Evidence of Two-Step Recursive
Pre-mRNA Splicing In Vitro
Ken Seldeen
Department of Biochemistry and Molecular Biology
University of Miami School of Medicine
A rotation student’s project in Dr. Mayeda’s laboratory
February 11, 2005
Approved by ____________________________________________________
Mentor
Akila Mayeda, Ph.D.
Ken Seldeen
ABSTRACT
Precise removal of introns from nascent gene transcripts, or pre-mRNAs, by
splicing is a crucial step during gene expression in eukaryotes. Large size genes
containing very long introns are prevalent in human genome, however, little is
known about the splicing mechanism of extremely long introns.
Recursive
splicing, i.e., the stepwise removal of introns by sequential re-splicing at spliced
junctions, was discovered in the long intron of a fruit fly (Drosophila
melanogaster) gene in 1988. We assume that the recursive splicing is one of the
reasonable splicing mechanisms for long introns in human (Homo sapiens)
genes, however, the evidence has not been reported to date.
To study the mechanism of recursive splicing, we have been trying to
reconstitute this phenomenon in vitro with minimal model pre-mRNA.
The
Mayeda lab previously constructed a modified -globin mini-gene, which contains
one recursive splice site (a 3’ splice site adjacent to a 5’ splice site). Using this
transcript as a minimal substrate, in vitro splicing with HeLa cell nuclear extracts
was performed.
(1) Besides the final spliced mRNA, we observed upstream
partial spliced product (between the authentic 5’ splice site and the RSS).
However, no downstream partial spliced product (between the RSS and the
authentic 3’ splice site) was detected even by sensitive RT-PCR assays. (2) Point
mutation of 3’ splice site sequence (AGGG) generates no upstream partial
spliced product. (3) Point mutation of 5’ splice site sequence (GUAU) in RSS
generates
more
upstream
partial
spliced
product.
(4)
The
substrate
corresponding to the upstream partial spliced RNA was further spliced to the final
mRNA in vitro. All these data strongly indicated that, at least, a portion of the
final spliced mRNA is generated in a two-step sequential process mediated by
recursive splicing at the RSS.
This is the first demonstration to reconstitute
recursive splicing in vitro, and our system provides a useful tool to identify and
characterize the trans-acting factors that are specific in recursive splicing.
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INTRODUCTION
Most of eukaryotic messenger RNA precursors (pre-mRNAs) are interrupted by
introns, which are not included in the final spliced mRNA.
Pre-mRNA splicing is
essential process to remove intron and generate mature mRNA, which is a template for
the translation.
Pre-mRNA splicing requires both small nuclear ribonucleoprotein
particles (snRNPs) and many non-snRNP protein factors. These factors assemble into
a large complex on the pre-mRNA known as a spliceosome, in where splicing reaction
catalyzes (reviewed in Krämer, 1996; Burge et al., 1999). The splicing machinery must
recognize exon/intron boundaries with high fidelity, so that cleavage and re-joining can
be made precisely at the right position. However, important signal sequences around
the intron/exon junctions, the 5’ and 3’ splice sites and branch site (reviewed in Burge et
al., 1999) are not highly conserved, and thus they are not sufficient for the accurate
splicing.
The
biochemical
steps of splicing involve
two consecutive transesterification
(Fig.
1;
reactions
reviewed
in
Fig. 1. Two-step catalytic process for pre-mRNA splicing.
A pre-mRNA with a single intron is shown at left, with two exons shown as
boxes and the intron shown as a line (modified from Burge et al., 1999).
Burge et al., 1999). The
first step is a cleavage that occurs at the 5’ splice site, and the phosphate at the 5’-end
of the intron links to the 2’-OH of an adenine nucleotide, which called the branch site.
The branch site is usually located 20–40 nucleotides (nt) upstream from the 3’ splice
site. The second step is a cleavage that occurs at the 3’ splice spice site, and the 5’phosphate of the down-stream exon joins to the 3’-OH of the upstream exon.
Eventually, two exons are re-joined and intron is released as a lariat form (reviewed in
Krämer, 1996; Burge et al., 1999).
In Eukaryotes, very large genes with long introns are very prevalent (International
Human Genome Sequencing Consortium, 2001; 2004; Venter et al., 2001).
For
instance, many genes that play important roles in development and human disease,
such as cystic fibrosis, retinoblastoma, muscle dystrophy, neurofibromatosis, are very
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Ken Seldeen
large with many long introns. Little is known about
mechanisms that facilitate the accurate and efficient
splicing of extremely long introns.
However, it is
technically very demanding to assay pre-mRNA
splicing of long introns.
Our project is one of the
possible approaches to elucidate splicing mechanism
of such long introns.
Recursive splicing, i.e., the stepwise removal of
intron by sequential re-splicing at spliced exon-exon
junctions, was discovered in the very long intron of
the Ultrabithorax (Ubx) gene of D. melanogaster (Fig.
2B; Hatton et al., 1998). The members of the Mayeda
lab have been examining possible recursive splicing
in intron 7 (109,574 bp) of the human dystrophin
(DMD) gene. During the course of this research, they
unexpectedly found evidence of a novel mechanism,
Fig. 2. Three splicing pathways for
long intron.
(A) Conventional one-step splicing.
(B) Recursive multi-step splicing,
which was originally found in D.
melanogaster. (C) Nested-intron multistep splicing. 5’ ss: 5’ splice site, 3’ss:
3’ splice site. 3’ ss/5’ss: re-splicing site
(RSS).
termed nested-intron splicing: i.e., two putative
nested-intron lariats have been detected in intron 7 by reverse transcription-polymerase
chain reaction (RT-PCR; H. Suzuki & A. Mayeda, unpublished data).
The nested splicing is the multiple sequential splicing of the internal intron followed
by the eventual authentic splicing via 5’ and 3’ splice sites (Fig. 2C). We assume short
introns can be easily spliced out by a conventional one-step splicing (Fig. 2A), thus if
there are many short nested introns, or potential 5’ and 3’ splice sites, within the long
intron, a series of multiple nested splicing can shorten gradually the whole long intron
until it becomes short enough to be spliced via authentic 5’ and 3’ splice site eventually
(Fig. 2C).
We predict that the nested splicing, together with possible recursive splicing, may be
a general mechanism for splicing of extremely long introns in human genes.
It is
conceivable that the involved mechanism and factors might be distinct from those of
authentic splicing, since the nested splicing should be a multi-step sequential splicing
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Ken Seldeen
event whereas the conventional splicing essentially completes its process by re-joining
of exons.
As a rotation student’s project, I focused on the possible recursive splicing in human
gene. To elucidate the mechanism of recursive splicing, it is crucial to reconstitute in
vitro splicing with a minimal model pre-mRNA that can be spliced in two-step through
one inserted recursive splice site.
Following the studies of previous two rotation
students, we eventually obtained the first evidence that recursive splicing takes place in
vitro.
MATERIALS AND METHODS
Transformation of E. coli
Plasmid (1 µg) was mixed into 200 µl of competent E. coli cells (strain DH5) and
incubated on ice for 30 min followed by immediate heat at 42ºC for 1 min. The mixture
was then put on ice for 2 min and added 150 µl SOC (2% Tryptone, 0.5% Yeast Extract
10 mM NaCl, 10 nM MgSO4, 10mM MgCl2). The transformed E. coli was spread on LB
agar plates containing 50 µg/ml ampicillin and incubated at 37ºC overnight.
Preparation and plasmids and templates
Single colony from the transformants was inoculated in LB medium with 50 µg/ml
ampicillin and incubate at 37ºC overnight for the preparation of plasmid. Plasmids were
prepared by plasmid midi-preparation kit according to the manufacturers protocol
(Qiagen). The plasmid DNA yield was determined by measuring the UV absorbance at
260 nm by spectrophotometer (Ultraspec 2100, Amersham).
To prepare template DNA for in vitro transcription, the plasmid was digested at 36ºC
for 1 h with BamHI using appropriate reaction buffer (New England Biolabs).
The
digested plasmid was checked by agarose gel electrophoresis to confirm the right
plasmid.
Digested DNA samples were analyzed on 1% agarose gels (mini-size).
Electrophoresis was performed at 100 mV for 20 min with 0.5 X Tris-acetate EDTA
(TAE) buffer. Agarose gel was stained with 0.5 µg/ml ethidium bromide and visualized
under UV light.
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Ken Seldeen
1/10 volume of 3 M sodium acetate and an equal volume of phenol chloroform were
added to the digestion reaction with restriction enzyme and shook by Vortex for 10 sec.
After micro-centrifugation for 5 min at 12,000 rpm (Tomy), the supernatant was saved to
a fresh tube. An equal volume of chloroform was added and centrifuged again for 5
min. The supernatant was transferred to a fresh tube, 2.5 volume of chilled ethanol was
added, and kept at –80ºC for 15 min to precipitate DNA. After micro-centrifugation at
12,000 rpm for 15 min at 4ºC, the supernatant was carefully removed and the pellet was
dissolved in water to concentration of 1 µg/µl.
Plasmid construction for model splicing substrates
Previous lab work showed that the cryptic 5’ splice sites upstream of the authentic 5’
splice site (in the first exon of (-globin mini-gene; Krainer et al., 1984) are activated in
the model substrate with only 5’ half of introns. To avid the activation of upstream two
cryptic 5’ splice sites, I created the GTAT point mutations in the original plasmid
constructs.
PCR was performed with 100 ng of each of the 4 plasmids (all from
previous lab work): pSP64-HUX2-AG/GT, pSP64-HUX2-AG/AT, pSP64-HUX2GG/GT, and pSP64-HUX2-AG/GT∆5’intron (modified plasmid with 5’ half of intron
removed). Custom DNA primers for PCR were purchased (Qiagen). The primers used
have been designed according to the sequence upstream and downstream of the first
exon: Mutation Primer-S 5’-GTGGGGCAAGaTGAACGTGGATGAAGTTGGTGaTGAGGCCCTG-3’; Mutation Primer-AS, 5’- CAGGGCCTCAtCACCAACTTCATCCACGTTCAtCTTGCCCCAC-3’. Lower-case bold letters represent location of the base mutations.
The reaction mixture (50 µl) contained: 100 pmol primers Mutation Primer-S and
Mutation Primer-AS (dissolved in 1 X TE buffer), 0.5 mM dNTP (4 kinds) mixture, 100
ng pSP64-HUX2-AG/GT, and 20 units Pfu Turbo polymerase with Pfu DNA
polymerase buffer (Stratagene). All the PCR conditions for each cycle were 94ºC for 30
sec, 60ºC for 30 sec, and 68ºC for 10 min, and a total 19 cycles were performed. After
PCR is completed, dNTP and primers were removed by S-300 microspin column
(Amersham) according the manufacturers protocol. The PCR product was digested
with 40 units of DpnI at 37C for 1 h, followed by phenol/chloroform extraction.
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Ken Seldeen
PCR product was treated with T4 DNA polynucleotide kinase (New England Biolabs)
and subcloned into pSP64 vector (Promega). Ligation was performed with T4 DNA
ligase (New England Biolabs) at 16ºC for 1–2 h. After ligation of the plasmid, 5 µl of the
reaction was directly used for the transformation of E. coli. The medium-scale plasmid
preparation was carried out to obtain plasmids termed pSP64-AGGT, pSP64-AGAT,
pSP64-GGGT, pSP64-2nd Splice. The concentration of the plasmid was measure by
UV absorbance. ~1 µg of each plasmid was digested with 2 units of BamHI and HindIII
at 37ºC for 1 h. Digested samples were checked by agarose gel electrophoresis to
confirm whether the plasmid contained the correct extended intron. Digested plasmid of
the previous construct was used as a control. DNA size markers used were 0.5 µg of
100 bp and 1 kbp DNA ladders (New England Biolabs). The plasmids were further
verified by sequencing (DNA Core Lab or Michigan University).
Preparation of 32P-labeled pre-mRNA
50
µg
of
the
pSP64-AGGT,
pSP64-AGAT,
pSP64-GGGT,
and
pSP64-
AG/GT∆5’intron plasmids were digested with BamHI (1.5 units) at 37C overnight. DNA
was extracted with phenol/chloroform and precipitated with ethanol. The DNA pellets
were air dried and dissolved in 1 X TE buffer (to final concentration of 1 µg/µl).
The linearized plasmids were used as template of run-off transcription in vitro
(Mayeda & Krainer, 1999b). The transcription reaction (25 µl) was performed with 0.5
mM ATP/CTP mixture, 50 µM GTP/UTP mixture, 2 mM GpppG cap analog (New
England Biolabs), 1 unit of RNase inhibitor (PRIME), 1 µg of DNA template, 25 µCi [32P]
UTP and 10 units SP6 RNA polymerase with SP6 polymerase buffer (New England
Biolabs). The reaction mixture was incubated at 40ºC for 1.5 h, followed by 10 min
incubation with 15 units RQ DNase (Promega) to degrade template DNA.
After
incubation, 150 µl H2O, 50 µl 7.5 M ammonium acetate and 150 µl Tris-saturated
phenol were added and immediately shook by vortex for 2–3 min.
After micro-
centrifugation for 5 min at 12,000 rpm, the aqueous phase was recovered to a fresh
tube. To precipitate RNA, 0.5 ml 100% ethanol and 15 µg glycogen were added and
kept at 4ºC for at least 10 min. After micro-centrifugation at 12,000 rpm for 15 min at
4ºC, the supernatant was carefully removed and the pellet was washed with 80%
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Ken Seldeen
ethanol. The RNA pellet was dried for 5 min under vacuum and resuspended in 100 µl
10 mM Tricine-HCl (pH 7.6).
The yield and concentration of RNA transcript was
estimated by TCA-insoluble scintillation counting.
In vitro splicing assays
Preparation of HeLa cell nuclear and cytosolic S100 extracts, and in vitro splicing
reaction were described previously (Mayeda & Krainer, 1999a; 1999b). Splicing buffer
mixture was made in a batch, according to the desired number of reactions: the content
per one reaction was 1 µl 12.5 mM ATP / 0.5 M creatine phosphate mixture, 1 µl 80 mM
MgCl2, 1.25 µl 0.4 M Hepes-KOH (pH 7.3), 5.0 µl 13% polyvinyl alcohol (which was
added last due to viscosity), 20 fmol of
32P-labeled
pre-mRNA, and H2O to 10 µl total.
Splicing reaction mixture (in 25 µl) was prepared on ice with 8 µl of HeLa cell nuclear
extract (or 8 µl S100 extract plus 10 pmol or 20 pmol of recombinant SF2/ASF). Buffer
D [20 mM Hepes-NaOH (pH 8.0), 100 mM KCl, 0.2 mM EDTA, 20%(v/v) glycerol, 0.5
mM PMSF, I mM DTT] was added to make a volume to 15 µl. Then 10 µl of splicing
buffer mixture was added, mixed gently, and incubated at 30ºC for 1–4 h or otherwise
stated.
The reactions were terminated by adding 175 µl of splicing stop solution [0.3 M
sodium acetate (pH 5.2), 0.1%(v/w) SDS, 62.5 µg/ml tRNA] immediately followed by
phenol extraction. After micro-centrifuged for 5 min at 12,000 rpm, the upper aqueous
layer was removed carefully and then RNA was precipitated with 0.5 ml ethanol (and
kept at –80ºC for at least 10 min). Human -globin pre-mRNA, transcribed from BamHI
cleaved pSP64-H6 (Krainer et al., 1984), was used as a positive control.
Analyses of splicing products
The ethanol-precipitated samples were micro-centrufuged for 15 min and ethanol
was carefully removed. The RNA pellet was dissolved in 3.5 µl RNA dye mixture [90%
(v/v) formamide, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% Bromophenol blue, 0.1%
Xylene cyanol FF], heated at 80ºC for 5–10 min, and immediately loaded onto 5.5%
polyacrylamide/7 M urea gel electrophoresis (denaturing PAGE). 100 bp and 1 kbp
DNA markers (New England Biolabs), which were radio-labeled with [32P] ATP and T7
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Ken Seldeen
polynucleotide kinase, were used as size markers.
The electrophoresis was done at constant voltage at
700 V for 100 min.
Gel was transferred to
presoaked 3M paper (Wattman) and then dried by
gel dryer at 85ºC for 1.5–2 h. Autoradiography was
done with intensifying screen at –80ºC for 2–3 h.
RESULTS
Preparation of model recursive splicing
Fig. 3. In vitro splicing pathway of the
original model pre-mRNA.
constructs
The original model -globin mini-gene contains a
strong RSS, which was derived from intron 7 of the
Dystrophin gene (G.R. Screaton & A. Mayeda,
unpublished).
In vitro splicing of this pre-mRNA
generates two spliced products, the partially spliced
product of the 5’ half of the intron (splicing between
the authentic 5’ splice site and 3’ splice site of the
RSS) and the final spliced product (Fig. 3). There
exist two possible pathways to generate this final
product, one from the pre-mRNA via authentic 5’
and 3’ splice sites (Fig 3, “Conventional Splicing”)
and the other from the partially spliced product (Fig
3, “Resplicing?”).
To demonstrate sequential
recursive splicing in two steps, it is important to
show, at least, the partially spliced pre-mRNA (455
nt) can be spliced to the final spliced product (365
nt). Preliminary in vitro splicing results of the first
spliced substrate (Fig 4, “First Splice Substrate”)
have shown that cryptic 5’ splice sites upstream of
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Fig. 4. mRNA substrates used for in
vitro splicing or are a product of in vitro
splicing. Observed In vitro splicing
marked by black lines. Red X’s mark
areas where splicing was not observed.
Ken Seldeen
the
authentic
5’
splice
sites
were
activated
(T.
Venkataraman & A. Mayeda). The unexpected activation
of the cryptic 5’ splice sites (without any mutation on
authentic 5’ splice site) may be due to the shortness of
the introns (88 nt). Thus, two cryptic 5’ splice sites were
abrogated with a single base change from GT to AT
using the site directed mutagenesis. This was successful
and no evidence of the use of the cryptic 5’ splice splices
was found during the following experiments.
In vitro splicing analysis
Transcripts from the plasmid constructs were splice in
vitro with HeLa cell nuclear extracts. The splicing
products were analyzed by denaturing PAGE followed by
autoradiography. The splicing process of the wild-type
(AGGT) pre-mRNA was observed over the time course
Fig. 5. Results of in vitro splicing
of the wild-type AGGT substrate.
Generated splicing products were
indicated by their schematic
structures (see Fig 4).
(1, 2, 3, 4 h; Fig 5). Besides unspliced pre-mRNA, two
discrete bands were visible that are corresponding to
the first spliced product and final spliced product. In
the wild-type substrate (WT), both these bands are
increasing over time, while unspliced pre-mRNA is
descreasing.
Using the same splicing conditions, mutant
substrates (MUT1 and MUT2) were also analyzed in
vitro over the time course (1, 3 h; Fig. 6). In MUT2
substarte, a band corresponding to the first spliced
product is missing. This is due to the mutation in the
3’ splice site of RSS that prevents a splicing in the 5’
half of intron.
In MUT1 substrate, the first spliced
product is more accumulated compared with that of
WT substrate.
This observation suggests that the
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Fig. 6. Results of in vitro splicing of
the mutant substrates.
Predicted
splicing products were indicated by
their schematic structures (see Fig 4).
Ken Seldeen
sequential second splicing in WT substrate is actually
takes place (besides conventional direct splicing to the final
product; see Fig. 3), because the accumulation of the first
spliced product could be due to the prevention of the
second splicing in this mutant (5’ splice site mutation in
RSS forces to prevent the second splicing).
Finally, it is necessary to demonstrate that splicing can
occur from the first spliced product to generate the final
spliced product. Therefore, in vitro splicing was performed
in the substrate corresponding to the first spliced product
(“First Splice Substrate” in Fig. 4).
The substrate was
spliced in vitro over the time course (1, 2, 3, 4 h) and we
detected increasing amounts of the final spliced product,
which has an identical mobility of the spliced product of
control -globin pre-mRNA (Fig. 7). It is also noted that
Fig. 7.
Results of in vitro
splicing of the first spliced
substrate.
Predicted splicing
products were indicated by their
schematic structures (see Fig
4).
bands just below the final spliced product are not the spliced products via cryptic 5’
splice sites but rather non-specific cleaved products, since the bands were generated in
constant levels throughout the reaction from 1 h through 4 h.
DISCUSSION
Two essential findings came from the research providing convincing evidence for the
recursive splicing model (Fig. 8A). (1) The first splicing takes place exclusively between
the 5’ splice site and the 3’ splice site of the inserted RSS (whereas the splicing
between the 5’ splice site of the inserted RSS and the 3’ splice site does not occur). (2)
The substrate corresponding to the first spliced product can be spliced to the final
spliced product. These two results indicate, although not prove, two-step recursive
splicing through inserted RSS (Fig. 8A). We cannot rule out the possibility that the
second splicing takes place independently, but not sequentially after the first splicing.
To provide definitive proof of the sequential splicing via RSS, it is essential to detect
the lariat RNA arising from the subsequent splicing of the first spliced product using the
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Ken Seldeen
wild-type (AGGT) substrate (Fig. 8A). Since
the first splicing does not occur from the 3’
half of intron, we can assume that the lariat
RNA is exclusively generated by the second
splicing from the first spliced product. We will
first try direct detection of these lariat RNAs
(either lariat intermediate or lariat intron) on
PAGE gel.
Since a higher percentage of
polyacrylamide (e.g., 9%) facilitates the
detection of lariat RNAs (whose gel mobility
are much lower than the linear RNAs), we will
use both usual 5% and higher 9% denaturing
page to detect these lariat RNAs.
If we
cannot detect by this direct method (because
of the low yield of these lariat products), then
we will use sensitive RT-PCR detection.
Previously, it was shown that lariat RNA can
Fig. 8. (A) The pathway of two-step recursive
splicing and expected generation of lariat
products. (B) The RT-PCR detection of lariat
RNA.
be selectively detected by RT-PCR across the branch site (Fig. 8B; Lorsch et al., 1995;
Vogel et al., 1997). If we could get expected size of RT-PCR product and the sequence
was verified, this would provide a final proof of our recursive splicing hypothesis.
ACKNOWLEDGEMENTS
I thank Dr. Hitoshi Suzuki for his helpful advice and patience in training me in the
various experiments needed to complete this project. I am grateful to my advisor Dr.
Akila Mayeda to support my research during my rotation period.
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REFERENCES
Burge, C.B., Tuschl, T. & Sharp, P.A. (1999). Splicing of precusors to mRNAs by the
spliceosomes. In Gesteland, R.F., Cech, T.R. and Atkins, J.F. (ed.), The RNA world, Second
edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp. 525-560.
Hatton, A.R., Subramaniam, V. & Lopez, A.J. (1998). Generation of alternative Ultrabithorax
isoforms and stepwise removal of a large intron by resplicing at exon-exon junctions. Mol. Cell
2, 787-796.
International Human Genome Sequencing Consortium. (2001). Initial sequencing and analysis
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International Human Genome Sequencing Consortium. (2004). Finishing the euchromatic
sequence of the human genome. Nature 431, 931-945.
Krainer, A.R., Maniatis, T., Ruskin, B. & Green, M.R. (1984). Normal and mutant human bglobin pre-mRNAs are faithfully and efficiently spliced in vitro. Cell 36, 993-1005.
Krämer, A. (1996). The structure and function of proteins involved in mammalian pre-mRNA
splicing. Annu. Rev. Biochem. 65, 367-409.
Lorsch, J.R., Bartel, D.P. & Szostak, J.W. (1995). Reverse transcriptase reads through a 2'5'linkage and a 2'-thiophosphate in a template. Nucleic Acids Res. 23, 2811-2814.
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extracts for in vitro splicing. Methods Mol. Biol. 118, 309-314.
Mayeda, A. & Krainer, A.R. (1999b). Mammalian in vitro splicing assays. Methods Mol. Biol.
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Venter, J.C., Adams, M.D., Myers, E.W., Li, P.W., Mural, R.J., Sutton, G.G., Smith, H.O.,
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D.H., Wortman, J.R., Zhang, Q., Kodira, C.D., et al. (2001). The sequence of the human
genome. Science 291, 1304-1351.
Vogel, J., Hess, W.R. & Borner, T. (1997). Precise branch point mapping and quantification of
splicing intermediates. Nucleic Acids Res. 25, 2030-2031.
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