by the deletion of only two nucleotides within the SL1 loop being sufficient to inhibit the second trans-esterification reaction of pre-mRNA splicing in vitro (O'Keefe and Newman 1998). Next, the splicing activity of U5 snRNAs lacking IL2 is severely hindered in vitro (Dix et al. 1998). Yet, we show here that the deletion of IL2 does not prevent the recruitment of U5 snRNPs to nascent transcripts. Finally, U5 snRNAs lacking IL1, which we show here as being unable to associate with nascent transcripts, are still able to catalyze splicing in vitro, albeit with a much lower efficiency that the wild-type U5 snRNA (Dix et al. 1998). Collectively, these data demonstrate a functional modularity of the U5 snRNP and an uncoupling of the elements that regulate its catalytic activity and its intranuclear trafficking.
This work is supported by a grant from the National Science Foundation.