Tip110 binding to U6 small nuclear RNA and its participation in pre-mRNA splicing
© Liu et al. 2015
Received: 6 April 2015
Accepted: 3 July 2015
Published: 23 July 2015
RNA–protein interactions play important roles in gene expression control. These interactions are mediated by several recurring RNA-binding motifs including a well-known and characterized ribonucleoprotein motif or so-called RNA recognition motif (RRM).
In the current study, we set out to identify the RNA ligand(s) of a RRM-containing protein Tip110, also known as p110nrb, SART3, or p110, using a RNA-based yeast three-hybrid cloning strategy. Six putative RNA targets were isolated and found to contain a consensus sequence that was identical to nucleotides 34–46 of U6 small nuclear RNA. Tip110 binding to U6 was confirmed to be specific and RRM-dependent in an electrophoretic mobility shift assay. Both in vitro pre-mRNA splicing assay and in vivo splicing-dependent reporter gene assay showed that the pre-mRNA splicing was correlated with Tip110 expression. Moreover, Tip110 was found in the spliceosomes containing pre-spliced pre-mRNA and spliced mRNA products. Nonetheless, the RRM-deleted mutant (ΔRRM) that did not bind to U6 showed promotion in vitro pre-mRNA splicing, whereas the nuclear localization signal (NLS)-deleted mutant ΔNLS that bound to U6 promoted the pre-mRNA splicing both in vitro and in vivo. Lastly, RNA-Seq analysis confirmed that Tip110 regulated a number of gene pre-mRNA splicing including several splicing factors.
Taken together, these results demonstrate that Tip110 is directly involved in constitutive eukaryotic pre-mRNA splicing, likely through its binding to U6 and regulation of other splicing factors, and provide further evidence to support the global roles of Tip110 in regulation of host gene expression.
In eukaryotic cells, gene expression begins in nucleus with transcription of protein-coding genes to primary RNA transcripts, or so-called pre-messenger RNA (pre-mRNA) or heterogeneous nuclear RNA (hnRNA) . The pre-mRNA are processed to become mature mRNA through a series of post-transcriptional modifications . One of them is the precise excision of noncoding sequences (introns) from pre-mRNA in the spliceosomes . The spliceosomes are formed through assembly of spliceosomal small nuclear ribonucleoprotein (snRNP) and recruitment of numerous splicing factors to pre-mRNA [1, 2]. Efficient and proper pre-mRNA splicing is a critical step in gene expression control [3, 4]. Dysregulation of splicing elements or splicing regulators and subsequent abnormal pre-mRNA splicing result in expression of aberrant protein products and development of diseases [3, 4]. For example, overexpression of splicing factor SF2/ASF has been shown to trigger malignant transformation .
snRNP are important components of the spliceosomes in which the pre-mRNA splicing occurs . There are five major snRNP U1, U2, U4, U5, and U6 in the spliceosomes containing small nuclear RNA (snRNA) U1 snRNA, U2 snRNA, U4 snRNA, U5 snRNA, and U6 snRNA, respectively . Those snRNA are transcribed in the nucleus and exported to the cytoplasm after acquiring a m7G-cap [1, 2]. The core snRNP are initially assembled in the cytoplasm to contain snRNA, survival motor neuron protein, Gem-associated proteins, and Sm proteins, followed by nuclear import . Once the core snRNP get into the nucleus, they enter the Cajal body, undergo further modifications such as RNA-targeted ribose methylation and pseudouridylation, recruit additional proteins to complete its final assembly for pre-mRNA splicing . One group of the best-defined proteins that snRNP recruit in the Cajal body are serine-/arginine-rich repeat (RS domain)-containing splicing factors called SR proteins . SR proteins are characterized by the presence of RNA recognition motifs (RRM) in the N terminus and the presence of RS domain in the C terminus. Most of the serine/arginine-rich (SR) proteins are found in the nucleus, specifically in nuclear speckles, and are known for their roles in alternative and constitutive pre-mRNA splicing [9, 10]. Besides SR proteins, there are a number of other pre-mRNA splicing factors in the nuclear speckles. Those pre-mRNA splicing factors are constantly moving between nuclear speckles and the nucleoplasm or other nuclear substructures . Nevertheless, nuclear speckles are often regarded as the sites for storage, assembly, and modification of the splicing factors as well as the supply sources of splicing factors for active transcription sites [12–14]. In addition, nuclear speckles have been shown to be directly involved in the pre-mRNA splicing .
Tip110, also known as p110nrb, SART3, or p110, has been proposed to serve several important biological functions. These include tumor antigen function [16–18], control of gene transcription [19–21], regulation of protein stability [22, 23], and stem cell pluripotency and differentiation [24–26]. In addition, Tip110 has been proposed to be directly involved in pre-mRNA splicing through interaction with RNPS1 , U4/U6 snRNP [27, 28], YB-1 , and C-MYC . In agreement with Tip110 roles in pre-mRNA splicing, Tip110 has two RNA recognition domains  and has been detected in the nuclear substructures Cajal bodies and nuclear speckles [14, 30, 31]. Further supporting Tip110 roles in pre-mRNA splicing is the recent findings that Tip110 directly binds to unphosphorylated C-terminal domain (CTD) of RNA polymerase II in the direct manner . In the current study, we set out to identify specific RNA targets, if any, that might be involved in interaction with Tip110 and account for Tip110 function in pre-mRNA splicing. We took advantage of the RNA–protein binding-based yeast three hybrid screening strategy and identified snRNA U6 from a hybrid RNA library as putative Tip110-binding partner. We then characterized the interaction and its role in pre-mRNA splicing.
Cell lines and cell transfection
293T and Hela cells were purchased from American Tissue Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, in a 37°C, 5% CO2 incubator. Cells were transfected using the standard calcium phosphate precipitation method.
Plasmids pTip110.His, pTip110s.His, and deletion mutants ∆NLS and ∆RRM, and pGEX-Tip110, pGEX-Tip110s, and deletion mutants ∆NLS and ∆RRM, and pAD.Tip110 were described elsewhere . The standard PCR cloning technique was used to construct pTip110∆LSM, with pTip110.His as the templates and primers 5′-GGA ATT CAC CAT GGC GAC TGC GGC CGA A-3′ and 5′-CCG CTC GAG TCA ATG ATG ATG ATG ATG ATG GGC AAC TGC AGG AGC CG-3′ (the restriction enzyme sites underlined). The Tip110 siRNA expression plasmid was constructed in the backbone of pSHAG-1 by annealing oligonucleotides 5′-CGG GAT CCG ACT CAG CCT CGG GTT CTG AA-3′ and 5′-CGG GAT CCA AAA AAT TGG ACT CAG CCT CA-3′, and inserting the annealed DNA at the Bam HI site of pSHAG-1. All recombinant plasmids and deletion mutants were verified by sequencing. The sources of the other plasmids used in the study are: pGEX-4T-3 from Amersham (Piscataway, NJ, USA), pSHAG-1 from Dr. Gregory Hannon, pIIIA.MS2 from Dr. Marvin Wickens, pT7.U6 from Dr. Iain Mattaji, pDM138 from Dr. Thomas Hope, pcRev from Dr. Bryan Cullen, and pSP64-Hβδ6 from Dr. Adrian Krainer.
Construction of a hybrid RNA library and yeast three-hybrid screening
A hybrid RNA library and yeast three-hybrid screening were performed as previously described , with some modifications. Briefly, genomic DNA was isolated from 293T cells and digested by Mse I, Tsp509I, Alu I and Rsa I, and then by T4 DNA polymerase in the presence of 100 μM dNTP. Digested DNA was fractionated on a 2% agarose gel to obtain DNA fragments of 50–150 bp. These small DNA fragments were then ligated into SmaI-linearized and calf intestine phosphatase-treated pIIIA.MS2 plasmid. The ligation mixture was transformed into GC5 competent bacteria for propagation and isolation of the library DNA. Yeast strain L40coat stably expressing LexA-MS2 coat fusion protein was first transformed with pAD-Tip110 plasmid and then with the RNA expression library. The selection of transformed yeast was performed on synthetic complete plates containing 0.5 mM 3-aminotriazole and no tryptophan, leucine, uracil and histidine, followed by X-gal filter assay for β-galactosidase expression. β-galactosidase-positive colonies were further cultured to isolate the hybrid RNA plasmids.
Recombinant protein purification
GST-Tip110, GST-Tip110s, Tip110 deletion mutants ∆NLS and ∆RRM, and GST proteins were expressed in and purified from E. coli BL21, as previously described . Briefly, expression plasmids were transformed into BL21 and induced with 1 mM isopropyl-d-thiogalactopyranoside for 2 h at 37°C. GST fusion proteins were purified using a Pierce GST fusion protein purification kit (Rockford, IL, USA). When necessary, GST was removed by treating the eluted protein with thrombin protease (10 units) (Invitrogen, Carlsbad, CA, USA) at room temperature for 18 h. The digested protein solution was dialyzed overnight in 4 l of phosphate-buffered saline and cleared of GST protein by additional incubations with fresh glutathione beads. The purified proteins were electrophoresed on a 8% SDS-polyacrylamide gel and stained with Gold–Blue (Pierce) to ensure the complete removal of GST protein as well as undigested fusion protein and other contaminated proteins.
RNA–protein gel mobility shift assay
U6 RNA was synthesized using an Ambion in vitro transcription kit (Austin, TX, USA). Briefly, pT7-U6 plasmid was linearized with Dra I and incubated with ATP, CTP, GTP and 32P-labeled UTP in the presence of T7 RNA polymerase at 30°C for 1 h, followed by removal of free nucleotides by an CentriSpin column (Princeton Separation, Princeton, NJ, USA). 32P-labeled-U6 RNA was then mixed with purified GST-Tip110 or its mutant proteins in a total volume of 20 μl containing 1 mM HEPES, pH 7.6, 1 μM MgCl2, 16 mM KCl, 2.5% glycerol (v/v), and 10 nM DTT. The mixture was incubated on ice for 15 min and then subjected to 5% nondenaturing polyacrylamide gel electrophoresis (acrylamide-bisacrylamide, 80:1) in the 0.5× TBE buffer. The gel was dried and exposed for autoradiography.
Preparation of pre-mRNA and in vitro splicing assay
Human β-globin minigene pre-mRNA was synthesized using an Ambion in vitro capped RNA transcription kit (Ambion). Briefly, pSP64-Hßδ6 plasmid was linearized with BamH I and incubated with ATP, CTP, GTP, cap anolog and 32P-labeled UTP at 37°C for 2 h. 32P-labeled pre-mRNA was then purified on a 5% denaturing polyacrylamide gel and suspended in 10 mM Tris.HCl, pH 7.9. The 32P-labeled pre-mRNA was then aliquoted and stored at −80°C. Splicing reactions were carried out in a 25 μl volume containing 12.8 μM MgCl2, 500 μM ATP, 20 mM creatine phosphate, 2.7% (w/v) polyvinyl alcohol, 1,000 U/ml RNasin, 12.8 mM HEPES, pH 8.0, 14% (v/v) glycerol, 62 mM KCl, 0.12 mM EDTA, and 0.7 mM DTT at 30°C for 2 h. The reactions contained 6 μl Hela nuclear extract (Promega, Madison, WI, USA) and 12.5 ng 32P-labeled pre-mRNA prepared above.
Monoclonal antibody production
Anti-Tip110 monoclonal antibodies were produced by Promab Biotechnologies, Inc. (Albany, CA, USA). Briefly, purified GST-Tip110 protein was used to immunize BALB/c mice. The mice were given an additional boost immunization after 3 weeks. Three days after the boost, the spleen cells were isolated from the immunized mice and fused with SP20 cells (ATCC) using polyethylene glycol (Sigma, St. Louis, MO, USA). Resulting hybridoma were screened to be specific anti-Tip110 antibody using ELISA. To purify the monoclonal antibody, the hybridoma cells were cultured in a production module of the miniPERM device (Viva Science, Inc.), and the antibody was further purified from the culture supernatant using a Protein A column and a AKTA prime-automated liquid chromatography system (Amersham, Piscataway, NJ, USA). The antibody protein purity was determined to be IgG2 with a purity of higher than 99%.
Purified Tip110 monoclonal antibody (20 μg) was incubated with 50 μl of 50% slurry protein A-Sepharose (Amersham) in a buffer containing 20 mM HEPES, pH 8.0, 100 mM KCl, 0.2 mM EDTA, 20% (v/v) glycerol, 0.5 mM PMSF, 1 mM DTT at 4°C for 4 h with continuous mixing. The protein A beads were then recovered by centrifugation and repeated washes with the same incubation buffer to remove excess anti-Tip110 antibody. An aliquot of the beads were then incubated with Hela nuclear extract at 4°C for 2 h with continuous mixing, and the supernatant was used as Tip110-depleted Hela nuclear extract. Tip110 protein on the immunoabsorbed beads was eluted by adding 20 μl of 4× SDS sample buffer, followed by boiling 5 min. The eluent and aliquot of the Tip110-depleted Hela nuclear extract were subjected to 10% SDS-PAGE gel and Western blot analysis to ensure efficient Tip110 depletion.
Pre-mRNA splicing-dependent CAT reporter gene assay
293T cells were plated in a 6-well plate at a density of 0.72 × 106 cells/well and 1 day later transfected with 1 μg pDM138, 1 μg pcRev, 1–5 μg pTip110.His or pTip110.siRNA. In all transfections, a CMVβGal plasmid was included to normalize transfection variations, and pcDNA3 was used to equalize the amount of transfected DNA. Forty-eight hours after transfection, the cells were harvested and subjected to 3 rounds of freeze–thaw in 0.25 M Tris.HCl, pH 8.0, and the lysates were collected for β-galactosidase and CAT activities. β-galactosidase was determined using a colorimtric assay , while CAT was determined by one-step simple phase extraction and scintillation counting as described .
Preparation of cell lysates and Western blot analysis
Cells were washed twice with ice-cold phosphate-buffered saline (PBS), and cell pellets were suspended in two volumes of whole cell lysis buffer containing10 mM NaHPO4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.2% sodium azide, 0.5% sodium deoxycholate, 0.004% sodium fluoride, and 1 mM sodium orthovanadate and incubated on ice for 10 min. Cell lysates were obtained by centrifugation, removal of the cell debris, and electrophoretically separated on 10% SDS-PAGE and analyzed by immunoblotting. Anti-His antibody was from Qiagen (Valencia, CA, USA), while β-actin was from Santa Cruz Biotech. (Santa Cruz, CA, USA). The blots were first probed with primary antibodies, followed by appropriate horseradish peroxidase-conjugated secondary antibodies, and then visualized with the ECL system (Amersham).
RNA isolation and RNA-Seq
Hela were transfected with human Tip110 siRNA or the universal control siRNA. Total RNA was purified from transfected cells using Trizol according to the manufacturer’s instructions (Life Technologies, Grand Island, NY, USA) and used for RNA-Seq. The RNA-Seq was performed at the Genomic Core of UT Southwestern Medical Center, Dallas, TX, USA. The RNA-Seq reads were aligned to the human genome sequences for further differential and spliceosome pathway analysis using the Panther Pathway Analysis software.
Total RNA was extracted using Trizol and used to synthesize cDNA using a ScriptII RT reagent kit (Promega). cDNAs were used for qPCR using a Power Sybr®Green PCR kit (Life Technologies) according to the manufacturer’s instructions. The qPCR program consisted of one cycle of 95°C for 10 min, 40 cycles of 95°C for 15 s and 60°C for 1 min.
Identification of Tip110 RNA ligands by the yeast three-hybrid cloning
Binding of Tip110 protein to U6 RNA
Promotion of pre-mRNA splicing by Tip110
Inhibition of pre-mRNA splicing by Tip110 depletion and its reversal by Tip110 add-back
Correlation between Tip110 expression and pre-mRNA splicing-dependent reporter gene expression
Requirement of Tip110 domains and pre-mRNA splicing
Presence of Tip110 in the splicing complexes
Effects of Tip110 on global pre-mRNA splicing
In this study we aimed to identify the RNA targets for Tip110 binding. We demonstrated that Tip110 specifically interacted with small nuclear U6 RNA through the yeast 3-hybrid screen and RNA EMSA (Figures 1, 2). Tip110 promoted pre-mRNA splicing in vitro (Figure 3) and in a pre-mRNA splicing-dependent reporter gene assay (Figure 5). Corroborated with these findings were that depletion of Tip110 from nuclear extract led to inhibition of pre-mRNA splicing and that add-back Tip110 restored pre-mRNA splicing (Figure 4). Moreover, RRM domain of Tip110 was shown to be important for U6 RNA binding; RRM, LSM, and NLS domains were all important for Tip110 function in pre-mRNA splicing (Figures 2, 6). Furthermore, Tip110 was detected in association with pre-mRNA- and spliced RNA-containing spliceosomes (Figure 7). Lastly, RNA-Seq analysis showed that Tip110 expression was linked to alternate pre-mRNA splicing of the majority of genes in human genome including several splicing factors (Figure 8). These results demonstrated that Tip110 regulated global pre-mRNA splicing likely through interaction with snRNA U6 and other splicing factors.
Biogenesis of the snRNP U1, U2, U4, U5, and U6 is a complex process. Newly synthesized snRNP undergo various modification and assembly, and then export from nuclear to cytoplasm where they mature and transport back into the Cajal bodies in the nucleus. snRNA/snRNP then recruit other splicing factors, undergo further assembly and maturation in the Cajal bodies [42–44]. Our current study identified U6 snRNA to be the putative Tip110-binding target through the yeast three hybrid cloning (Figure 1) and further confirmed the binding specificity through the gel shift mobility assay (Figure 2). These results are consistent with the previous reports that the U6 snRNP is targeted to the CB by specific binding to Tip110, followed by Tip110-induced assembly of U4/U6 snRNP for recycling of U4 and U6 snRNPs [27, 28, 41, 45]. Tip110 has been shown to localize not only in Cajal bodies, and is also required for the induction of CB formation as well [14, 31, 46]. These studies together suggest that Tip110 not only targets U6 snRNP to Cajal bodies through direct interaction, but also is required for snRNP U6 assembly into mature U4/U6.U5 tri-snRNP to exit Cajal bodies. When we further defined the Tip110 regions that were responsible for the snRNA U6 binding, we made a series of deletion mutants which including the C-terminal (1–350aa), deletion of NSL (∆600–670aa), deletion of two RRM (∆740–850aa). The C-terminal truncated protein had no binding activity at all, the deletion of NSL didn’t affect the binding, but deletion of the two RRM could abort the binding activity to about 80% (Figures 2, 6). Thus, the two RRM domains are very important to maintain the U6 binding activity of Tip110.
After exiting Cajal bodies, mature snRNP enter nuclear speckles. Nuclear speckles are sites for pre-mRNA splicing that serve as storage and/or modification sites for splicing factors . Our previous digital fluorescence microscopic imaging revealed that Tip110 was detected within the nuclear speckle structure . Tip110 may shuttle from Cajal bodies to nuclear speckles, likely along with the mature spliceosomes for pre-mRNA splicing, as both Cajal bodies and nuclear speckles are enriched in the snRNP U1, U2, U4, U5, and U6 . Tip110 was shown to bind to RNPS1 at its C terminus and could be involved in constitutive and alternative pre-mRNA splicing . RNPS1 is expressed in nuclear speckles and found in the active spliceosomes and functions as a general activator of pre-mRNA splicing [49, 50]. Our current study showed that Tip110 played important functions in pre-mRNA splicing (Figures 3, 4, 5) and was present in the spliceosomes (Figure 7). Therefore, it is likely that Tip110 interaction with RNPS1 and co-localization in nuclear speckles structure are also important for Tip110 function in pre-mRNA splicing. Transcription and pre-mRNA splicing are believed to be coupled by the C-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) . CTD phosphorylation is required for the stimulatory effect of the CTD on splicing [51, 52]. We have previously shown that Tip110 bound to the C-terminal domain of unphosphorylated RNA polymerase II in a direct and specific manner and Tip110 expression was associated with increased phosphorylation of serine 2 of the heptapeptide repeats within the RNAPII C-terminal domain . Thus, Tip110 association with unphosphorylated RNAPII CTD provides another line of evidence to support direct involvement and function of Tip110 in pre-mRNA splicing.
To further determine the significance of Tip110 involvement in pre-mRNA splicing, we performed RNA-Seq analysis of the alternate pre-mRNA splicing transcripts. As expected, Tip110 knockdown led to alterations in a large number of the gene transcripts including four known genes in pre-mRNA splicing (Figure 8). Tip110 knockdown significantly decreased expression of PRPF4, PRPF38b, SNRPB2, and ZRSR2. PRPF4 is a component of the U4/U6 di-snRNP and the U4/U6.U5 tri-snRNP and is important for maintaining the pre-mRNA splicing efficiency . SNRPB2 is also important for maintaining the pre-mRNA splicing efficiency . ZRSR2 is a splicing factor engaged in the initial steps of pre-mRNA splicing, including 3′ splice-site recognition . PRPF38b interacts (directly or indirectly) with at least one of the U6 snRNA-containing snRNP complexes and is required for pre-mRNA splicing . These findings suggest that Tip110 regulation of these key pre-mRNA splicing factors, is, if not more, as important as its interaction with snRNA U6 in pre-mRNA splicing. Tip110 is barely detectable in normal tissue and cells and highly elevated in tumors and differentiating stem cells [20, 25]. Thus, it is possible that Tip110 function in pre-mRNA splicing is evident only in cancerous cells and tumors through which elevated Tip110 expression causes changes in pre-mRNA splicing and gene expression and subsequent stem cell differentiation and malignant transformation.
YL, JF and ZW performed experiments; YL and JH designed experiments and analyzed data; YL and JH wrote the manuscript. All authors read and approved the final manuscript.
We would like to thank Dr. Marvin Wickens for pIIIA.MS2 plasmid, Dr. Iain Mattaji for pT7.U6 plasmid, Dr. Thomas Hope for pDM138 plasmid, Dr. Bryan Cullen for pcRev, Dr. Adrian Krainer for pSP64-Hβδ6 plasmid, and Dr. Gregory Hannon for pSHAG-1 plasmid.
Compliance with ethical guidelines
Competing interests The authors declare that they have no competing interests.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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