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Construction and characterization of RRP6 deletion in Saccharomyces cerevisiae

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Construction and characterization of RRP6 deletion in Saccharomyces cerevisiae

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Content CONSTRUCTION AND CHARACTERIZATION OF
RRP6 DELETION IN SACCHAROMYCES CEREVISIAE
by
Pi-chu Kaylene Lin
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY & MOLECULAR BIOLOGY)
December 2003
Copyright 2003 Pi-Chu Kaylene Lin
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UMI Number: 1420380
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES, CALIFORNIA 90089-1695
This thesis, written by
Li'n , pNcjpu K&yl-en-e.
under the direction o f h & r thesis committee, and
approved by all its members, has been presented to and
accepted by the Director o f Graduate and Professional
Programs, in partial fulfillment o f the requirements fo r the
degree o f
Director
p ate D ecem b er 1 7 , 2003
Thesis_Committee
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ACKNOWLEDGMENTS
I would like to thank Dr. Daniel Broek for his support and advice.
I would like to thank Dr. Zoltan Tokes for Ms encouragement and help.
I would like to give my special appreciation to Dr. Raymond Mosteller for his
understanding and tremendous help. His insightful advice during our discussions of
my experiments was a great help to me.
I thank the past and present members of Dr. Broek’s laboratory for all their help and
suggestions. They are Xiaodong Shu, Balaka Das, Weicheng Wu, John Johnson, and
Youngeun Yoo.
I also like to thank many friends’ generosity in giving me helpful technical support.
They include Eileen, Erika, Eva, Jonathan, Dan, Mihaela, Yufen, Faye and the
members of Dr. Amy Lee’s lab.
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TABLE OF CONTENTS
Acknowledgments ii
List of Figures iv
Abstract v
CHAPTER 1: INTRODUCTION 1
1.1 Processing of Eukaryotic rRNA Processing 1
1.2 Processing, Export and Degradation of Eukaryotic mRNA 4
1.3 3’-5’ Exoribonuclease, Exosome and RRP6 9
CHAPTER 2: RRP6 DELETION IN S. CEREVISIAE 14
2.1 Construction of RRP6 Deletion 14
2.1.1 Plasmid Method to Construct a RP6 Deletion 14
2.1.2 PCR Method to Construct a RRP6 Deletion 17
2.1.3 LiOAc Yeast Transformation 20
2.2 Verification of RRP6::His-3 21
2.2.1 Screening by Using RCR/RFLP and Sequencing 21
2.2.2 Structural Analysis of RRP6::His-3 by Southern Blot 29
2.2.3 RT-PCR Analysis of RRP6::His-3 mRNA 33
2.3 Characterization of RRP6::His-3 35
2.3.1 Morphology Analysis of RRP6::His-3 35
2.3.2 Temperature Sensitive Growth Phenotype 38
2.3.3 Growth Curves 40
CHAPTER 3 CONCLUSION AND DISCUSSION 42
CHAPTER 4 MATERIALS AND METHODS 46
REFERENCES 54
iii
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LIST OF FIGURES
Figure 2.1 A Diagram of the RRP6 Locus 16
Figure 2.2 A Diagram of Construction of RRP6 Deletion by PCR 18
Figure 2.3 PCR Method to Generate RRP6::His-3 19
Figure 2.4 A Diagram of PCR Primer Pairs for.Screening 22
Figure 2.5 PCR Screening for RRP6::His-3 23
Figure 2.6 RFLP Analysis of RRP6::His-3 24
Figure 2.7 RFLP Analysis of RRP6::His-3 Candidates 26
Figure 2.8 PCR/RFLP Verification 27
Figure 2.9 Map of pGEM Vector 28
Figure 2.10 Structural Analysis of the RRP6::His-3 by Southern Blot 30-2
Figure 2.11 RT-PCR 34
Figure 2.12 The Morphology Analysis of RRP6::His-3 Cells 36-7
Figure 2.13 Temperature Sensitive Growth Phenotype of RRP6::His-3 39
Figure 2.14 The Growth Curves 41
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ABSTRACT
Most nascent transcripts synthesized from eukaryotic genes are immature and require
a series of processing events to produce functional RNAs. The RNAs processing
needs the activity of endonucleases and exonucleases as well as the recruitment of a
number of nuclear proteins. The series of processing steps are functionally coupled
with the nuclear transport and highly regulated. An aberrant pre-RNA is not
transported to the cytoplasm but sequestered in its transcription site. Rrp6p is a 3’-5’
exoribonuclease. Through its collaboration with the nuclear exosome complex and
various nuclear proteins, Rrp6p mediates 3’ end processing of many RNAs, and
regulates the nuclear transport system and the nuclear mRNA degradation. To have
a better understanding of the functions of Rrp6p, we constructed a RRP6 deletion,
RRP6::His-3 in the yeast SP1 and identified some of its growth phenotypes.
Although the disruption of RRP6 in the yeast is not lethal, it does show aberrant
growth characteristics. The RRP6::His-3 does slow cell growth and is sensitive to
temperature. When grown on plates, the colonies appear to be small and dense. In
liquid medium, the cells of RRP6::His-3 tend to clump together and cell numbers in
stationary phase are much higher than the normal. We conclude that certain
regulatory growth signaling mechanisms may have failed in these mutant cells.
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CHAPTER 1
INTRODUCTION
Transcription, the first step in gene expression, is a process that is highly regulated.
Most nascent transcripts synthesized from eukaryotic genes are not mature and must
undergo extensive processing steps to produce functional RNAs. The steps in
maturation of RNAs include: endonucleolytic and exonucleolytic cleavages, 5’ 7-
methylguanosine capping, 35 cleavage/polyadenylation, intron removal/exon splicing
and editing. When RNAs become mature and functional, they are actively exported
to the cytoplasm as a complex of ribonucleoproteins (RNP). The RNAs processing
is functionally coupled with the nuclear transport. As a matter of fact, each stage of
gene expression from transcription in the nucleus to protein synthesis and mRNA
degradation in the cytoplasm is closely linked, and these multiple steps of the RNA
processing and export are the evolutionary machinery for gene regulation. Rrp6p, a
3’-5’ exoribonuclease, is a component of the nuclear exosome complex which plays
an important role in the quality control of RNAs processing, the nuclear transport as
well as the nuclear mRNA degradation.
1.1 Processing of Eukaryotic rRNA
Three of four ribosomal RNAs in eukaryote are transcribed as a single precursor,
called 35S, which contains two internal transcribed spacers, ITS1 and ITS2 and two
external transcribed spacers, 5’ETS and 3’ETS. The removal of these transcribed
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spacers from the transcript to generate functional 25S, 18S and 5.8S rRNAs is the
major task in pre-rRNA processing pathway. rRNA processing requires the
activities of endonucleases and exonucleases, and the mediation of a number of
nuclear proteins, small nucleolar RNAs and the ribonucleoprotein (Briggs et al.,
1998; Venema and Tollervey, 1996; Butler, 2002). The multiple steps of rRNA
processing start with cleavages at sites AO and Al in the 5’ external transcribed
spacer (5’ETS) and site B2 from 35S pre-rRNA to form 32S pre-rRNA (Morrissey
and Tollervey, 1995). Classes of trans-acting factors that comprise a variety of small
nucleular ribonucleoprotein particles (snoRNAs) are required for the processing of 5’
region of the pre-rRNA. Depletion of any snoRNAs, e.g. U3, U14, snRlO or snR30
shows an inhibition in cleavage at sites AO, Al and A2 in the 5’ region of pre-rRNA,
which consequently prevents the formation of mature 18S rRNA (Morrissey and
Tollervey, 1995; Kressler et al., 1999; Venema and Tollervey, 1999). The yeast
gene encoding Rntlp is homologous to E. coli RNase III (RNT1). Through its
cooperation with snoRNAs, recombinant Rntlp shows its endonuclease activity and
cleaves at AO site in the 5’ ETS. In the absence ofRntlp, cleavage at AO site in the
55 ETS and 3’ ETS of pre-rRNA is abolished (Abou et al., 1996).
Early studies have identified few trans-acting factors important to 3’ ETS processing,
such as the product of RNA 82 gene (Piper et al., 1983). The artificial rRNA
minigenes in yeast demonstrate that the mutant ma 82 suppresses the endonuclease
activity and retards the 3’ end processing in pre-rRNA (Kempers-Veensfra et al.,
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1986). Yeast RNase III (Rntlp) also plays an important role in processing of 3’ ETS.
The stem loop structure at 3’ ETS is the site which is anchored and cleaved by Rntlp,
(Allmang & Tollervey, 1998). In the mutant, mtl-1, processing of 3’ ETS is blocked
and results in accumulation of undigested 3’ end intermediates of 27S pre-rRNAs
and 25S rRNA (Abou et al, 1996). There are two species of 5.8S rRNA, the major
form of 5.88s and the minor form of 5.8S l- These are synthesized from two
alternative processing pathways. The cleavage by RNase MRP and Rrp5p at A3 in
ITS1 generates the short form of 5.8S rRNA; whereas, the cleavage atBIL creates
the long form of 5.8S rRNA. In this step, the cleavage in ITS1 separates 20S from
27S pre-rRNA (Henry et al, 1994). The stem-loop structure at A3 and B1L is also
the target for RNP (Allmang and Tollervey, 1998; Briggs et al., 1998). MRP is a
RNP, and is structurally homologous to the ubiquitous RNP RNase P, whose activity
is mostly found in the nucleus (Schmitt et al., 1993; Morrissey and Tollervey, 1995).
Defective RNase MRP in the yeast S. cerevisiae synthesizes only 5.8S l, but not
5.8Ss owing to the failure in B1L cleavage during the processing of ITS 1 (Schmitt
and Clayton, 1993).
The processing of ITS2 is still quite elusive, but it is known that the final step for the
maturation of both the short form and long form of 5.8S rRNA is the removal of 3’
end extended ITS2 nucleotides of 5.8S pre-rRNA by the activity of the 3’-5’
exonuclease (Chu et al., 1994; Henry et al., 1994; Schmitt and Clayton, 1993;
Morrissey and Tollervey, 1995). The 3’-5! exoribonuclease activity determines the
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fate of 5.8S whether it becomes mature 5.8S. Rrp4p, a 3’-5’ exoribonuclease, is a
component of the exosome complex. The immunoprecipitation study on Rrp4p first
verified its role in 3’-55 exoribonuclease activity in vitro. The 3’- 55 exoribonuclease
activity of Rrp4p is required for the removal of 3’ extension of 5.8S pre-rRNA and
the synthesis of a functional 5.8S RNA. In RRP4 mutant strain, Rrp4-1, the mature
5.8S rRNAs fail to be produced, but loads of 5.8 S rRNA intermediates are found in
the transcription site (Mitchell et al., 1996).
1.2 Processing, Export and Degradation of Eukaryotic mRNA
mRNAs are also synthesized in the nucleus as a precursor and require a series of
maturation processes before being transported to cytoplasm for protein synthesis.
The mature structures of mRNAs include: the 5’ end of methylated guanosine cap,
polyadenylation of 3’end, and spliced and/or edited exons (Proudfoot et al., 2002).
Each part of the processing events is important for generating a functional mRNA.
However, in yeast, the 3’-5’ ribonuclease activity for the processing of poly(A) tails
has captured more attention than that of mRNA splicing in the maturation and export
of mRNA because yeast contains only a small number of introns (Vasudevan and
Peltz, 2003). Processing factors, mal4, mal5 and poly (A) polymerase (papl) are
important molecules participating in 3’ mRNA maturation. The 3’ end of mRNA is
synthesized by several processing complexes, and some examples are Rnal4p and
RnalSp (CstF77 and CstF64 in humans), which are implicated in cleavage and
coupled polyadenylation (Minvielle-Sebastia et al., 1994).
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Most eukaryotic mRNAs are polyadenylated, and the length, of the poly (A) tails is
highly defined. The modified length of 3’ end poiy(A) tail not only confers stability
to mRNA but also has implications with the control of gene expression. In early
development, gene expression can be regulated by altering the length of 35 end
poly(A) tail of maternal mRNAs (Wickens et al., 1997). Elongation of the poly(A)
tail is controlled by several proteins, including CFIA, CFIB, polyadenylation factor
I(PFI), poly(A) polymerase (PAP) and poly(A) binding protein I (PabI). The poly(A)
tails of a yeast nascent mRNA is 55-90 nucleotides (nt) in length, while mammalian
poly(A) tails are 150-250 nt in length (Brown and Sachs, 1998; Zhao et al., 1999).
Rnal4p and Rnal5p mediate the activities of both pre-mRNA cleavage and
transcription termination. The activities of cleavage and termination are suppressed
in Rnal4p and RnalSp mutants, which have characteristically long 3’-extended pre-
mRNA, that are soon degraded by the nuclear exosome (Torchet et al., 2002).
Mutation of any of these 3’-end processing factors results in retention of mRNA in
the nucleus (Brodsky and Silver, 2000; Hilleren et al., 2001). In yeast, cleavage
factors IA, IB and II (CFIA, CFIB and CFII) are in charge of nuclear cleavage and
are recruited by the signals from the sequences within the 3' UTR of the pre-mRNA
(Zhao et al., 1999). In S. cerevisiae, a faulty polyadenylation signal also results in
nuclear retention of pre-mRNA (Long et al., 1995).
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Many studies have found that splicing of pre-mRNA is coupled with mRNA export
(Luo and Reed, 1999; Zhou et al., 2000; Kataoka et al., 2000). Aly is the mRNA
factor found to be coupled with the spliced mRNA protein complex (mRNP) for
mRNA export. In the frog Xenopus, the conserved DEAD-box helicase UAP56
inhibits mRNA export. Overexpression of UAP56 inhibits Aly from reacting with
the spliced mRNP, and thereby brings a halt to mRNA export (Luo et al., 2001). In
S. cerevisiae, DEAD-box RNA helicase Sub2p and Yralp are two proteins involved
in mRNA export (Jensen et al, 2001; Strasser and Hurt, 2000; Lei and Silver, 2002).
Evidence from chromatin immunoprecipitation (CHIP) assay shows that Hprlp
recruits mRNA export factors Yral and Sub2p to assist active genes during
transcriptional elongation. A deletion of Hprlp leads to an accumulation of poly(A)
mRNA in the transcription locus and mRNA export is abolished owing to defects in
recruiting the essential mRNA export factors (ZenMusen et al., 2002).
The mRNA processing acquires a surveillance system to ensure good quality of the
mRNA products to be exported. Any flaw in pre-mRNA would result in
sequestration of nuclear mRNA from export. Quality control of mRNA processing
and nuclear transport is exosome/Rrp6p-mediated (Vasudevan and Peltz, 2003).
Rrp6p plays an important role in this surveillance system, which coordinates with
many processing factors for regulating the nuclear mRNA export. Mutation in
helicases, Yral or Sub2p in hprl null cells is lethal. However, mutation of Rrp6p in
yral mutants reverses quasinormal mRNA levels (ZenMusen et ah, 2002). Hprlp and
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THO protein are involved in transcription elongation. HSP104 transcripts found to
be 3 ’ end truncated in THO complex mutant and the defect causes the transcripts to
be sequestrated in the nuclei. However, when RRP6 is deleted from THO complex
mutant strain, mRNA level of HSP104 is reestablished and the full-length transcripts
can be released from the nucleus, revealing that RRP6 plays a regulatory role in
nuclear transport (Libri et al., 2002). The 3’ untranslated regions (UTRs) of mRNAs
are found to be an important regulatory element for 35 end poly(A) processing,
which interact with a variety of nuclear proteins (Beelman et al., 1996).
Nuclear pore complexes are proteins located in the nuclear envelope that can
selectively transport macromolecules between the nucleus and cytoplasm. The
heterodimer Mex7p/Mtr2p recruits numerous repeat nucleoporins, such as GLFG,
FXFG and FG, to form nuclear pore complex subcomplexes and carries the mRNP
through the nuclear pore (Strasser et al., 2000). Exchange of macromolecules
between the nucleus and cytoplasm is an active transport and signal-mediated
process (Mattaj and Englmeier, 1998). Nucleoporins (Nups) are a complex of
proteins, which build up to construct the nuclear pore complexes (NPCs) play an
important role in surveillance nuclear import and export (Rout et al., 2000). hi S.
cerevisiae, the mutation on any mRNA export factors, including Mex67p, Mtr2p,
Glelp, Nupl59p, Dbp5p and Rip Ip causes a tremendous accumulation of pre-
mRNAs in transcription sites, and these poly (A) tails of mRNAs are much longer
than those of wild strains (Jensen et al, 2001).
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All the defective, surplus or unnecessary mRNAs in cytoplasm are targets for
degradation. In E. coli, two major types of 3’-5’ exoribonuclease enzymes, RNase II
and polynucleotide phosphorylase (PNPase) are responsible for RNAs degradation in
vitro. PNPase regenerates RNAs phosphorolytically to nucleoside 5’ diphosphates,
while RNase II hydrolyzes RNA to nucleoside 5’ monophosphates and the latter
mechanism is applicable for more than 95% of poly (A) hydrolytic activity in E. coli
(Deutscher and Reuven, 1991; Deutscher, 1993). Various types of RNases are
involved in RNA processing, turnover and degradation reactions. Since they are so
important in maintaining physical function of cells that a disruption of both RNasell
and PNPase is lethal in cells (Donovan and Kushner, 1986). In S. cerevisiae,
degradation of cytoplasmic mRNAs involves in two pathways: a major 5’-3’
pathway and a minor 3’-5’ pathway. The major 5’-3’ pathway starts with a
deadenylation by a Poop2p-Cc4p-Caflp poly (A) nuclease complex, followed by the
removal of 5’ 7-methylguanosine cap by the decapping enzyme Dcplp, and finally
degradation of unprotected mRNA by 5’-3’ exonuclease Xmlp and other associated
proteins (Daugeron et al., 2001; Beelman et al., 1996). The minor 3’-5’ pathway is
achieved by the exosome, a complex of 3’-55 ribonuclease. The degradation of
mRNAs slows down when a defect occurs in either pathway; however, it becomes
lethal when the both pathways fail (Mitchell et al., 1997). Besides the major 5’~3’
pathway and the minor 3’-5’ pathway, there is an alternative degradation pathway for
mostly defective mRNA in the yeast called the nonsense mediated mRNA decay
(NMD) pathway or mRNA surveillance, where mRNAs contain defective open
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reading frames (ORFs) or premature termination codon (PTC) are the targets of
degradation (Hentze and Kulozik, 1999).
1.3 3 ’-55 Exoribonuclease, Exosome and RRJP6
The exosome is a complex of 3’-5’ exoribonucleases. 3’-5’ exonuclease activity had
not been demonstrated in eukaryotic proteins until the in vivo study of
immuoprecipitation studies of Rrp4p in yeast showing that Rrp4p possesses the 3’-5’
exonuclease activity (Mitchell et al., 1996). Rrp4p is verified to be a part of a
nutienzyme ribonuclease complex, called exosome. Rrp4p, Rrp41p, Rrp42p, Rrp43p,
and Rrp44p are the first five important exosome proteins that were identified in S.
cerevisiae (Mitchell et al., 1997). The characteristic of 3’-5’ exoribonuclease
activities of exosome is highly conserved in eukaryote. The homologues of its
proteins can be found in humans, where the expression of human Rrp4p gene is able
to compensate the rrp4-l mutation in yeast. The exosome of unicellular parasite
Trypanosoma brucei is smaller than that of yeast, and it comprises at least eight
exosome subunit homologes, which are all required for 5.8S rRNA maturation.
However, the exosome complex is not found in E. coli (Mitchell et al., 1997; Estevez
et al., 2001).
The exosome resides in both the nucleus and cytoplasm. The nuclear exosome
functions in maturation of many pre-RNAs and degradation of pre-mRNAs which
have defective poly(A) tails; whereas, the cytoplasmic exosome is in charge of the 3’
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to 5’ degradation of mRNAs (Helleren et al., 2001; Anderson and Parker, 1998). All
known substrates for the exosome are RNA protein (RNP) complexes. It is most
likely that the proteins associated RNP complex mediates the exonuclease activities
of the exosome. Numerous RNP complex associated proteins modulate the
exonuclease activities of the exosome depending on the situation. 3’-5’ degradation
of mRNA requires Ski2p, Ski3p and Ski8p, while processing of 5.8SrRNA does not
require these Skip proteins, only the Ski2p-realated protein Mtr4p (Anderson and
Parker, 1998). The exosome also plays a role in the 3’ processing of many snRNAs
and snoRNAs. The final maturation of the 3’-extended pre-U3 small nucleolar RNA
needs snoRNP proteins and the action of 3’-5’ exonucleases, the exosome complex
(Kufel et al., 2000). By mutating the core element of the exosome and a variety of
RNAs processing associated proteins, the steps of processing in different RNAs
including tRNAs, snRNAs, snoRNAs, SRP RNAs and 5S rRNAs are unfolded. The
nuclear exosome is required for 3’ end processing of many RNA species, including
U4 snRNA and snoRNAs. The nuclear proteins Rrp6p and Mtr4p are required for
processing of 5.8S rRNA, snRNA and snoRNA by exosome, while Ski2p but not
Rrp6p or MTR4 play a role on mRNA degradation by exosome. This suggests that
the nuclear form and cytoplasmic form of exosome are regulated by different RNAs
processing associated proteins, and their functions are also distinct (van Hoof et al.,
2000). The autoantigen PM/Scl complex is the human homologue of the yeast
exosome. PM/Scl autoantibodies are usually found in sera from patients with
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idiopathic inflammatory myopathy (IIM), scleroderma, and the PM/Scl overlap
syndrome (Brouwer et al., 2002).
The Prp6p, a 3’-5’ exoribonuclease, is a component of the nuclear exosome. Rrp6p
plays an important role in 3’ end processing of 5.8 rRNA. Molecular structure of
RRP6 shows its highly homologous to the human PM-Scl 100-kDa autoantigen and
to Escherichia coli RNase D, a 3’-5’ exoribonuclease. The homologous core regions
among these organisms indicate the conservation of protein function. Domain I, II
and V of Rrp6p may contain important motifs for the 3’-5’ exonucleolytic reaction
because they are homologous to domain Exol, ExoII and ExoIII, which are required
for the 3’-5’ deoxyriboexonuclease activity of DNA polymerase I (Briggs et al.
1998). Maturation of 3’ end transcripts is a multiple step process, each step
requiring different components of exosome. During 5.8 rRNA and boxC+D
snoRNAs synthesis, the core exosome is first required before RRP6 (Torchet et al.,
2002; Mitchell et al., 1997). Disruption of RRP6 leads to an accumulation of
extended 5.8 S pre-rRNAs, suggesting that Rrp6p plays an important role in removal
of ITS2 from 5.8 S precursors (Briggs et al. 1998).
RRP6 is a gatekeeper in mRNA export, and it prevents defective RNAs from being
released in their transcription sites. A 3’ end defected heat shock HSP104 transcripts
in THO complex mutant or sub2 mutant cannot be exported but retained in the
nucleus. However, when RRP6 is deleted from these strains, the defective
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transcripts are transported to the cytoplasm due to the absence of surveillance Rrp6p
(Libri et al., 2002). Poly(A) polymerase (encoded by PAP1) is the enzyme that
required for poly (A)tail synthesis. Mutation in PAP1 is lethal in yeast owing to a
serious defect in poly (A) tails of mRNAs. However, adding RRP6 inhibitor or
deletion of RRP6 from papl-1 strains are able to partially recover the poly (A)
mRNA (Burkard and Butler, 2000; Vasudevan and Peltz, 2003).
Rrp6p is only located in the nucleus, and it collaborates with various nuclear proteins
for mRNA degradation. The C-terminal portion of Rrp6p exists putative nuclear
localization signals implies that Rrp6p is present in the nucleus and/or the nucleolus
(Briggs, et al., 1998). Northern blot and graphical analyses of the rate of mRNAs
degradation in TCM1, whose RRP6 is deleted, showed that mRNA degradation rate
is not affected but levels of poly(A) mRNA increased, suggesting that Rrp6p does
not participate in mRNA decay in cytoplasm. The subcellular localization of Rrp6p
also demonstrates the fact that Rrp6p is located in the nucleus. The yeast two-hybrid
screen shows that Rrp6p interacts with pap-1 and the hnRNA protein Npl3p.
Knockout of both Npl3-1 and RRP6 is found to be synthetically lethal and suggests
that Rrp6p and Npl3p are functionally linked (Burkard, and Butler, 2000). Das and
his colleagues in their recent studies defined a nuclear mRNA degradation (DRN)
system and showed that the importance of RRP6 function in this pathway. They
utilized a mutant yeast strain Nupl 16-A, which is defective in exporting any of its
RNAs out of the nucleus, causing the retention of nuclear mRNA and resulting in
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rapid mRNA degradation. The authors knocked out RRP6 from rnipl 16-A yeast
strain, and surprisingly they found that RRP6-A suppressed the rapid nuclear mRNA
turnover rates, which proves RRP6 is required in the DRN pathway (Das et al., 2003).
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CHAPTER 2
RRP6 DELETION IN S. CEREVISIAE
Gene deletion is a fundamental tool to understand the functions of a gene. The
disruptive gene in an organism may reflect on its phenotype, thereby the roles and
characteristics of the gene may be determined. To identify how RRP6 gene works,
we constructed a complete gene disruption mutant of RRP6 in the yeast
Saccharomyces cerevisiae by using homologous recombination. The strategy is that
the core element of RRP6 DNA is deleted and cloned into the yeast SP1 cells, which
have normal chromosomal copy of gene. As recombination occurs, the mutant
RRP6 gene replaces the normal one in the yeast cells; therefore, the RRP6 gene is
completely disrupted.
2.1 Construction of RRP6 Deletion
2.1.1 Plasmid Method to Construct a RRP6 Deletion
First, we used a plasmid vector as a tool to construct the RRP6 deletion. We utilized
the cDNA of SP1 yeast strain as the template to amplify an intact 2200 bp full length
RRP6 PCR product. This RRP6 PCR product was designed to have the Xba I and
EcoR I flanking end on each side; so it can be constructed into a 3000bp pBluescript
vector. Within RRP6 DNA of the RRP6/pBS construct, there are two Bgl II sites,
from which a 1227 bp RRP6 core element can be excised and replaced by a 1700 bp
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Histidine DNA (Figure 2.1). After molecular cloning, the construct of KBJP6
deletion was integrated into the SP1 yeast strain.
However, this attempt was aborted, as a result of the vectors, which we obtained
were difficult to be manipulated. We used Restriction Fragment Length
Polymorphism (RFLP) analysis to test the pBS vector before the construction. The
correct structure of pBS has one Hind III site but has no Bgl II site. The first pBS
vector we obtained failed to pass the first RFLP test because it could not be digested
by Hind III. The second pBS vector we obtained passed the RFLP test; therefore, we
utilized it to construct the RRP6 deletion (though we found the size of the vector was
a little small). We continued the construction until a discrepancy occurred as we
verified the construct with PCR/RFLP during the final step. We realized that these
pBS vectors might contain unknown structures, thus we quit using the plasmid
method.
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Bgl II Bgl II
RRP6
Histidine
Figure 2.1 A diagram of the RRP6 gene deletion.
There are two Bgl II sites within RRP6 DNA, from
which a 1227 bp RRP6 core element can be excised
and replaced by a 1700 bp Histidine DNA.
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2.1.2 PCR Method to Construct a RRP6 Deletion
We changed our strategy to generate the complete gene disruption of RRP6 by a
PCR method (Baudin et a!., 1993). We first, used genomic DNA of SP1 as template
to produce three PCR products: two short RRP6 fragments, which corresponded to
the 293 bp 5’ end of RRP6 gene and the 304 bp 3’ end of RRP6 gene; and one 1684
bp Histidine gene. The Histidine gene PCR product was designed to have two
flanking ends, which had the sequences that can complementarily anneal to the two
short RRP6 fragments (Figure 2.2). After the first run PCR, the three expected PCR
products were obtained, and then we performed the second run PCR to link the first
three PCR fragments and generate a RRP6 deletion (Figure 2.3). We verified the
mutant RRP6 construct by PCR/RFLP analysis and also automatic sequencing using
Sequenase 2.0 before integrating it into SP1 yeast genome.
17
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RRP6 deletion
RRP6 Histidine RRP6
B
R1A
RRP6
R IB
HI
R2A
RRP6
-R2B
Histidine
<«- H2
Figure 2.2 Construction of a complete gene disruption
of RRP6 by PCR method. (A) The putative PCR
product of RRP6 deletion. (B) Two primer pairs, R1A
/RIB and R2A/R2B, generate two 300bp fragments,
which correspond to the 5’ end and 3’ end of RRP6
gene. (C) The primer pairs H1/H2 create a 1686 bp
Histidine gene fragment with two flanking ends that
can anneal with the two short RRP6 fragments.
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M 1 2 3 4
1.5
1
0.3
Figure 2.3 PCR method to generate RRP6 deletion.
PCR products were run on 1% agarose gel. Lane 1,
R1A/R1B corresponds to 293 bp 5’ end of RRP6.
Lane 2, R2A/R2B corresponds to 304 bp 3’ end of
RRP6. Lane 3, H1/H2 corresponds to 1684 bp
Histidine gene. Lane 4, the PCR product, RRP6
deletion (2281 bp), was generated from linking the
first three PCR fragments.
19
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2.1.3 LiOAc Yeast Transformation
The RRP6 deletion PCR construct was integrated into SP1 yeast genome by using
the effective method of Lithium Acetate (LiOAc) yeast transformation. The SP1
yeast strain has a single mutation on the Histidine gene, which prevents production
of Histidine amino acid. Therefore, we used Histidine-lacking media to select
desired RRP6 deletion transformants (RRP6::His-3). The method was that we
streaked the transformation onto the complete synthetic medium (SC) (Sherman et al,
1986) lacking the His-3 amino acid plates and incubated for 5 days at 30 °C. The
single colonies of transformants that grew on the SC plates were picked and re
streaked onto YPD plates. We repeated the streaking at least three times to eliminate
the wild type yeast background. We performed four methods to screen and verify
the desired RRP6::HIS-3 mutant included: PCR, PCR/RFLP, automatic sequencing
and Northern blot analysis.
A little trouble we encountered while manipulating the yeast was that we could not
find a proper incubator with shaker to grow the yeast cells in liquid medium.
Though the optimal temperature for yeast grow is 30 °C, there was only a 26 °C
incubator available, which always took us more time to grow the cells
20
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2.2 Verification of RRP6::His-3
2.2.1 Screening by Using PCR, RFLP and Sequencing
After yeast transformation, we obtained less than 15 colonies from the SC-His-3
plates. But, after re-streaked for three times, we obtained hundreds or thousands
colonies. We used the yeast DNA mixiiprep method to extract yeast genomic DNA,
and then ran PCR with various PCR primer pairs to screen for the RRP6: :His-3
transformants (Figure 2.4). As the matter of fact, we made a blunder at the very
beginning of PCR screening. We only used PCR primer pairs to screen the region
within the RRP6 deletion construct that we integrated into the yeast genome. This
was incorrect because even though a yeast transformant might contain the full length
RRP6 deletion construct, the construct might not be integrated in the right locus of
the yeast chromosome. Figure 2.5 shows the PCR products that we thought were the
desired transformants, but after the PCR products were digested with restriction
enzymes, Bagl II and Hill, we realized that they were not the expected RRP6
deletion because the patterns were exactly like the wild type RRP6 (Figure 2.6).
21
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RRP-6
\* H
(PHO08) i^_ Construct of RRP6 deletion
RRP-6
h H
Yeast genome
RRP6-F-out
RRP6-F-out
HI
HIS-Forward
(ALG6)
His-3 gene
J_L
Yeast genome
Full length RRP6 gene
RRP6-R-out
H2
His-3 gene
RRP6-R-out
HIS-Reverse
Figure 2.4 PCR screening for RRP6::His-3. A
diagram shows various PCR primer pairs that are
used to select transformants for being properly
integrated into SP1 yeast genome.
22
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M M I
Kb
1 |
2
1
► ll
V ' * :ri l
- i l
• * ...l i t
Construct of RRP6 deletion
Yeast genome
His-3 gene
Yeast genome
h H
RRP-6
K H
RRP-6
R1A ►
* R2B
Figure 2.5 PCR screening for the presence of RRP6
deletion and His-3. 1-6 are transformants from SC-His-3
plates. 7 is plasmid with RRP-6 deletion as a positive
control. Within each group, lane 1-4, are the PCR
products produced by primer pairs R1A/R2B, H1/H2,
R1A/H2, H1/R2B, respectively. Note that the results of 1
and 5 seem to be consistent with 7, but R1A/R2B primer
pairs fail to detect whether the RRP6 deletion is correctly
residing in yeast genome.
23
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M M A B D
Kb
3
1
2
* 3
1
0.5
>
i W: Ijj|l
— M
W
w . -
M M P6
1 * 1
BgHI Bglll
Wild type RRP6“ >
Bglll
RRP6::His-3-^
w
H IT T W T H
Figure 2.6 RFLP analysis of RRP6::His-3 for the
presence of RRP6 deletion and His-3. A and B are
PPR6::His-3 candidates selected from PCR screening.
C, plasmid RRP6 deletion and D, wild type RRP6 are
two controls. Within each group, lane 1, PCR product;
lane 2, PCR product digested with Bgl II; lane 3, PCR
product digested with Hind III. Note that results show
that A and B are not PPR6::His-3 but wild type RRP6
which can not be cut by Hind III. *RRP6::His-3
contains four Bgl II sites which are not shown.
24
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From this experience, we learned that we have to use PCR primer pairs that is
outside the RRP6 deletion construct so that we can select not only for the full length
RRP6 with Histidine insert, but also specify that the deletion construct has been
integrated into the correct locus of the yeast chromosome (Figure 2.4). We screened
hundreds of yeast colonies by PCR with primer pairs outside the RRP6 deletion
construct. The obtained RRP6 deletion candidates were confirmed by PCR/RFLP
analysis (Figure 2.7 and Figure 2.8). Furthermore, all the selected RRP6 deletion
were cloned into the pGEM vector (Promega) (Figure 2.9) and confirmed with
automatic sequencing.
25
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M l 2* 3 4 5 6 7 8 9 10* 11 12 13
n n n n n n n n n n n n n
Kb
4.3
2
Wild type RRP6-» 2.2 Kb
RRP6::His-3-»
— w —
HTTT HITT
Figure 2.7 PCR/RFLP analysis of the candidate
RRP6::His-3 for the presence of His-3. Lane 1-11, are
the transformants which had been screened by PCR for
being integrated onto correct locus of yeast
chromosome. Lane 12, RRP::His-3 and lane 13, wild
type RRP6 are controls from plasmid DNA. Within
each group, the first lane is the selected PCR product
and the second lane is the PCR product being digested
with Hind III. Note that the results of 2 and 10 are
consistent with that of RRP6::His-3.
26
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Figure 2.8 PCR/RFLP confirmation of two RRP6::His-3
candidates for the presence of RRP6 deletion and
Histidine gene. A and B are selected RRP6:: His-3
candidates for being correctly integrated onto yeast
genome. C, RRP6:His-3 and D, wild type RRP6 are
controls from plasmid DNA. Within each group, the
first lane is the selected PCR product; the second lane is
the PCR product being digested with H III; the third lane
is the PCR product being digest with Bgl II. Note that
the results of A and B are consistent with that of
RRP6::His-3.
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V. 2707;
1
'.S
fiC Q ■
mil I
A fc ‘ 1
JaaSl
Figure 2.9 Map of pGEM vector from pGEM-T
Vector System II (Promega). The pGEM vector was
used in this experiment to clone the RRP6 DNA, the
RRP6 deletion PCR construct and PCR products of
three mutants, RRP6::His-3.
28
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2.2.2 Structural Analysis of RRP6::His-3 by Southern Blot
The three RRP6::His-3 candidates established by homologous recombination were
analyzed for the RRP6 core deletion and for the 1601 bases substitution by Histidine
gene by Southern blot hybridization analysis. The genomic DNAs from the three
RRP6::His-3 candidates, LPC1, LPC2, LPC3 and the wild type SP1 yeast control
were isolated. The intact DNAs were digested with restriction enzyme Hind III, and
were fractionated onto agarose gel. Capillary-transfer was used to transmit the
DNAs fractionated from the gel onto nylon membrane. Then, the DNAs on the
membrane were hybridized with a radiolabeled full length RRP6 probe.
The correct constructs of RRP6::His-3 could be verified because the RRP6 gene
resides on a 9.2 Kb Hind III fragment in the wild-type locus (GenBank accession no.
NC_001147). When digested by Hind III, the wild type RRP6 control yields an
intact fragment of 9.2 Kb in size, while within the RRP6 deletion, there are two Hind
III sites at coordinate 6228 and 6415, which will yield three fragments of 6.2 Kb, 3
Kb and 0.2 Kb in size (Figure 2.10. A). The autoradiography from the Southern blot
analysis shows the structures of the three RRP6::His-3 candidates, LPC1, LPC2 and
LPC3 as well as the wild type yeast control, SP1 are in agreement with our
expectation (Figure 2.10.B).
29
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Figure 2.10 Structural analysis of the RRP6::His-3 by
Southern blot. The DNAs were digested with Hind III,
fractionated onto agarose gel, capillary-transferred onto
nylon membrane, and were hybridized with a radiolabeled
full length RRP6 probe. (A) A diagram shows that in the
wild type SP1, RRP6 resides on a 9.2 Kb Hind III fragment
(GenBank accession no. NC_001147), while RRP6::His~3
introduces two Hind III sites coordinated 6228 and 6415
will yield three fragments of 6.2, 3.0, and 0.2 Kb. (B) The
autoradiogram shows the results of the digestion with Hind
III of the wild type control SP1 and the RRP6:: His-3
LPC1, LPC2 and LPC3. Note that all mutant strains
contain 6.2 Kb, 3 Kb which is indicative of the loss of the
core element of RKP6 and the presence of Histidine.
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Figure 2.10 A
Yeast genome with wild type
RRP6 (9.2 Kb)
HIII HIII
After digestion with
HTII 19.2 Kb)
Yeast genome with
RRP6::His-3 19.4 Kb)
± = ^ - = 1 "r " m ~ n = l
HIII HIII HIII HIII
After digestion with
HIII (6.2 Kb)_____________
------------- j (.Q 2 K b )
■ (3 Kb)
nzzziizzziz]
31
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Figure 2.10 B (continued)
LPC1 LPC2
Kb
g 2
6.2 *
3 * nip
32
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LPC3 SP1 M
Kb
< 10
g
■
lllll
2.2.3 RT-PCR Analysis of RRP6::His 3 mRNA
Since 3 ’ end formation is an important step for mRNA maturation and export, RRP6
deletion results in accumulation of processing intermediates and hyperadenylated
mRNA in transcription site (Brodsky and Silver, 2000; Pei and Silver, 2000). We
presumed that in RRP6 deletion strains, the expression of RRP6 mRNA was void,
while mRNA level of other genes may not be affected, but the protein levels might
decline or become non-functional. To test this hypothesis, first, we examined
mRNA levels of the three RRP6::His-3 by performing RT-PCR and examining
whether the gene was completely knocked out. We expected to see the RRP6
mRNA express in SP1, but not in the mutants. After obtaining cDNA, we used
RRP6 primer pairs and HI-3 primer pairs to test the presence of these two genes (Fig
2.11). The results in Fig 2.11 showed that there was expression of His-3 mRNA,
though RT-PCR conditions were not quantitative, so we could not compare the
difference in expression level. Disappointingly, there was not detectable RRP6
mRNA in both RRP6 deletion mutants and wild-type control. After a few trials, we
were convinced that it is a difficult task to isolation mRNA from yeast cells. Perhaps,
RRP6 mRNA was traces in SP1. Note that the RRP6 cDNA, which was kindly
offered by one of our lab colleagues also yielded little RRP6 PCR product.
33
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Kb
2
IS
RRP6 primer pairs His-3 primer pairs
< ^ ® ft ®
~ a ® l . a * >
1 .£ 2 a .>
§"5 ^ ^ 5 § ti
_ o o u § S ^ u o u | § a
h h b h < g a j p ^ P H e ^ p ^ J S « «
E Z ) J J
1.5 — • * * • H *4 R R P6
4 H is-3
0.5
Figure 2.11 RT-PCR analysis of RRP6 and His-3
mRNA levels in wild-type SP1 and three RRP6::His-3
mutants, LPC1, LPC2 and LPC3. Plasmid DNA of
RRP6 deletion construct and genomic DNA of SP1
were used as positive controls.
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2.3 Characterization of RRP6 Deletion
2.3.1 Morphology Analysis of RRP6::His-3
The RRP6::His-3 mutant, LPC1, LPC2, and LPC3 and wild-type SP1 yeast strains
were cultured in liquid YPD medium with 2% glucose and 1% penicillin at 26 °C.
The cells were collected twice at the optical density reached to A600nm=0.5 and
A600nm=1.4, respectively, and the cells were observed under the bright field
microscopy with 200X and 600X magnification.
The morphology of the cells in term of color, size, shape and distribution were
compared between the mutants and the wild type. We found that there was not much
difference among the logarithmically growing cells (data not shown). However, as
the cells grew close to the stationary phase, the cells in the RRP6::His-3 mutants
tended to clump tog her, while those of the wild type SP1 dispersed. As shown in
Figure 2.12.A, each SP1 budding cell separated from its parental cell when it
developed into full size. It was hardly found two inseparable mature SP1 cells;
whereas, in the three mutants, it was more frequent to find several inseparable cells
aggregated into a cluster (Figure 2.12.A). More elongate and pear-shaped cells were
developed in the mutants; whereas, it occurred less frequently in SP1 cells (Figure
2.12.B).
35
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LPC2
LPC3
& 5 0
o
t o „ a V * f
% % l
■ 0 1 %
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Figure 2.12.B (600X) (continued)
SP1 LPC1
m cx ^
c
€
< r\ Y : ^ f ■
r -c
C 1
l i .
< % F
r * .
. . n
LPC2 LPC3
^ » > rf- * V - r ?
°~V- ^
W *
c *
Figure 2.13 The morphology analysis of RRP6::His-3 cells
versus the wild-type SP1 cells, which were collected at
A600iun=1.4 and observed under the bright field microscopy.
(A) cells observed with 200X magnification and clusters with
more than 10 cells were circled and (B) cells observed with
600X magnification.
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2.3.2 Temperature Sensitive Growth Phenotype
Some previous studies demonstrated that the temperature is an environmental factor
that transmits genetic traits or affects the growth phenotypes of a yeast strain (Briggs
et al, 1998; Libri et al., 2002). In this study, we examined the effect of temperature
on growth phenotype of RRP6::His-3 mutants and wild type SP1 by growing them
on YPD and Histidine-lacking synthetic complete plates at four different
temperatures, 14 °C, 25 °C, 30 °C and 37 °C for five days. Two separated
experimental trials, both YPD and SC(-)His plates gave us the same results though
SC(-)His plates took a longer time for cells to growth on (data not shown). As
shown in Figure 2.13, no cell was able to grow at 14 °C; on the contrary, all cells
could grow vigorously at permissive temperatures 25 to 28 °C. As the temperature
was altered to 37 °C, the numbers of colonies of both the mutants and SP1 dropped
dramatically and LPC2 couldn’t survive at such stringent temperature at all. We
compared the size of the colonies between SP1 and the three mutants at temperatures
25 to 28 °C. The mean size of SP1 colonies was 0.14±0.25 cm3; while the mutant
colonies could hardly develop to 0.04 (0.038±0.014) cm3. The mean size of SP1
colonies was statistically significantly larger than those of the mutants (p<0.001),
and the distribution of the wild type SP1 colonies was also much dispersed than
those of the mutants (Figure 2.13).
38
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Figure 2.13 The temperature sensitive growth phenotype
of RJRP6::His-3. The RRP6 deletion mutants, LPC1,
LPC2, LPC3, and the wild-type SP1 were grown on YPD
plates at 14, 25, 28, and 37 °C, respectively for 5 days.
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2.3.3 Growth Curves
The cells of S. cerevisiae divide by budding. A cell bud can exponentially increase
in the number and its doubling time is equal to the mean-division cycle, which can
be as short as one hour depended on the strain, medium and environmental factors.
The results of two recent studies showed that RRP6 and RRP4 mutants were
apparently impaired (Allmang, et al, 1998; Torchet, et al., 2002). To examine the
growth rate of the mutant yeast versus the wild type, we grew the RRP6::His-3
mutant and SP1 cells in liquid YPD in the presence of glucose. We found that the
growth rates of the three mutants were much slower than that of SP1. However,
when SP1 reached to the stationary phase, the three mutants kept increasing
uncontrollably till the cells become depleted of nutrients and lysed to death (Fig.
2.14.A & B). We were not sure whether the phenomenon was true though we
repeated the experiment three times. It was not a favorable experiment because the
cells did not grow at their optimal temperature by limiting the setting of the
incubator, and the readings of the spectrophotometers were not stable or reliable.
40
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A B
Yeast cell growth
3.50
,00
2.50
2.00
1.50
.00
0.50
0.00
0 10 20 30
hours
Yeast cell growth
2.50
2.00
SP1
LPC1
LPC2
LPC3
8 1 - 5 0
1.00
0.50
0.00
0 20 40 60
hours
Figure 2.14 The growth rates of the mutants versus the
wild-type yeast. The yeast cells were grown in liquid YPD
medium in the presence of glucose, and the concentrations
of the yeast cells were measured at A=600nm by
sp ectrophotometry.
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CHAPTER 3
CONCLUSION AND DISCUSSION
We have observed some growth phenotypes ofRRP6::His-3 mutants, LPC1, LPC2
and LPC3 in comparison with those of the wild-type yeast SP1. First, we verified
that RRP6::His3 mutation while not lethal, slows cell growth. Our finding is in
accordance with the previous studies (Briggs et al., 1998; Torchet et al., 2002; Libri
et al., 2002).
The cellular morphology of RRp6::His-3 is distinct from the wild type SP1.
The phenotypes between the mutants and the wild type does not show much
difference initially; but as they get closer to the stationary phase, there are lumpy,
elongated or pear-shaped cells appeared more frequently in the mutants. The pear-
shaped cells look similar to those forming zygote of haploid cells and the clumping
cells look like the diploid colonies. However, SP1 is a haploid strain, we assure that
these elongated, pear-shaped cells and inseparable cell clusters are the deformed
cells of the mutants. Perhaps, these distorted and clumpy cells are the results of
endogenous toxic residues in cells. Recall that RRP6 deletion may cause defect in 3 ’
end of nuclear pre-RNAs or can result in releasing of defective mRNA from the
nucleus to cytoplasm, and both of which are detrimental to the cells (Briggs et al.,
1998; Torchet et al., 2002; Vasudevan and Peltz, 2003).
42
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RRP6 deletion affects the phenotype of yeast colonies grown on both YPD and SC-
His-3 plates. The colonies of mutants grow slower and are smaller in size compared
with those of the wild type. In contrast with the mutant colonies, which grow
abundantly and crowded, those of the wild type are more disperse. We believe this
phenomenon to be the effect of pheromones, the substances released from cells to
communicate with one another. We assume RRP6::His-3 cells may fail to secret
certain pheromones due to blocked regulatory pathways. We know that RRP6 deters
various formation of 3’end RNAs and Impairs the nuclear export, but little is known
about how RRP6 deletion has been done to affect other signaling pathways.
Apparently, temperature did affect the growth phenotype of the RRP6::his-3.
Although we cannot detect any difference in structural analysis among the three
mutants, we did find the growth phenotype of LPC2 appears to be very distinct from
LPC1 and LPC3. LPC1 and LPC3 do not look much different in all phenotype tests.
The growth phenotype of LPC2 appears to be severely aberrant. Its growth is very
slow and completely ceases at 37 °C. This evidence shows that deletions other than
RRP6 deletion may have caused one or more genes destruction in LPC2 during yeast
transformation. LPC1 and LPC3 as well as wild type SP1 cannot grow at stringent
temperatures, 14 °C, but they show some growth at 37 °C. The temperature sensitive
phenotype of RRP6 mutation has been demonstrated by some studies (Briggs et al,
1998). In Briggs’s study, they compared the control strain, which can grow at low
temperature in contrast to their mutant RRP6. Unfortunately, the wild type SP1 does
43
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not have this property and it cannot grow at low temperature, so we are unable to do
such comparison. Since we know that the cells growth was dramatically affected by
37 °C incubation, it will be interesting to show cell growth of mutant RRP6 v.s. SP1
at the elevated temperature by plotting their growth curves.
We have obtained cell growth curves at permissive temperature 26 °C for the RRP6
mutants and SP1. The cell growth in mutants is impaired so they grow slower than
the wild type initially. However, the mutant cell growth shows uncontrollable
increase after SP1 virtually reaches its stationary phase, and the concentration of
cells is much higher than that of SP1 in their stationary phases. As the mutant cells
grow too crowded, they start to go self-lysis and die. Apparently, as food source
running to depletion, the mutant cells somehow loss their ability of detection the
environmental change and fail to control their growth rate.
We intended to test RRP6 mRNA level in the three RRP6::His-3 by RT-PCR
because we presume there is no expression of RRP6 mRNA in the deletion mutant.
But, we failed to demonstrate this because we could not get good quality mRNA.
We still believe that since RRP6 deletion leads to default in surveillance of various
RNAs maturation and the nuclear export systems, many gene expression flaws may
be found in protein levels. It would be interesting to test levels or functions of
proteins to see how the absence of RRP6 affects the cells.
44
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Finally, the molecular structure of RRP6 and how it cooperates with other nuclear
molecules in doing its work is still elusive and there are many mysteries of RRP6
remain to be revealed.
45
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CHAPTER 4
MATERIALS AND METHODS
4.1 Yeast Strain, Medium and Genomic DNA Extraction
A single colony of SP1 yeast strain was picked from an YPD plate, and incubated in
10 mi of liquid YPD included 2% (w/v) glucose and 1% (v/v)
penicillin/streptomycin overnight in 300 rpm of agitation at 26 °C (Note that 30°C is
the optimal temperature for yeast growth. Throughout the experiments, 26 °C was
used for yeast growth in liquid medium owing to the limitation of the incubator
setup). Cells were harvested when A600=0.35 and were centrifuged for 5 min at
2500 rpm. Yeast genomic DNA was extracted according to the protocol of Hoffman
and Winston (1987). Cells were brought to vortex mixing in acid-washed glass
beads, 0.2 ml of 2% Triton X-100, 1% sodium dodecyl sulfate, 100 mM NaCl,
lOmM Tris-Cl (pH 8), 1 mM Na2 EDTA and 0.2 ml of phenolxhloroformdsoamyl
alcohol (25:24:1). The mixture was centrifuged for 5 min and the aqueous layer was
transferred to mix with 1 ml of absolute ethanol in a new tube. The pellet was
collected by 2 min of centrifugation, and was re-suspended at 37 °C in a 0.4 ml of
TE and 3 pi of 10 mg/ml RNase A solution (Qiagen). After 5 min incubation, 10 pi
of 4 M ammonium acetate and 1 ml of absolute ethanol was added into the mixture
and mixed by inversion. The DNA pellet was collected by 2 min of centrifugation
46
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and supernatant was discarded. The air-dried peilet was re-suspended in 50 pi of
1XTE buffer.
4.2 Using Plasmid to Construct a RRP6 Deletion
2200 bp full length of RRP6 DNA was amplified by using primer pairs (5’-
GCGGGATCCATGACTTCTGAAAATCCGGATGT-3 which contained Xbal
flanking end) and (5’-GCCGAATTCTCACCTTTTAAATGACAGATTCTTA-3 ’,
which contained EcoRl flanking end), thereby the PCR products could be inserted
into the Xbal and EcoRl sites of the pBluescript vector. The PCR reaction mixture
was made up in a 30 pi volume containing 2 pi of cDNA, 0.5 pg of each
oligonucleotide primer, IX Taq polymerase buffer, 6 mM of dNTP and 0.15 unites
of Taq DNA polymerase (Qiagen). The main cycling parameters included: 30 cycles
with 95°C for 45s, 55°C for 45s, 72°C for 2min 30s. Then, the pBluescript vector
was linearized with restriction enzymes EcoRl and Xbal (Promega) and RRP6 was
cloned into the vector. Within RRP6 gene of the construct, there are two Bagl II
cutting sites from which a 1227 bp of RRP6 core element was excised. Right after
the Bagl II digestion, the 5' phosphates of the linear construct was polished with the
Alkaline Phosphatase, Calf Intestinal (CEP) (New England Biolabs) to prevent the
liner construct from recircularization. The excised region of RRP6-pBS was
replaced by 170Qbp of His-3 gene, and consequently the RRP6 gene was deleted.
47
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4.3 Construction of RJRP6 Deletion by PCR
The strand of RRP6 deletion was constructed by applying PCR method. Using
genomic DNA of yeast stain SP1 as template, three fragments of DNA, HIS-3,
R1A/R1B and R2A/R2B were synthesized. HIS-3 was produced by using a pair of
PCR primers (5 ’ -TC TT C AGT ATTT AGGCGAG AC GCTTTGTCTT C ATT C- 3 ’) and
(5’ -CC AAGTTT GCT A AGGC AGGCGATCTCG GCCTTTTCG-3 ’), wherein
complementary oligo nucleotides to RRP6 gene were tagged on each 5’ end, and
were subjected to link to R1A/REB and R2A/R2B. R1A/R1B corresponds to a short
fragment of 5’ end of RRP6 was produced by the pairs of PCR primer (5’-
GCATCATCGTTAGCCAGTCA-3’) and (S’-GCCTAAATACTGAAGATCG-3’),
while R2A/R2B corresponds to a short fragment of 3’ end of RRP6 was synthesized
by the pairs ofPCR primer (5 ’-CTGCCTTAGCAAACTTGGAGC-3 ’), and (5’-
CGCTAGATGATGGGTCGAAT-3 ’). The PCR to synthesize RRP6 deletion was a
total volume of 30 pi, containing 0.7pg of R1A/R1B PCR products, 0.55pg of
R2A/R2B PCR products, 0.25pg of His-3 PCR products, IX Taq polymerase buffer,
6mM of dNTP, and 0.15 unites of Taq DNA polymerase (Qiagen) for TA cloning or
TrueFideligy DNA polymerase (Continental Lab Products) for the yeast
transformation.
4.4 Molecular Cloning and DNA Sequencing
PCR products were cloned into pGEM vector by using pGEM-T Vector System II
(Promega) according to manufacturer’s instruction. DNA was incubated with T4
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ligase and pGEM vector overnight at 4 °C, and was transformed into competent cells
DH5a.
4.5 RFLP and Automatic DNA Sequencing
The desired DNA fragments were confirmed by RFLP. The restriction enzymes,
Bagl II and Hind III (Promega or New England Biolabs), were used for enzyme
digestion following the manufacturers’ instructions. For automatic DNA sequencing,
DNA was run on agarose gel. The desired fragments were cut out from the gel and
purified using the GENECLEAN® kits (Bio 101), and then sequenced by Sequenase
2.0 (United States Biochemical).
4.6 LiOAc Transformation of Yeast
A single SP1 yeast colony was picked from an YPD plate and incubated in 10 ml of
liquid YPD, included 2% (w/v) glucose and 1% (v/v), overnight at 26 0 with 300 rpm
shaking. Cells were harvested when A600=0.35 and were centrifuged for 5 min at
2500 rpm. Cells were washed once with sterile water to eliminate the remainder of
medium, re-suspended in 2 ml of 0.1 M LiOAc in TE and incubated for 4 hr at 26 °C
with shaking. 5 pi (Ipg) of DNA from PCR products was incubated with 15 pi of
lOmg/ml sonicated-denatured salmon sperm DNA and 200 pi of LiOAc treated cells
for 30 min at 26 °C with shaking. Then, 1.2 ml of 0.1 M LiOAc/TE/40%PEG 4000
was added and mixed with the cells by inversion. After 30 min incubation at 26 °C
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with shaking, the mixture was brought to heat shock for 15 min at 42 °C. After
being washed with 1 ml of TE, the cells were collected and re-suspended in 150 pi of
TE. To selectively maintain cultures, 150 fil of each transformation was struck onto
complete synthetic medium (SC) (Sherman et al, 1986) lacking the His-3 amino acid
plates and incubated for 5 days at 30 °C. Single colonies of transformants were
picked and re-struck onto YPD plates at least 3 times to eliminate the wild type yeast
background.
4.7 Yeast DNA Miniprep
A single yeast colony from yeast transformation was picked and cells were cultured
overnight in 10 ml of YPD included 2% glucose and 1 %penicillin/streptomycin at
26°. The cells were harvested and collected by centrifuging at 2000 rpm for 5
minutes. Then, the cells were brought to vortex mixing in 200 pi of acid-washed
glass beads and 200 pi of breaking buffer (2% Triton, 1% SDS, 0.1M NaCl, lOmM
Tris, pH8, ImM EDTA), and 200pl phenol/CHC13. After centrifugation, the upper
aliquot portion was collected and extracted again with CHC13. The upper aliquot
was obtained and DNA was participated with 750 pi absolute alcohol for 10 min.
The DNA pellet was collected by centrifugation for 10 minutes, washed with 70%
alcohol. After DNA was air dried, it was dissolved in 40pl of 1XTE buffer.
50
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4.8 PCR to Screen for RRP6::His-3 Mutant
The mutant yeast strain RRP6::His-3 was screened from the yeast transformants by
PCR analysis. A single colony of yeast transformants was grown in liquid YPD with
2% glucose and 1 %penicillin/streptomycin to A600=G.35 and DNAs were extracted
with Yeast DNA Miniprep. Four pairs of oligo nucleotides were used to screen for
expected fragments: RRP6-outer-forward (5 ’-ATGACTTCTGAAAATCCGGATG-
3’) and RRP6-outer-reverse (5 ’ -TCACCTTTTAAATGACAGATT-3 ’), His-forward
(5’-GAGCACTCGATCTTCCCAG-3’) and His-reverse (5’-
GTTGTAGCCGCCGTTGTTG-3 ’), R1A (5 ’-GCATCATCGTTAGCCAGTCA-3 ’)
and His-Reverse (5’-GTTGTAGCCGCCGTTGTTG-3’), His-Forward (5’-
GAGCACTCGATCTTCCCAG-3 ’) andR2B (5’-CGCTAGATGATGGGTCGAAT-
3 ’)• PCR reaction was performed with 2pl yeast miniprep DNA as template,
included lOOpmol of each primers, IX Taq polymerase buffer, 6mM of dNTP, and
0.15 units of Taq DNA polymerase (Qiagen). The main cycling parameters included:
94 °C for 2 min, 30 cycles of 94 °C for 45 s, 56 °C for 45s and 72 °C for 2 min 30s,
and extension at 72 °C for 5 min.
4.9 Southern Blotting Analysis
The genomic DNA was isolated from yeast strain SP1 and three RRP6::His
transformants. The intact DNA was digested with restriction enzyme Hind III (New
England Biolabs) at final concentration of 250 units/ml overnight at 37 °C in a total
51
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volume of 80 pi, and additional 250 units/ml of Hind III enzyme was added to boost
digestion for 5 hr at 37 °C. The digested DNAs were fractionated on a 0.7% (w/v)
agarose gel in 1XTAE (v/v) 20 V for 12 hours. The DNA was capillary-transferred
onto nylon membrane (Bio-Rad) with 0.4 N NaOH for 48 hr. The membrane was
dried with Whatman paper and UV crossed link at 1200. The purified full length of
RRP6 PCR product was used as probe for the structural analysis. RRP6 DNA was
radiolabeled using Prime-It II random primer labeling kit based on manufacturer’s
instructions (Stratagene) and purified through a ProbeQuant G-50 micro column
(Amersham). The membrane was hybridized with the probe overnight at 42 °C.
After being washed twice in 2xSSC, 0.5%SDS for 30 min each, and twice in 0.2 SSC,
0.5% SDS for 30 min each, the membrane was subjected to autoradiography.
4.10 RNA Isolation and RT-PCR
Yeast cells were grown in liquid YPD with 2% glucose and 1% penicillin-
streptomycin medium at 26 °C to A600= 0.35. Total RNA was extracted by using
RNeasy Mini Kit (Qiagen) according to manufacturer’s protocol. The cDNA was
synthesized in a 20 pi reaction mixture, containing 2 pg of total RNA, 2 pi 10X
buffer RT, 2 pi 5 mM each dNTP, 2 pi 10 pM oligo(dT), Ipl lOU/pl RNase inhibitor,
and 1 pi Omniscript RT (Qiagen). The PCR reaction mixture was made up in a 10
pi volume, containing 2 pi of cDNA, 0.5 pg of each oligonucleotide primer, 5 pi
2XHotStarTaq Master Mix (Qiagen) for 40 cycles with 95 °C for 45s, 55 °C for 45s,
72 °C for 2min 30s.
52
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4.11 Temperature and Cell Growth Analysis in Plates
Single colonies from yeast SP1 and RRP6::His-3 (LPC1, LPC2, and LPC) were
struck onto four sets of YPD and four sets of SC-His3 plates and each set of plate
was grown at 14 °C, 25 °C, 28 °C and 37 °C, respectively for 5 days. The
experiment was repeated twice. The conditions of cell growth were compared
among four yeast strains and the sizes of colonies were measured and analyzed using
the Student’s t-Test.
4.12 Growth Analysis in YPD Medium
Single colonies from yeast SP1 and RRP6::His-3 (LPC1, LPC2, and LPC3) were
picked and grown in tubes with 10ml YPD medium in the presence of 2% glucose
and 1% penicillin-streptomycin at 26 °C overnight. The concentrations of yeast cells
in the four tubes were measured, adjusted to be equal and aliquoted into 17 tubes
(total 17X4 tubes). Approximately every three hours, one yeast tube from each set
was collected into 4 °C until all tubes were collected. The concentrations of cells
were read at A600 by spectrophotomety.
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Asset Metadata

Creator Lin, Pi-Chu Kaylene (author)

Core Title Construction and characterization of RRP6 deletion in Saccharomyces cerevisiae

School Graduate School

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,biology, molecular,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Broek, Daniel (committee chair), Mosteller, Raymond (committee member), Tokes, Zoltan A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-311678

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