PremRNA Splicing in the Nuclei of Xenopus Oocytes Kyong Hwa Moon Xinliang Zhao and YiTao Yu

Summary

Xenopus oocytes have been utilized in a number of laboratories as an experimental system to study a variety of biological processes. Here, we describe its application to functional studies of spliceosomal small nuclear RNAs (snRNAs) in pre-messenger RNA (pre-mRNA) splicing, a process that occurs extremely efficiently in Xenopus oocytes. A DNA oligonucleotide complementary to an snRNA of interest is injected into the oocyte cytoplasm. The oligonucleotide subsequently diffuses into the nucleus and hybridizes to the target snRNA, thereby triggering snRNA degradation via endogenous RNase H activity. By the time the endogenous snRNA is depleted, the DNA oligonucleotide itself is degraded by endogenous deoxyribonuclease (DNase) activity. In principle, this procedure enables one to quantitatively deplete any snRNA of choice. Subsequently, a rescuing snRNA that is constructed in vitro may be injected into the snRNA-depleted oocytes to restore the splicing function. After reconstitution, a radiolabeled splicing substrate is injected into the nuclei of the oocytes. These oocyte nuclei are then manually isolated and used to prepare both nuclear RNA for splicing assays and nuclear extract for spliceosome assembly assays. The ability of an injected rescuing snRNA to reconstitute splicing can therefore be tested. Because all types of rescuing snRNAs (e.g., mutant snRNAs, snRNAs with or without modified nucleotides) can be constructed readily, the results obtained from this procedure provide valuable information on the function of a particular snRNA of interest in pre-mRNA splicing.

Key Words: Depletion-reconstitution; microinjection; nuclear isolation; pre-mRNA splicing; spliceosome; U2 snRNA; Xenopus oocytes.

1. Introduction

Most eukaryotic protein-coding genes are interrupted by introns (1). Initially copied into pre-mRNAs, the introns must be removed before the mature mRNAs can be produced and transported to the cytoplasm, where they direct the translation of proteins (2). The removal of the introns, a process termed pre-mRNA splicing, occurs in a large mul-ticomponent complex consisting of five small nuclear RNAs (snRNAs), including U1, U2, U4, U5, and U6, and a number of proteins (3,4).

From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu © Humana Press Inc., Totowa, NJ

For over two decades, several laboratories have developed a few different experimental systems to study pre-mRNA splicing (see, e.g., refs. 5-16). The most widely used are the yeast genetic system (5) and the cell-free in vitro system involving yeast cell extracts (6) or HeLa nuclear extracts (7-10). Several other systems, such as the Xenopus oocyte microinjection system (13,14,17), are largely underutilized. For several reasons, we believe that Xenopus oocytes provide an excellent experimental system for studying pre-mRNA splicing, and that its usage should be increased. First, pre-mRNA splicing occurs extremely efficiently in stage VI Xenopus oocytes (13,14,17), thus enhancing our ability to analyze this process. Second, because the oocytes are kept intact and alive during the entire course of splicing, this system closely recapitulates in vivo circumstances. Third, because Xenopus oocytes are large and contain a massive volume of cellular contents (18), including the spliceosomal components (19,20), they also constitute an excellent system for biochemical analysis. Finally, the spliceosomal snRNAs can be readily targeted for degradation by endogenous RNase H activity following microinjection with complementary deoxyribonucleic acid (DNA) oligonucleotides (14,17,21-23). Injecting rescuing snRNA into the depleted oocytes can then restore the function of degraded snRNA. These features offer an excellent opportunity to study, in great detail, the function of snRNAs in pre-mRNA splicing (13,14,17).

In this chapter, we describe how the Xenopus oocyte system can be utilized to study specifically the function of U2 snRNA in pre-mRNA splicing. This procedure involves several basic steps, including three injections, nuclear isolation, and splicing assay (Fig. 1). In the first injection, an antisense U2 DNA oligonucleotide is injected into the cytoplasm of Xenopus oocytes. The oligonucleotide subsequently diffuses into the nucleus and hybridizes with the endogenous U2 snRNA, thereby triggering the degradation of the U2 snRNA via endogenous RNase H activity. When the endogenous U2 snRNA is depleted, the DNA oligonucleotide is also degraded via endogenous deox-yribonuclease (DNase) activity. In the second injection, a rescuing U2 snRNA (wildtype U2 or modified U2) is injected into the cytoplasm of U2-depleted oocytes. Because the U2 transcript contains a 5' GpppG cap and an Sm-binding site, it is transported to the nucleus (24), where it restores U2 function (if the injected U2 is functional). In the last injection, a radiolabeled pre-mRNA, such as the standard adenovirus pre-mRNA, is injected directly into the nuclei of the reconstituted oocytes to monitor pre-mRNA splicing and the effectiveness of U2 rescue. One hour after nuclear injection, the oocyte nuclei are isolated, total nuclear RNA are extracted, and pre-mRNA splicing is assayed (a denaturing gel analysis). The nuclear extract can also be prepared from the oocyte nuclei for spliceosome assembly assays (a native gel analysis). The entire procedure, along with additional preparatory steps, is described in detail in this chapter.

2. Materials 2.1. Reagents

2. Gentamicin sulfate (Sigma; store at 4°C)

Xenopus Oocyte

Antisense U2 DNA Oligo

1st Injection

Rescuing U2 (Wt-U2 or 5FU-U2)

2nd Injection

Radiolabeled pre-mRNA

3rd Injection

Nuclear Isolation RNAs

^ Splicing Assay

Splicing Gel

Fig. 1. The Xenopus oocyte microinjection system. The depletion-reconstitution procedure consists of three sequential microinjections followed by nuclear isolation and splicing gel analysis. In the first injection, 50.6 nL of an antisense U2 DNA oligonucleotide (3 |lg/|lL) complementary to the branch site recognition region of U2 snRNA are injected into the cytoplasm of Xenopus oocytes, which are then incubated at 18°C overnight. During the incubation, the oli-gonucleotide readily diffuses into the nucleus and base-pairs with the endogenous U2 snRNA. This base-pairing induces the endogenous RNase H activity that degrades the U2 strand of the RNA/DNA hybrid. Soon after the depletion of U2 snRNA, the DNA oligonucleotide is also degraded. In the second injection, 50.6 nL of the newly transcribed wild-type U2 snRNA or 5FU-substituted U2 snRNA (50 ng/|L) are injected into the cytoplasm of the U2-depleted oocytes. The oocytes are then incubated overnight to allow sufficient time for the completion of U2 reconstitution. Because the U2 snRNAs contain a 5' GpppG cap and an Sm-binding site, they are quickly assembled, 5' hypermethylated, and transported into the nucleus (24). In the third injection, 13.8 nL of radiolabeled pre-mRNA (500,000 cpm/|L) are injected directly into the nuclei of the reconstituted oocytes. After 1 h, the nuclei are isolated, and total nuclear RNA is recovered and analyzed on a denaturing gel.

6. GpppG (guanosine-5'-triphosphate-5'-guanosine), GTP (guanosine-5'-triphosphate), ATP (adenosine-5'-triphosphate), CTP (cytidine-5'-triphosphate), UTP (uridine-5'-triphos-phate) (Amersham; store at -20°C).

7. 5-Fluorouridine 5' triphosphate (5FUTP; Sierra Bioresearch; store at -20°C).

8. [a-32P]GTP (guanosine-5'-[a-32P]-triphosphate) (NEN; store at -20°C).

Fig. 2. The plasmids containing a Xenopus wild-type U2 gene (A) or a standard adenovirus pre-mRNA sequence (B) are shown.

2.2. Equipment

1. Nanoject II (cat. no. 3-000-204; Drummond Scientific Company).

2. Zoom stereo microscopy (Olympus SZ-STB1).

3. Small scissors.

4. Forceps (Dumont).

5. Suturing kit.

7. Flaming/Brown Micropipette Puller (Sutter Instrument Co., model P-97).

8. 7-in. Drummond 100 replacement tubes (cat. no. 3-00-203-G/XL; Drummond Scientific Company).

9. Scintillation counter (cat. no. LS 6000SC; Beckman).

2.3. Solutions

1. 2X Benzocaine buffer: dissolve 0.3 g powdered benzocaine (cat. no. 1080-01; J. T. Baker) in 10 mL ethanol first and then add deionized water to 500 mL.

2. OR2 (oocyte Ringer's medium) buffer: 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.5. Sterilize using a 0.22- to 0.45-|lm filter.

3. Collagenase type I solution: dissolve collagenase (cat. no. 234153; Calbiochem) in OR2 buffer to bring the final concentration to 1 mg/mL (make fresh).

4. 10X Modified Barth's solution (MBS) Buffer: 0.8 M NaCl, 10 mM KCl, 8 mM MgSO4, 24 mM NaHCO3, and 10 mM HEPES, pH 7.4 (add 700 |L 1M CaCl2 into 100 mL 10X MBS buffer before use).

5. 5:1 Isolation buffer: 83.0 mM KCl, 17.0 mM NaCl, and 6.5 mM Na2HPO4, and 3.5 mM KH2PO4 (add 500 |L of 1M MgCl2 and 50 |L of 1M dithiothreitol (DTT) into 50 mL 5:1 isolation buffer before use).

6. 10X T7 transcription buffer: 0.4M Tris-HCl, pH 7.5, 100 mM NaCl, 60 mM MgCl2, and 20 mM spermidine.

7. G50 buffer: 20 mM Tris-HCl, pH 7.5, 300 mM NaAc, 2 mM EDTA (ethylenediamine-tetraacetic acid), and 0.3% sodium dodecyl sulfate (SDS).

3. Methods

3.1. Synthesis of Xenopus U2 snRNA and Adenovirus Pre-mRNA

Two types of RNA, including U2 snRNA (functional and nonfunctional) and radiolabeled pre-mRNA, are needed in the following procedure. To synthesize these RNAs, an Sma I-linearized pT7-U2 plasmid, containing a Xenopus U2 snRNA gene under the control of a T7 promoter, and a BamH I-linearized pT7-Ad plasmid, containing a standard adenovirus pre-mRNA sequence under the control of the T7 promoter, are used as templates for T7 transcription in vitro (Fig. 2).

3.1.1. T7 Transcription of U2 snRNA In Vitro

1. At room temperature, set up a 100- to 200-|lL transcription reaction containing 1.2 mM each of GpppG, ATP, CTP, and UTP (for functional wild-type U2) or 5FUTP for altered U2 snRNA with all uridines substituted with 5-fluorouridines (5FU), 0.3 mM GTP, 0.005 | Ci/|L [a-32P]GTP (see Note 1), 40 mM Tris-HCl, pH 7.5, 6 mM MgCl2, 2 mM spermidine, 5 mM DTT, 0.1 |g/|L of Sma I-linearized T7-U2 plasmid, and 4 U/|L T7 phage polymerase.

2. Remove 1 | L of the reaction and determine the radioactivity (cpm) using a scintillation counter (see Note 1).

3. Incubate the rest of the reaction (99-199 |L) at 37°C for 1 h.

4. Extract U2 snRNA transcript with PCA (Tris-HCl [pH 7.5]-buffered phenol:chloro-form:isoamyl alcohol [50:49:1]) and precipitate it with ethanol.

5. Resolve the precipitated U2 snRNA sample on a 6% polyacrylamide-8M urea gel (Fig. 3, lane 1).

6. Identify U2 band using autoradiography.

7. Excise the trace-labeled U2 band and transfer the gel slice to a new 1.5-mL microfuge tube.

8. Add 450 |L of G50 buffer and quickly freeze the sample in dry ice for 5 min.

9. Elute U2 snRNA at room temperature overnight.

10. Extract eluted U2 snRNA with PCA and precipitate it with ethanol (see Note 2).

Fig. 3. In vitro transcription of U2 snRNA and adenovirus pre-mRNA. Trace-radiolabeled wild-type U2 snRNA (lane 1) and 5FU-sustituted U2 snRNA (lane 2), as well as radiolabeled standard adenovirus pre-mRNA (lane 3) are transcribed in vitro. After transcription, RNAs are resolved on a 6% polyacrylamide-8M urea gel and exposed to a PhosphorImager screen for 5 min for wild-type U2 snRNA and 5FU-U2 snRNA and 30 s for pre-mRNA. The wild-type U2 snRNA, 5FU-U2 snRNA, and pre-mRNA bands (indicated on the right) are identified and excised, and RNAs are recovered. The numbers on the left are size markers of MspI-digested pBR322 DNA.

Fig. 3. In vitro transcription of U2 snRNA and adenovirus pre-mRNA. Trace-radiolabeled wild-type U2 snRNA (lane 1) and 5FU-sustituted U2 snRNA (lane 2), as well as radiolabeled standard adenovirus pre-mRNA (lane 3) are transcribed in vitro. After transcription, RNAs are resolved on a 6% polyacrylamide-8M urea gel and exposed to a PhosphorImager screen for 5 min for wild-type U2 snRNA and 5FU-U2 snRNA and 30 s for pre-mRNA. The wild-type U2 snRNA, 5FU-U2 snRNA, and pre-mRNA bands (indicated on the right) are identified and excised, and RNAs are recovered. The numbers on the left are size markers of MspI-digested pBR322 DNA.

11. Determine the radioactivity (cpm) using a scintillation counter and quantitate the amount of U2 snRNA transcript (see Note 1). Typically, the reaction produces about 3 to 6 |lg of 5' capped wild-type U2 snRNA (Fig. 3, lane 1) or 5' capped 5FU-U2 snRNA (Fig. 3, lane 2). Both U2 snRNAs are made in parallel to test their ability to reconstitute pre-mRNA splicing.

12. Dissolve the U2 snRNA transcript in RNase-free water at 50 ng/|L.

3.1.2. T7 Transcription of Adenovirus pre-mRNA In Vitro

1. At room temperature, set up a 10- to 25-|lL transcription reaction containing 1.2 mM each of GpppG, ATP, CTP, UTP, 0.3 mM GTP, 2 |Ci/|L [a-32P]GTP, 40 mM Tris-HCl, pH 7.5, 6 mM MgCl2, 2 mM spermidine, 5 mM DTT, 0.1 |g/|L of BamH I-linearized T7-Ad plasmid, and 4 U/| L T7 phage polymerase.

2. Carry out the reaction and gel purify the pre-mRNA according to the procedure described for U2 snRNA transcription in Subheading 3.1.1. Suspend the pre-mRNA in RNase-free water at 500,000 cpm/| L (Fig. 3, lane 3).

Fig. 4. The frog should be cut at the lower abdomen as indicated.
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