Finalthesis

UNIVERSITY OF CALIFORNIA
SANTA CRUZ
An investigation into small RNAs’ role in the genomic gymnastics that take
place during macronuclear development in Oxytricha trifallax
A thesis submitted by
CAMERON FERGUSON
March 2013
With Professor Alan Zahler

The role of small RNAs in Oxytricha’s Genome Gymnastics
2
Table of Contents
Abstract ...................................................................................................................3

Introduction ............................................................................................................5

Background .............................................................................................................8

Oxytricha trifallax ...................................................................................................8

Tetrahymena thermophila Scan-RNA model........................................................11

Conjugation-specific long RNAs ..........................................................................12

Conjugation-specific short RNAs..........................................................................13

Short RNAs interaction with a PIWI-like protein .................................................17

Summary................................................................................................................20

Specific Aim..........................................................................................................21

Results....................................................................................................................22

Generation of a usable ciliate lysate......................................................................22

Anion exchange FPLC ..........................................................................................22

Size exclusion FPLC .............................................................................................25

Discussion ..............................................................................................................27

Successful generation of ciliate lysates .................................................................27

Successful isolation of 27macRNAs and their associated proteins via anion
exchange and size exclusion chromatography.......................................................27

Future Aims...........................................................................................................29

Acknowledgments.................................................................................................31

Materials and Methods ........................................................................................32

Creation of Ciliate Strains .....................................................................................32

Growth of Ciliates .................................................................................................32

Mating of Ciliates..................................................................................................32

Generation of Ciliate Lysate..................................................................................33

Extraction of RNA from Ciliate Lysates...............................................................34

Fast Protein Liquid Chromatography (FPLC).......................................................34

Anion Exchange ..............................................................................................34

Size Separation ................................................................................................35

Dialyzing Pools .....................................................................................................36

RNA extraction of Pools .......................................................................................36

Ethanol Precipitation .............................................................................................37

Phosphatase Treatment of RNA ............................................................................37

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P Labeling of CIP-treated RNA..........................................................................37

Gel running of 32
P labeled RNA............................................................................38

Immuno-blotting....................................................................................................38

Silver stain.............................................................................................................39

Works Cited ..........................................................................................................41

Abstract
All organisms use a set of instructions, typically composed of DNA, to build
everything that they need to live and grow. Understanding how these instructions are
created and proliferated is essential in understanding how life works; this project aims to
elucidate a small part of that deeper understanding. In most organisms, including humans,
genetic instructions are passed from generation to generation in a form that is
immediately usable. Tiny single celled organisms called ciliates have a unique way of
passing instructions down generations. Instead of passing a fully functional set, they pass
a set of instructions that are interspersed with junk DNA. Tetrahymena thermophila’s
non-functional set of instructions contains 30% junk and each instruction is in the correct
order. Tetrahymena must delete the junk and push the resulting fragments together to
form their functional instructions. Oxytricha trifallax, a closely related species, faces a
more complicated problem in forming their functional instructions. Their non-functional
set of instructions contains 95% junk and most instructions are scrambled. That means
that instead of the non-functional set going (Coding region 1)-junk-(Coding region 2)-
junk-3-junk-4, it might go 3-junk-1-junk-4-junk-2. Oxytricha must not only delete a
larger portion of their DNA but they must move the remaining pieces into the correct
order to form their functional instructions. This makes them a more interesting model for
investigating how organisms pass instructions to future generations.
This project looks at a collection of short copies of the functional set of
instructions, called 27nt RNAs, which are thought to play a role in Oxytricha’s formation
of their functional instructions. My project's goal is to isolate proteins that interact with
the 27nt RNAs. I isolated a small number of proteins that associate with the 27nt RNAs,
one of which is a protein previously discovered to interact with 27nt RNAs. Subsequent

4
identification of the other proteins contained in these samples may provide clues to the
role the 27nt RNAs play in the genetic remodeling necessary for Oxytricha to form a
functional set of instructions.

5
Introduction
Understanding the way organisms generate and pass on their genetic information
is essential to understanding life. Most organisms contain their genetic information in the
form of DNA, stored in long strands called chromosomes. The chromosomes are
maintained, for most organisms, inside of a nucleus. A cell’s nucleus is used for two
functions. A nucleus is said to have (I), a germ-line function when it is used to pass
genetic information down generations and (II), a somatic function when it is running all
cellular processes. Most organisms have just one nucleus that is used as both a germ-line
and somatic nucleus. Ciliates however, do things differently. Instead of having one
nucleus they have two nuclei. One nucleus, called a micronucleus, has a germ-line
function. The second nucleus, called a macronucleus, has a somatic function.
The macronucleus is formed from a micronucleus during conjugation, a sexual
phase that is a part of the ciliate lifecycle. During conjugation, ciliates exchange genetic
information and each ciliate forms two new micronuclei. One of the newly formed
micronuclei will remain intact, functioning as a germ-line nucleus. The other nucleus is
converted into a macronucleus that functions somatically. Furthering the understanding
of this conversion is the goal of this project.
The difference between a micronucleus and a macronucleus is that the
micronucleus contains junk DNA. The junk DNA prevents the micronucleus from having
somatic function. To form the somatically functioning macronucleus, all of the junk DNA
is removed. This large-scale deletion to form a different nucleus is a unique way of
separating nuclear function.
While all ciliates undergo deletion of junk DNA, there are differences in the way
different ciliates put the remaining DNA back together. The ciliate species Tetrahymena

6
thermophila merely eliminate a minority of the micronucleus, while maintaining the
order of the information, to form their macronucleus. Oxytricha trifallax however, delete
the majority of their micronucleus, and rearrange the individual fragments into a new
order to form their macronucleus. While the mechanism behind how Tetrahymena form
their macronucleus is mostly known, Oxytricha’s more complicated macronuclear
formation, involving rearrangement of DNA, remains mostly unknown. Investigation into
the unique processes Oxytricha undergo to delete and reorder the DNA in their nuclei
may lead to the discovery of novel proteins and processes, previously undiscovered in
Tetrahymena.
A species of RNA has been discovered that probably guides this transition from a
germ-line micronucleus to a somatic macronucleus. That RNA plays a critical role in
cellular events in all species has been known for quite a while, but the extent of its role is
only recently being revealed. RNA has long been known to be part of the catalytic unit of
the ribosome, along with playing crucial roles in chromosome regulation in mammals
(Crick 1968; Jaenisch, et al. 2002). RNA has recently been discovered to play a role in
double strand break repair of DNA in yeast, where it is used to pass genetic information
through direct interaction with chromosomes (Resnick, et al. 2007). Tetrahymena have
been shown to use conjugation-specific RNAs to compare their two nuclei, allowing
them to mark junk DNA regions so they can delete them to form their somatic nucleus
(Aronica, et al. 2008). Due to the discovery of similar RNAs present in Oxytricha, along
with the close relationship between the two species, it is possible that Oxytricha use a
similar mechanism to form their macronucleus (Zahler, et al. 2012).
While RNA plays crucial roles in cellular function and development, it rarely acts
alone. RNA works in tandem with proteins. Sometimes RNA functions as a catalyst, as

7
discovered in the ribozyme of Tetrahymena, while proteins act as scaffolds (Celander and
Cech 1991). Other times RNA is used to bring proteins to specific locations, where the
proteins catalyze some sort of change, as is the case in Tetrahymena (Mochizuki, et al.
2002). Conjugation-specific RNAs (csRNAs) in Tetrahymena are used to compare its two
genomes and bring proteins to the junk DNA regions on a micronucleus, causing the
micronucleus to become a macronucleus. Due to Oxytricha’s close relation to
Tetrahymena, and the discovery of csRNAs in Oxytricha, it is possible that a similar set
of proteins may exist in Oxytricha. However, additional proteins are probably present in
Oxytricha due to their need to rearrange the pieces of good DNA after junk DNA
excision. Discovery of the proteins that interact with csRNAs may give clues to the role
that these RNAs play in Oxytricha’s macronuclear formation.
With the above as background, the goal of this project can be described as
follows: to isolate proteins that interact with the previously identified conjugation-
specific RNAs. Identification of these proteins will clarify how Oxytricha generate and
maintain their genetic information, fleshing out our idea of how life works.

8
Background
Oxytricha trifallax
The ciliated protozoan Oxytricha trifallax has a remarkable genetic make up,
composed of two separate and very different nuclei (Prescott 1994). One is a
transcriptionally silent germ-line nucleus called the micronucleus (MIC). The MIC is
passed from generation to generation but is not used for any transcription, most likely due
to the composition of its chromosomes. The chromosomes on the MIC are similar to
typically eukaryotic chromosomes: they are long DNA strands, containing multiple
genes, which are flanked by telomeres. The genes on the chromosomes however, are
broken up into many different pieces called Macronuclear-Destined Sequences (MDS),
which are interspersed with non-gene coding regions called Internally Eliminated
Sequences (IES). Not only do IESs interrupt the MDSs, but also the MDSs are sometimes
scrambled, existing in the incorrect order and orientation (Figure 1) (Greslin, et al. 1989).
The ciliate's other nucleus is called the macronucleus (MAC), and is responsible for all
transcription (Prescott 1994). It is composed of thousands of chromosomes, called
nanochromosomes, of which the majority (~90%) contain: a single gene, a small amount
of non-protein coding DNA, and telomeres (Swart, et al. 2013; Prescott, et al. 1995).
Ciliates have three different phases in their lifecycle, one of which is a basic
sexual process called conjugation. When ciliates conjugate, they exchange haploid
micronuclei (MIC) that fuse to form new diploid MICs . These subsequently undergo
meiosis. One of the new MICs is then converted into a new somatic macronucleus
(MAC) (Prescott 1992) (Figure 2). The transition from MIC to MAC involves 4 basic
steps described in Figure 3. At some point during this transition the ciliates original MIC
and MAC are deleted. This transition is correlated with the production of a series of short

9
27nt RNAs transcribed off the paternal MAC (Zahler et al., 2012). These RNAs have
been hypothesized to play a role in protecting macronuclear-destined sequences from
elimination in the developing macronucleus (Fang, et al. 2012).
Figure 1- A depiction of the genomic gymnastics that Oxytricha must undergo to form a functional gene. a: The
micronuclear version of the actin I gene of Oxtricha. Eight internally eliminated sequences, ranging from 11-110bp, are
interrupting the 9 macronuclear-destined sequences. Telomere addition sites (TASs) are denoted by an arrow. b: A
model for intramolecular recombinations. X’s denote DNA recombination events. c: The final macronuclear version of
the actin I gene after excision of flanking DNA and telomere addition. (Adapted from Greslin and Prescott, 1992)
a
c
b

10
Figure 2- A ciliates genetic
recombination events during
mating. a: A sketch of a ciliate
containing two micronuclei (MIC)
and two macronuclei (MAC). b:
Diploid MIC are replicated via
meiosis to generate 4 haploid MIC,
one of which is then exchanged
with another ciliate. c: The
maternal haploid MIC and the
paternal haploid MIC fuse to
generate a new fused diploid MIC.
d: The fused MIC undergoes
mitosis, to generate two fused MIC.
At this point, all old, un-fused DNA
is eliminated. e: One of the
remaining MIC starts to become a
MAC. f: The MAC and MIC
undergo mitosis, and each MAC
then amplifies each chromosome to
around 1900 copies. (Adapted from
Prescott 1992)
Figure 3- The transition
from MIC to MAC of a
hypothetical gene is shown
in 4 steps. Step 1 involves
excision and deletion of
IESs (green) while retaining
MDSs (orange). Step 2 is
the unscrambling and
reordering of MDSs into the
functional gene order. Step
3 is the addition of
telomeres (black) and step 4
is amplification of the
nanochromosome to around
1900 copies.
!
!
!
!
!
!Mic! 4! 1! 3!
4! 1! 3!
4!1! 2! 3!
1!
2!
3!
4!1! 2! 3!
4!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
4!1! 2! 3!
Mac!
An Oxytricha stained by the Feulgen technique to show the two
compact, transcriptionally inactive micronuclei and the two
DNA-rich, transcriptionally active macronuclei. Bar represents
20 ~m.
°
(a)
Mi
Ma
(b) (c)
(d) (e) (f)
FIG[]
Simplified diagram of nuclear events during mating in
Oxytricba. The two micronuclei (Mi) are equivalent, and the
two macronuclei (Ma) are equivalent. Only one micronucleus is
shown undergoing meiosis. Open small circles: diploid
micronuclei. Black or hatched small circles: haploid micronuclei.
Part-black, part-hatched circles: new diploid micronuclei, or
developing macronucleus. (a) A vegetative cell. (b) Mating cells,
with meiotic products of one micronucleus in each cell. (c) A
new diploid micronucleus in a cell that has mated. (d) Mitosis of
the new micronucleus and degradation of all other nuclei.
(e) Development of 6ne of the new micronuclei into a
macronucleus. (f) Division of the micronucleus and the newly
developed macronucleus to produce a mature, vegetative cell.
DNA replication bands are shown in the macronuclei.
Reproduced, with permission, from Ref. 26.
molecules, each with an averag
although some, such as the mo
are differentially amplified fu
copies 2. Altogether, a macronu
tains about 2.5 x 107 gene-sized
Internal eliminated sequences
micronuclear genes
Since macronudear gene
micronuclear genes during dev
genes should have counterp
exactly to the base pair in m
the 746 bp sequence of the m
0. nova occurs perfectly in
copy 15. However, in addition t
dence, the micronuclear copy
an additional 130 bp distribute
the gene (Fig. 4). A block o
coding region (open reading
block of 49 bp of different
gene beyond the 3' end of th
scribed part of the trailer, and a
different sequence interrupts th
of the C2 gene. During genome
ment these three sequence b
eliminated sequences, or IESs
functional macronuclear gene.
ments of the C2 gene that a
to make the macronuclear ge
segments are designated as
sequences, or MDSs. All the 20
far in Oxytricha nova and E
IESs, usually several to many.
per unit length of DNA, the
must contain over 50 000 IE
removed and destroyed during
ment. IESs are single-copy AT-
in size from 14 to 548 bp. Th
less randomly scattered in ge
pattern in relation to each oth
they separate.
TIG DECEMBER1992 VOL.8 NO. 12
i4C
MIC
MAC

Tetrahymena thermophila Scan-RNA model
The ciliate species Tetrahymena thermophila has a similar genetic make up to that
of Oxytricha but varies in several ways (Table 1). Most notably, Tetrahymena’s
micronuclear version of each gene is not scrambled and Tetrahymena discard only 30%
of the DNA from the MIC to make a new MAC (Cassidy-Hanley, et al. 2005; Coyne,
Stover and Miao 2012).
Information comes from the following sources: a
(Raikov 1982); b
(Prescott 1994); c
(Cassidy-Hanley, et al. 2005); d
(Coyne, et al. 2012); e
(Swart, et al. 2013); f
(Eisen, et al. 2006); g
(Coyne, et al. 2008)
A mechanism called the Scan-RNA model describes how Tetrahymena convert a
germ-line micronucleus into a somatic macronucleus during sexual conjugation
(Mochizuki and Gorovsky 2004) (Figure 4). The scan RNA model proposes that
Tetrahymena use a variety of RNA transcripts to compare their two nuclei, enabling them
to identify regions they must delete to form their macronucleus. First they generate long
non-coding RNA (ncRNA) transcripts of both of their nuclei. Next, the long ncRNA
transcripts of the micronucleus are processed by a dicer-like protein (Dcl1p) into short
28nt RNAs (Mochizuki and Gorovsky 2005). Then, a PIWI-like protein (Twi1p)
transports these short RNAs to the parental macronuclear long nc-RNA transcripts
(pmac-ncRNA) (Mochizuki, et al. 2002). Here the short RNAs ‘scan’ the pmac-ncRNA
searching for complementary regions, earning the short RNAs the name Scan RNAs
(scnRNAs) (Mochizuki and Gorovsky 2004). ScnRNAs that do not have a
!
Table 1. Genomic comparison of Oxytricha and Tetrahymena.
Micronucleus
Genome
Size
Chromosomes Scrambled? Gene coding ‘junk’ DNA
Oxytricha 1GBa
Few, longb
Yesb
4%b
96%b
Tetrahymena 220MBc
5c
Noc
70%d
30%d
Macronucleus
Genome Size Genes Chromosomes Ploidy
Oxytricha ~50MBe
~18,400e
~15,600e
~1900b
Tetrahymena 105 MBf
24,700g
~225f
45f

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complementary sequence on the macronuclear RNA transcript must subsequently
correspond to micronuclear specific sequences (which are IESs). The scnRNAs that
correspond to IESs are transported to the developing macronucleus, while scnRNAs that
have complementary sequences in the parental macronuclear transcripts are degraded
(Figure 4). ScnRNAs then interact with the long ncRNAs transcribed from the
developing macronucleus and cause chromatin remodeling around their complementary
sequences, which leads to eventual deletion of these segments (Liu, et al. 2007). The
exact mechanism by which the scnRNAs guide deletion of IESs for the formation of a
macronucleus will be discussed later.
Figure 4- The scan RNA Model
for Tetrahymena thermophila. a:
A long RNA transcript is
transcribed from the maternal
MAC, while short scnRNAs are
produced by Dcl1p from a long
RNA transcript made from the
MIC. b: ScnRNAs are transported
to the maternal MAC where they
scan the maternal MAC’s long
RNA transcript (pmac-ncRNA). c:
ScnRNAs that don’t match up with
the long RNA of the maternal MAC
are transported to the developing
MAC. d: ScnRNAs that match to
sequences in the developing MAC
lead to deletion of MIC specific
sequences in the developing MAC.
(Figure from Nowacki, et al. 2008)
Conjugation-specific long RNAs
In Tetrahymena, long non-coding RNAs (ncRNAs) are transcribed from both the
parental MIC and MAC during conjugation (Mochizuki and Gorovsky 2004). The
micronuclear ncRNAs are processed by Dcl1p (Dicer homolog) into short ‘scan’ RNAs.
The long macronuclear ncRNAs are scanned by the short micronuclear ncRNAs to enable
selection of short ncRNAs that correspond to IESs. Laura Landweber et al. recently
discovered a set of long RNAs present during conjugation that provides support for a
G12CH16-Landweber ARI 5 August 2011 14:19
Maternal MAC
MIC
a
Maternal MAC
MIC
b
Maternal MAC
Developing MAC
c
Maternal MAC
Developing MAC
d
Maternal
transcripts
scnRNA
Transposon IES
Figure 5
The scan RNA model for programmed genome rearrangements. (a) Endogenous short RNAs (scnRNAs,
blue and orange dashed lines) are produced in the meiotic micronucleus (MIC). (b) These scnRNAs are
exported to the maternal macronucleus (MAC), where they may scan the genome via interactions with long
maternal transcripts (blue wavy lines). The scnRNAs at this point correspond to the entire germline genome,
including transposons and internal eliminated sequences (IESs, both in orange). (c) scnRNAs (orange dashed
lines) that cannot pair with homologous sequences are then selectively transported to the developing
(zygotic) MAC, where they target histone methylation on homologous DNA sequences. (d ) The process in
panel c results in the speciﬁc elimination of micronuclear sequences that are absent from the maternal MAC.
Tiny red lines indicate telomeres.
forms. Jahn et al. (49, 50) suggested that these
circular forms in Oxytricha and Euplotes may
derive from very large genomic regions located
outside physical clusters of MDSs in the MIC.
More recent studies of Tetrahymena have
provided additional evidence for the associa-
tion of heterochromatin with DNA eliminated
during macronuclear development. Studies
of Pdd (programmed DNA degradation)
proteins, which are located in the developing
macronuclei and form large heterochromatic
structures that appear during DNA elimina-
tion (23, 77, 78, 97, 98, 124), have shown
by hybridization that these heterochromatic
regions contain IESs. Moreover, Pdd proteins
contain chromodomains, which are conserved
in heterochromatin-associated proteins over
a wide range of organisms (60). Notably,
chromodomains do not directly bind DNA but
rather interact with RNA (2). This observation
further suggests a role for RNA in the process
of heterochromatin formation and DNA
elimination in Tetrahymena.
In general, studies of various ciliate species
suggest that DNA elimination is preceded by a
major reorganization of chromatin structure in
the developing MAC. It seems clear that het-
erochromatin structure must be formed prior
378 Nowacki ·Shetty ·Landweber
byUniversityofCalifornia-SantaCruzon09/16/11.Forpersonaluseonly.

13
scan-RNA model in Oxytricha (Nowacki, et al. 2008). Landweber et al. found a long
non-coding RNA template of parental MAC chromosomes (pmac-ncRNA) present during
early development (~5hr) that disappears later in conjugation (~50hr). RNA interference
against putative long RNA templates caused improper MAC formation, suggesting that
the long RNA templates play a role in successful MAC formation. Injection of
alternatively rearranged MAC chromosomes led to these alternatively rearranged MAC
chromosomes being present in the ciliates offspring. These results led Landweber et al. to
suggest a model for how these pmac-ncRNAs may guide correct rearrangement of MDSs,
explained in Figure 5 (Nowacki, et al. 2008). This mechanism for rearrangement in
Oxytricha is not present in Tetrahymena because Tetrahymena does not have to
unscramble its genome. Tetrahymena does not need a guide RNA to put all the
macronuclear-destined sequences (MDSs) in the correct orientation, it only needs to
delete its internally eliminated sequences (IESs), which it does using short RNAs.
Figure 5 – Landweber’s model for how long RNA templates may help guide the genetic unscrambling during
macronuclear development. a: A long RNA template (pmac-ncRNA) is transcribed from the parental MAC. b: The
pmac-ncRNA template is transported to the developing macronucleus. c: the MIC chromosome begins to rearrange
using the pmac-ncRNA template as a guide. d: De novo telomere addition
Conjugation-specific short RNAs
In Tetrahymena short 28nt RNAs (scnRNAs) are produced early in development
from the entire micronucleus (Chalker and Yao 2001). These scnRNAs then form

14
complexes with Twi1p proteins that transport the scnRNAs to the macronucleus
(Mochizuki, et al. 2002). At some point during this transportation, the scnRNAs are 2`-O-
methlyated by Hen1p (Kurth and Mochizuki 2009). This methylation is required for
stable accumulation of scnRNAs and proper DNA elimination. ScnRNAs are then
selectively degraded to retain only scnRNAs that match IESs. Ema1p (putative RNA
helicase) mediates an interaction between the scnRNA-Twi1p complexes and the long
ncRNA transcripts of the parental macronucleus (pmac-ncRNA) (Aronica, et al. 2008). It
has been proposed that the scnRNA-Twi1p complexes, with help from Ema1p, scan the
pmac-ncRNA and bind to complementary sequences. Bound scnRNA-Twi1p complexes
are degraded. This results in accumulation of scnRNAs corresponding to IESs
(Mochizuki, et al. 2012). The remaining scnRNA-Twi1p complexes are transported to the
developing macronucleus where, in conjunction with Ema1p, they interact with the non-
coding RNA transcript of the developing macronucleus (nmac-ncRNA). The scnRNA-
Twi1p complexes, which are now only complementary to IESs, associate with these IESs
on the nmac-ncRNA. Knockouts of Ema1p, Twi1p, and Ezl1p (a histone
methyltransferase) showed greatly reduced H3K9/K27 methylation and the resulting
progeny died. This suggests that scnRNA-Twi1p complexes localize to chromatin sites
where they in turn recruit Ezl1p (Liu, et al. 2007, Aronica, et al. 2008). Ezl1p, along with
other proteins, methylates H3K9 and K27. It has been suggested that methylation of H3
causes the deletion of IESs by some unknown mechanism (Liu, et al. 2004 and 2007).
ScnRNAs are complementary to IESs and have been shown to interact with a
histone methyltransferase. This suggests a mechanism in which scnRNAs mark regions
for deletion in the developing macronucleus by guiding histone methylation (Cheng, et al.
2010). Histones form 4 unit structures called nucleosomes that, in Tetrahymena, interact

15
with 202bp of DNA (Gorovsky, et al. 1978). IESs in Tetrahymena are all larger then
202bp, so if a nucleosome is in fact used to mark IESs it could effectively mark just the
IES and not any surrounding coding regions. This suggests the following mechanism for
IES deletion: (I) scnRNAs localize at IESs due to their complementarity, (II) scnRNAs
interaction with Ezl1p causes methylation of nearby histones, (III) the nucleosome
containing the now methylated histone marks that portion of DNA for deletion simply by
containing a modified histone, (IV) this modified nucleosome may in turn recruit other
protein factors that excise and delete the DNA contained within it.
This mechanism currently fails to explain how Tetrahymena manage such precise
excision. Nucleosomes are rarely perfectly stationary and IESs are not all exactly 202bp,
so using them alone to guide excision would not be precise. Instead, I propose that
nucleosome marking merely gives the organism a general location and other DNA/RNA
factors must play a role in order to obtain the necessary specificity.
This mechanism of histone methylation to mark regions for deletion is not
possible in Oxytricha, because IESs have been found to be as small as 11bp. So, any
nucleosome modified to mark that IES would cause marking and eventual deletion of the
flanking regions of that IES, which are coding regions that need to be retained. This
mechanism also requires a species of short RNAs to be IES-specific in order to modify
histones corresponding to IESs. As is described below, this is not the case in Oxytricha.
Alan Zahler at UCSC discovered in Oxytricha a species of 27nt RNAs that are
MAC specific (Zahler, et al. 2012). These RNAs (27macRNAs) are transcribed
exclusively during mating (Figure 6). Sequencing showed that 27macRNAs had a
uniform distribution across MAC chromosomes and spanned MDS-MDS junctions. The
27macRNAs spanning MDS-MDS junctions did not include IESs, suggesting that they

16
are transcribed from the parental MAC, which is different than the MIC-transcribed
scnRNAs in Tetrahymena. The 27macRNAs were further
classified via beta-elimination to show that they contain no
3’ end modification, in contrast to the 2`-O-methyl 3` ends
of scnRNAs. The method of sequencing used by Zahler et
al. would not work if the 27macRNAs did not contain a 5’
monophosphate. Landweber also independently discovered
the 27macRNAs by sequencing RNAs that associated with a
PIWI protein (described below) (Fang, et al. 2012). She
then injected synthetic 27macRNAs corresponding to
specific IESs into conjugating ciliates, which caused
inclusion of those IESs. In contrast to Tetrahymena’s
scnRNAs that mark regions for deletion, 27macRNAs:
correspond to MDSs, are transcribed exclusively during
mating, and have the ability to cause inclusion of IESs.
This implies that 27macRNAs mark DNA for retention, not
deletion.
The slight yet important differences (Table 2) of
Oxytricha’s short conjugation specific RNAs from that of Tetrahymena’s might be
explained by the differences in the genetic makeup of each ciliate (Table 1). The short
RNAs of each organism seem to act upon the smallest portion of DNA. Oxytricha’s
27macRNAs probably interact with the 5% of the genome they retain for the new MAC.
Tetrahymena’s scnRNAs however mark the 30% of the genome that they delete from the
developing MAC. The location of 27macRNA biogenesis suggests that they probably do
Figure 7- 15% polyacrylamide-BIS PAGE gel
loaded with P32 labeled. Lanes 1-4 are vegetative
RNA extract. 27nt RNAs are not present in these
lanes. Lanes 5-11 is RNA extracted at certain
time points after mating. 27nt RNAs appear at
around 24 hr and slowly taper off . Lane 12 is
RNA extracted from mock mating in which XC9
ciliates were exposed to mating conditions
without their mating pair (XC2) (Figure from
Zahler, et al. 2012)

17
not cause methylation of histones near IESs as they are not complementary to IESs.
Instead, they may play a role in causing methylation of histones near MDSs. This is also
supported by the fact that, as has been previously described, IESs in Oxytricha are too
small to be marked exclusively by histones. The apparently different role of 27macRNAs
suggests that they interact with different proteins than the scnRNAs. This project
attempts to isolate those different proteins, as well as similar ones, with the hope that
subsequent identification of the proteins will provide clues into the role of the
27macRNAs in Oxytricha’s macronuclear development.
*Exact role of sRNA is unknown; this is an untested hypothesis of b.
Information from the following sources: a
(Zahler,
et al. 2012); b
(Fang, et al. 2012); c
(Mochizuki, et al. 2012); d
(Mochizuki, et al. 2002); e
(Kurth and Mochizuki 2009)
Short RNAs interaction with a PIWI-like protein
The Tetrahymena scan-RNA model describes a PIWI-like protein, Twi1p, which
interacts with the short 28nt RNAs (scnRNAs), acting as a sort of shuttle, transporting the
scnRNAs from the parental MAC to the developing MAC (Mochizuki, et al. 2002).
Twi1p continues to be associated with the scnRNAs throughout macronuclear
development, and is essential for correct IES excision. Experiments with Twi1p knockout
ciliates resulted in considerable reduction, although not complete elimination, of
scnRNAs as well as death of any progeny (Mochizuki and Gorovsky 2004). Twi1p is not
necessary however for vegetative growth (Mochizuki, et al. 2002).
Hans Lipps et al., working with a species closely related to Oxytricha, Stylonychia
mytilus, discovered a PIWI-like protein that localizes to different nuclei during
conjugation (Postberg, et al. 2008). Stylonychia’s genetic makeup is very similar to
Table 2. sRNA differences between Oxytricha and Tetrahymena
Derived
from
Length 5’ end 3’ end Mark for Act on %
of genome
Interact
with
Oxytricha MACa
27nta
Mono Pa
No Moda
Retention*,b
10a
Otiwi1b
Tetrahymena MICc
28ntd
2`-O-CH3e
Deletiond
30b
Twip1d

18
Oxytricha’s, with a scrambled micronucleus composed of a lot of junk DNA
(Ammermann, et al. 1974). Lipps et al. used an antibody specific to human PIWIL1,
along with a secondary antibody with a luminescent tag, to visualize the movement of
PIWI proteins during conjugation. A PIWI-family protein moves from the parental
macronucleus to the developing macronucleus, closely matching the way Twi1p moves
around Tetrahymena’s nuclei during mating (Mochizuki, et al. 2002).
Landweber et al. repeated this experiment in Oxytricha after discovering a set of
PIWI-like proteins she called Otiwi (Fang, et al. 2012). Otiwi1 appears to be the most
important of the 13 Otiwi proteins during development and it interacts with 27macRNAs
during conjugation. Using the PIWIL1 antibody that Lipps used, Landweber et al.
demonstrated that Otiwi1 moves from the parental MAC to the developing MAC during
development, in a similar manner to Twi1p in Tetrahymena (Figure 7). Knockout of
Otiwi1, via injection of antisense DNA oligos that match the Otiwi1 coding sequence,
caused a decrease in the production of 27macRNAs as well as death of any progeny.
These results closely match results for Twip1 in Tetrahymena.

19
Figure 8 - Otiwi1 moves from the paternal MAC to the developing MAC during conjugation. Otiwi1 is absent
early in conjugation (A) but appears in mid to late pair formation (B) and begins to enter the cytoplasm and developing
MAC a little later (C). At 24 hours (D) Otiwi1 is only present in the developing nuclei. Otiwi1 expression disappears
later in development (E). Immuno-staining of Otiwi1 was done with the rabbit anti-PIWIL1 antibody in conjunction
with a mouse-anti-rabbit Alexa 488 2˚ antibody. DAPI was used to stain nuclei, while α-tubulin was stained using
mouse anti-α-tubulin 1˚ antibody in conjunction with goat-anti-mouse Alexa 568 2˚ antibody. AN stands for anlage.
(Figure from Fang, et al. 2012)

20
Summary
Several aspects of the scan-RNA model from Tetrahymena appear to be present in
Oxytricha, despite what appears to be an “evolutionary sign change” for the role of the
short RNAs (Fang, et al. 2012). Long RNA transcripts have been discovered that appear
to be transcribed from the macronucleus. A species of short 27nt RNAs are also
transcribed from the MAC exclusively during mating. A PIWI protein, Otiwi1, has been
shown to co-localize with 27macRNAs as well as move from the parental MAC to the
developing MAC.
All of the aforementioned discoveries have corollaries in the scan RNA model of
Tetrahymena. However, two differences in the genetic make up of each ciliate suggest a
distinctly different mechanism. First, the difference in minimum IES length between
Tetrahymena, 300bp, and Oxytricha, 11bp, suggests that the role of histone modification
must be different. In Tetrahymena, histone modification could effectively mark just IESs
due to the 202bp of DNA the nucleosome, which contains the modified histone, interacts
with. A similar mechanism in Oxytricha however would cause the nucleosome to mark
regions of MDSs along with the IES due to their small IES size. The second genetic
difference is that the transition from MIC to MAC for Oxytricha involves more than just
deletion of the IESs, which is all that Tetrahymena do. MDSs in the MIC of Oxytricha
are scrambled and occasionally found in the incorrect 5`à3` orientation compared to that
of the functional gene (see Table 1). As such, the MDSs must be unscrambled and
reordered as well as ligated together to form the correct nanochromosome. Due to these
slight yet important genetic differences, several novel processes and proteins are likely
involved to help guide the unscrambling of MDSs as well as mark MDSs for retention.

21
Specific Aim
My project aims to identify additional proteins that associate with 27macRNAs
using Fast Protein Liquid Chromatography (FPLC). By following 27macRNAs, as
opposed to specific target proteins, unexpected proteins may be identified. This may give
us clues to the role the 27macRNAs play in the genome gymnastics of Oxytricha. I first
generated a ciliate lysate that maintained the integrity of the proteins and RNAs. I then
used a variety of columns on an FPLC machine to isolate 27macRNAs and their
associated proteins.

22
Results
Generation of a usable ciliate lysate
In order to run FPLC, we needed to generate a ciliate lysate
that maintained the integrity of Oxytricha RNAs and proteins. As no
protocol existed for Oxytricha lysate production, I adapted a protocol
from a previous graduate student, Sam Gu, who generated a lysate of
C. elegans (Gu, et al. 2007). Our attempts to concentrate ciliates in
Buffer B (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2, 1 mM
DTT and 0.5 mM PMSF) caused premature lysing, so we decided to
concentrate ciliates in a special Pringshiem solution that contains no
Ca(NO3)2. After ciliates were successfully concentrated in a Corex
tube, a 5X version of Buffer B was added prior to sonication. Buffer B
contains DTT and PMSF, two chemicals known to inhibit proteases
(Cleland 1964) (Chong, Garrard and Bonner 1974). This lysate
however failed to maintain the integrity of the 27macRNAs. We
decided to add the RNase inhibitors, RNasin and Vanadyl complex, to
stop RNA degradation (Figure 8).
Anion exchange FPLC
RNA is a negatively charged molecule, so we started FPLC
with a column that binds negatively charged molecules: a Mono Q
10/100 GL anion exchange column (column volume 7.94ml). After
!
60nt!
40nt!
30nt!
20nt!
50nt!
10nt!
27nt!
1!!!!2!!!!3!!!!4!!!!5!!!!6!
Figure 9- 32
P endlabeled RNA on a 15%
polyacrylamide gel. A 27nt band is seen in
lanes 2, 4, and 5. Degradation products are
seen in lanes 2 and 6 and to a lesser extent
in lane 4.
Lane Sample
1 Decade Marker
2 24hr Total RNA extract
3 48hr Total RNA extract
4 Lysate + RNasin
5 Lysate + RNasin + Vanadyl
6 Lysate only

23
several runs we decided to switch to a Mono Q 5/50 GL anion exchange column (column
volume = 0.98ml), which is the same type of column but has a smaller column volume, as
we were considerably under loading, in terms of protein concentration relative to column
capacity, the 10/100. A smaller column volume means that changes in salt concentration
take a smaller eluent volume to affect the whole column. This results in less dilution of
proteins in the peaks. The change from a column volume of ~8ml to ~1ml caused our
peak of interest to be condensed from a 6ml region to a 1-2ml region. After several
experiments using linear gradients from 50mM KCl to 2M KCl, a general location for the
27macRNAs was identified to be between 100mM KCl and 300mM KCl although a
minor subset of 27macRNAs did elute later (data not shown). We also changed to using
fresh lysates as opposed to ones prepared and frozen at -70˚C as this appeared to reduce
RNA degradation (data not shown). The FPLC method was then changed from a linear
gradient to a step gradient to further isolate the location of the 27macRNAs. In the FPLC
run displayed below, salt concentration is stepped from 50mM KCl to 100mM KCl and
run for 3 column volumes. Then the salt is stepped to 300mM KCl and run for another 3
column volumes before being raised to 2M KCl for 4 column volumes to wash off any
molecules that had yet to elute. After the FPLC was run, fractions were chosen based on
the chromatograms UV graph (Figure 9). Fractions underneath peaks in the UV graph
were pooled and analyzed for RNA content, via 32
P end-labeling, and for protein content,
via immuno-blot with anti PIWIL antibody and silver stain, as described in Materials and
Methods (Figure 10).

24
Figure 10 – Chromatogram of 2ml of fresh lysate run over a Mono Q 5/50 GL column. Numbers correspond to pools
chosen for analysis. All pools were processed as described in Materials and Methods and subjected to 32
P labeling, immuno-
blot with α-PIWL1 antibody, and Silver stain analysis. Yellow line stands for %B, or percent of Buffer from pump B. Pump
B contained the 2M KCl version of Buffer B, so 5% B corresponds to ~100mM KCl.
Figure 11 - Protein and RNA analysis of pools from the above Mono Q column. A: A silver stain was performed on
the 7 pools, with samples run on a 10/7 SDS-PAGE gel. To the left of the gel is a diagram of the PageRuler Plus ladder
(Thermo Sci) that was run in this and all other 10/7 SDS-PAGE gels. B: The 7 pools and a Pre-fractionation sample
were RNA extracted, CIP treated, 32
P labeled, and run on a 15% polyacrylamide-urea denaturing gel in 1X TBE.
Shown is a 1-hour phosphoimage exposure. Isolated 27nt bands are seen in Pools 4, 5 and 6. 27nt, along with other size
RNAs, are also present in the Pre sample and Pool 8. C: The 7 pools were run on a 10/7 SDS-PAGE gel in SDS
Running Buffer. The resulting gel was transferred to a PVDF membrane and subjected to immuno-blot with α−PIWIL1
antibody. Pools 4 and 5 show strong banding of Otiwi1 along with fainter bands in 3 and 7.
!
!
10nt!
50nt!
40nt!
30nt!
20nt!
27nt!
!1!!2!!!3!!4!!!5!!!6!!!7!!!!!
Pre!
B
C
A

25
Size exclusion FPLC
To further purify the 27macRNAs and their associated proteins we ran pools
containing 27macRNAs, generated from our Mono Q column, over a Superdex 10/300
GL sizing column. This was done to estimate the size of the protein complex that
associates with 27macRNAs, as well as to separate out any smaller or larger unrelated
proteins. Free RNAs should elute at the end of the column due to their small size, while
RNAs associated with proteins will elute sooner. Pool 4 from the above FPLC run was
loaded on the Superdex column (Figure 11). Pools were again chosen based on the UV
graph and analyzed for RNA content, via 32
P end-labeling, and for protein content, via
immuno-blot with α-PIWIL1 antibody and silver stain (Figure 12).
Figure 12 - Chromatogram of 500µl of a 27macRNA containing pool (Pool 4 of Figure 9) run over a Superdex
10/300 GL column. Buffer C was used as the liquid phase. Numbers correspond to pools chosen for analysis. All pools
processed as described in Materials and Methods but without dialyzing. Pools analyzed via 32
P labeling, immuno-blot
with α-PIWIL1 antibody, and silver stain.

26
Figure 13- Protein and RNA analysis of pools from the above Superdex column. A: A silver stain of the pools
from the Superdex column run on a 10/7 SDS-PAGE gel. All lanes loaded with 30µl. Far left lane is the PageRuler Plus
ladder (Thermo Sci) whose bands correspond to the ladder image below. Multiple bands are only seen in lanes 4 and 5.
B: 200µl of each Pool was CIP-treated and 32
P labeled prior to running on a 15% polyacrylamide-urea denaturing gel.
18-hour exposure shown. Lane 5 contains the only identifiable RNA, with a band at ~27nt. C: An immuno-blot with α-
PIWIL1 antibody of a 10/7 SDS-PAGE gel run with the 7 samples. PageRuler Plus Ladder has been lined up
representative to its location on the gel. Lane 5 contains the only band at approximately 100kDa.
!!
10nt!
50nt!
40nt!
30nt!
20nt!
27nt!
1!!2!!3!4!!5!!6!7!
!!1!!!!!!!!!2!!!!!!!!!3!!!!!!!!!!4!!!!!!!!!!5!!!!!!!!!!6!!!!!!!!!7!
A
C
B
!!

27
Discussion
Successful generation of ciliate lysates
The first step of this project was to generate a usable lysate that maintained the
integrity of Oxytricha proteins and RNAs. The lysate protocol we developed generated a
lysate that, if fractionated immediately, maintained the 27macRNAs while avoiding
excessive degradation (Figure 8). Immuno-blotting with an α−PIWIL1 antibody along
with silver staining of FPLC pools confirmed that the lysates also retained proteins
(Figure 10 and 12). We conclude that we have successfully developed a lysate protocol
suitable for RNA and protein isolation of Oxytricha trifallax.
Successful isolation of 27macRNAs and their associated proteins via anion
exchange and size exclusion chromatography.
The next step was to isolate the 27macRNAs and their associated proteins from
other RNAs and proteins. Surprisingly just one column was necessary to isolate the
27macRNAs from any other detectable RNAs (Figure 10). The 27macRNAs elute at a
relatively low salt concentration (100mM to 300mM KCl) compared to that of free RNA,
which should elute at high salt concentration. The 27macRNAs therefore appear to be
associated with a protein or complex of proteins with a slight positive charge, giving the
whole RNA-protein complex a slightly negative charge. The 27macRNA-containing
pools also contained Otiwi1, which was previously shown to associate with 27macRNAs
(Figure 10, C) (Fang, et al. 2012). This suggests that the Mono Q column not only allows
for the separation of the different RNA species but also their associated proteins. These
pools, as shown by silver stain, remain fairly complex in terms of protein content (Figure

28
10, A) so we decided that further fractionation of these pools was necessary prior to
analyzing them via mass-spectrometry.
We then ran the pools containing 27macRNA/Otiwi1 on a Superdex size
exclusion column (Figure 11). This column should separate molecules by size, causing
larger proteins to elute faster than smaller ones. Free RNAs should elute almost last due
to their small size. Pool 5 from the Superdex contained our 27macRNAs (Figure 12, B).
The silver stain of this pool displayed 6 protein bands, suggesting that 6 different sizes of
proteins are present in the same pool that contains 27macRNAs (Figure 12, A). Our
immuno-blot with α−PIWIL1 shows that one of these proteins is Otiwi1, which is known
to interact with 27macRNAs (Figure 12, C). Our results suggest that we have managed to
isolate several proteins that associate with 27macRNAs. It is not surprising that the
27macRNAs associate with more than just Otiwi1, as the scnRNA-Twi1p complexes,
present in Tetrahymena, associate with many different proteins throughout development
(Mochizuki, et al. 2012). Alternately other proteins with similar chromatographic
properties may simply elute at the same time as the 27macRNAs without actually being
related to them. This needs to be examined via further experimentation
We also see a separate group of 27macRNAs and Otiwi1 proteins that elute at a
higher salt concentration (Pool 7, figure 10 B, C). These RNAs and proteins may have
eluted eventually at 300mM KCl but the disappearance of the Otiwi1 signal in Pool 6 of
Figure 10, C suggests this is not the case. Instead we believe that the 27macRNAs and
Otiwi1 protein may form complexes with many different proteins and we have only
isolated the most positively charged of the complexes. This makes sense when compared
to the Tetrahymena scnRNA model, in which scnRNA-Twi1p complexes associate with a
wide range of different proteins at different points of macronuclear development

29
(Mochizuki, et al. 2012). Given that not all ciliates are in the same exact stage of
conjugation during lysing we would assume that 27macRNAs and Otiwi1 proteins would
not be isolated to a single location and protein complex. Instead they may associate with
different proteins throughout development causing them to elute in multiple places on an
anion exchange column.
Future Aims
Now that we have successfully isolated the 27macRNAs and several associated
proteins, one of which is known to interact with 27macRNAs, we will analyze a sample
via mass-spectrometry, of which the most popular method is Multi-Dimenisonal Protein
Identification Technology (MuDPIT) (Wolters, et al. 2001). MuDPIT analysis will
provide us with a list of peptide fragments that, when compared with the predicted
proteolytically cleaved products of the Oxytricha proteome, will provide a list of proteins
that are potentially present in our sample. Some candidate proteins we expect to identify
are RNA helicases and histone methyltransferases, as well as other novel proteins that
may be involved in genome rearrangement. We shall generate antibodies for the proteins
we identify in order to run Co-immunoprecipiation assays (Co-IP). Using Co-IPs and
sequencing we will be able to confirm that a protein associates with 27macRNAs or
Otiwi1 and doesn’t just happen to elute at the same time. We will then attempt to
synthesize proteins that associate with 27macRNAs, using CellFree Science’s cell-free
protein translation system, for use in functional assays (Endo, Sawasaki and Takai 2010).
We also intend to continue trying to isolate 27macRNAs using a Mono S cation
exchange column from pools generated from the Mono Q column, because our current
Mono Q-Superdex sample may not contain a high enough concentration of proteins for

30
mass-spectrometry. We believe using a Mono S after a Mono Q will provide us with the
least amount of protein and RNA loss, while giving us a pure enough sample for mass-
spectrometry. Several attempts were made at doing this, however it appears the column
we were using was non-functional (data not shown). We are currently in the process of
obtaining another Mono S column.
Another FPLC method we are going to try is affinity chromatography. Affinity
chromatography may allow us to quickly find proteins that associate with Otiwi1. In
affinity chromatography the columns contain a matrix of beads connected to a specific
ligand. We intend to attach Otiwi1-27macRNA complexes to the beads and subsequently
run lysates over them. Otiwi1 will be generated using the CellFree Science’s cell-free
system. Proteins that associate with the Otiwi1-27macRNA beads probably also associate
with Otiwi1 or 27macRNAs within Oxytricha. These proteins will also be identified via
mass-spectrometry.
The original goal of this project was to isolate proteins that associate with
27macRNAs. After the start of this project it was discovered that a PIWI-like protein
called Otiwi1 interacts with the 27macRNAs (Fang, et al. 2012). The goal of the project
was then refined to focus on the isolation of additional proteins that associate with both
the 27macRNAs and Otiwi1 protein. So far we have managed to isolate 27macRNAs
from other detectable RNA species while retaining multiple proteins, including Otiwi1.
Subsequent identification of these proteins that associate with 27macRNAs via mass-
spectrometry should provide clues into the role 27macRNAs play in the genomic
gymnastics that take place during macronuclear development in Oxytricha trifallax.

31
Acknowledgments
I first and foremost want to thank Dr. Alan Zahler for giving me the opportunity
to work in his lab. Working in his lab has cemented my desire to become a research
scientist, and opened my eyes to the possibility of becoming a professor. Dr. Zahler’s
method of allowing independent work enabled me to grow both as a researcher and a
person. I consider the amount of knowledge and experience I have gained as priceless
and look forward to continuing my work in his lab.
I also wish to thank Dr. Melissa Jurica for the use of her FPLC machine along
with the many tips about how to move forward after failed columns. Without her this
project would be nowhere near close to completion. I also wish to thank her and Dr.
Susan Strome for inspiring me to obtain a research position in the first place.
I want to thank my lab mates who have helped with countless experiments. I have
had lots of fun working with all of you. Many thanks go to Athena Lin for training me in
the basics of ciliate maintenance and for generating ciliate lysates. I also want to thank
Zach Neeb for his advice and training in running 32
P gels. Thanks also to Diana Summers
for helping me get this thesis started and Matt Ragle for teaching me how to run SDS-
PAGE gels.
Last but not least I want to thank everyone who helped me with this thesis. Many
thanks go to Dr Alan Zahler, Dr Hinrich Boeger, and my father Toby Ferguson, for
sitting down and looking over my thesis with me. I also wish to thank Ken Boyd and
Kylie Kenny for editing my thesis.

32
Materials and Methods
Creation of Ciliate Strains
Robert Hammersmith supplied JRB310 and JRB510 strains of Oxytricha trifallax
(Zoller, et al. 2012). These strains were mated together and individual mating pairs were
separated in a 24 well plate. Each mating pair was grown up separately and eventually
cross-mated to find additional mating pairs. ALXC9 and ALXC2 were found to mate
with ~80% efficiency. However due to ALXC2s propensity for self-mating another cross
mating was performed in order to identify a better mating set. A separate mating pair
composed of ALXC9 and another isolate, CF3s, were found to have a high mating
efficiency (~85%). CF3s and ALXC9s do not self-mate, allowing for fine control of
mating initiation. Ciliates are stored in cyst state at -200˚C and are thawed when needed.
Growth of Ciliates
Ciliates are grown in Pyrex baking dishes containing 300ml of a vegetative
growth media called Ciliate salts (0.1mM CaCO3, 0.08mM KCl, 0.029mM CaHPO4,
0.0166mM MgSO4). Ciliates are fed Chlorogonium elongatum (UTEX Collection Strain
B203), a strain of algae. Cultures are transferred into clean dishes daily.
Mating of Ciliates
Mating strains are individually filtered through cotton to remove food. The
ciliates are then extracted from their vegetative growth media using a sheet of 10 micron
nitex, which allows the ciliate salts, algae, and bacterial food, to flow through but not the
ciliates. The ciliates are washed off the nitex into a mating media called Pringsheim salts
(0.11mM Na2HPO4, 0.08mM MgSO4, 0.85mM Ca(NO3)2, 0.35mM KCl). Ciliates are

33
then counted in two rows of 5, 20µl drops of a 1:100 dilution. Each strain is then diluted
to 1600 ciliates per ml before being mixed in equal volumes. After 48 hours the ratio of
ciliates with and without anlage are counted. Anlage are vacuole-like shapes within
mated ciliates that are the developing nuclei.
Generation of Ciliate Lysate
Ciliates are first mated as described above. At 24 hours post mixing the ciliates
are filtered through cotton. They are then removed from their mating media, Pringshiem
salts, using nitex. The ciliates are washed off the nitex with 15ml of a special Pringsheim
salt solution lacking Ca(NO3)2 into 15ml Corex centrifuge tubes. The Corex tubes are
centrifuged in a Beckman roter at 14.25xg for ~2 min. 1ml of concentrated ciliates are
removed from the bottom of the Corex tubes using a Pasteur pipet, and placed in a 1.7ml
micro-centrifuge tube. The concentrated ciliates are viewed under a microscope to
confirm that they have not prematurely lysed. Next 250µl of 5X Buffer B (50 mM Tris-
HCl, pH 7.5, 50 mM KCl, 5 mM MgCl2, 1 mM DTT and 0.5 mM PMSF) is added to the
lysate. Buffer B was adapted from Sam Gu’s protocol for generating lysates of C. elegans
(Gu, et al. 2007). We added 5µl of RNasin (Promega), an RNase inhibitor, and 139µl of
Vanadyl complex (for a final concentration of 10mM) (Sigma), which inhibits a variety
of RNases, to prevent RNA degradation. The ciliates are then lysed in a Cup-Horn
sonicator for 3, 25-second runs, and successful lysing is confirmed by observation under
a microscope. The solution is then centrifuged at 15339xg for 15 minutes in a 4˚C micro-
centrifuge. The supernatant is put in its own micro-centrifuge tube, while the pellet is re-
suspended in 1X Buffer B. 100µl of each is saved for testing of the lysate, with the rest
either used immediately or stored for future use at -70˚C. Each ciliate lysate was tested

34
for retention of 27macRNAs by extracting RNA, which was subsequently 32
P labeled,
and run on a polyacrylamide-urea denaturing gel, with all processes described below. We
only use the supernatant for FPLC runs as it was found to contain the 27macRNAs.
Extraction of RNA from Ciliate Lysates
200µl of mirVana Lysis/Binding buffer from the mirVana miRNA isolation kit
(Ambion) was added to 100µl of lysate. The protocol for total RNA extraction using the
kit was followed. RNA, eluted in 43µl of deionized distilled water (ddH2O), was stored at
-70˚C to prevent degradation.
Fast Protein Liquid Chromatography (FPLC)
Lysates were spun at 15339xg to remove any precipitated proteins. Lysates were
then loaded onto 0.2 micron Corning Costar SPIN-X filters (Sigma) and centrifuged at
15339xg. The resulting flow-through should contain no particulate matter that would
interfere with the matrix of the columns. The AKTA FPLC system (Amersham
Biosciences) was used in conjunction with the UNICORN software suite. The UNICORN
software suite enables computer controlled column chromatography. Chromatography is
a technique to separate molecules by varying characteristics, such as: size; charge; or
affinity for specific ligands.
Anion Exchange
Lysates were run over a Mono Q 5/50GL anion exchange column that has a
column volume of 0.98ml (Amersham Biosciences). A Mono Q 10/100 GL column, with
a column volume of 7.94ml, was also used. Anion exchange columns contain a matrix of
positively charged beads that cause negatively charged molecules to stick to the column.

35
The salt concentration of the buffer is then slowly increased causing molecules of
increasing negative charge to elute. Eventually the salt is raised to a high enough level to
elute even the most negatively charged molecules. For my FPLC runs using Mono Q
columns, Buffer B without PMSF (50mM KCl) along with a 2M KCl version of Buffer B
without PMSF was used in a linear gradient from 50mM KCl to 1M KCl, before stepping
to 2M KCl to elute everything else. The column is run at a flow rate of 1.5ml per min,
which keeps the pressure below the maximum for the column (4.0 mPa). 1ml fractions
were captured in a 96 well 2ml riplate in a row pattern. Each fraction was stored in a
micro-centrifuge tube at -70˚C. Samples of fractions corresponding to peaks were pooled
together, typically at 200µl total volume, and tested for their RNA and protein
composition as described later.
Size Separation
Lysates and 27macRNA-containing fractions from Mono Q columns were run
over a Superdex 10/300 GL Sizing column (colume volume = ~24ml). Sizing columns
separate molecules via size by containing a matrix of perforated beads. Each bead
contains holes of multiple sizes. Smaller molecules can pass through more of the holes
and so have to travel farther to reach the end of the column. Larger molecules are
excluded from the majority of holes and have a shorter distance to the bottom of the
column so they elute the fastest. Buffer C (20 mM HEPES-NaOH, pH 7.5, 50 mM KCl, 5
mM MgCl2, 1 mM DTT) was used as the liquid phase for the chromatography at a flow
rate of 0.25ml per minute for 1.5 column volumes. 0.5ml fractions were collected in 96
well 2ml riplates in a row pattern. Each fraction was stored in a micro-centrifuge tube at -

36
70˚C. Samples of fractions corresponding to peaks were pooled together, typically at
200µl total volume, and tested for their RNA and protein composition as described later.
Dialyzing Pools
Each FPLC run generated a chromatogram. The UV graph of a chromatogram
show the concentration of protein in a given fraction. Using the UV graph as a guide,
different groups of fractions, called pools, were chosen for analysis. Pools with a high
salt concentration (>0.5M KCl) were dialyzed into the 50mM salt version of their original
buffer (I.E. Buffer B or C, not their 2M or 1M KCl counter parts), to prevent the salt from
interfering with RNA extraction. The high salt pools were loaded onto 0.5ml Ultracell
30K membrane microfuge filter units (Millipore Amicon Ultra) along with the 50mM
KCl version of their respective buffer to a final volume of 0.5ml. The filter units were
centrifuged in a 4˚C microcentrifuge at 15339xg for 6 min or until approximately 50µl
remained in the filter unit. The flow through was discarded and 450µl of the 50mM KCl
buffer was added to the filter. The filter units were centrifuged again at 15339xg for 5
minutes. Then the filter units were placed upside down in a fresh collection tube and
centrifuged at 78xg for 2 minutes to capture the now dialyzed sample. 50µl of the 50mM
KCl buffer was then added to aid in recovery of the RNA.
RNA extraction of Pools
RNA was extracted from each pool by adding a 1:1 ratio of Acid-Phenol
Chloroform (Ambion). Each pool was then vortexed for ~20 seconds before being
centrifuged at 15339xg on a desktop micro-centrifuge for 5 minutes. The top aqueous
layer was removed and put in a fresh micro-centrifuge tube. Chloroform (IBI scientific)
was added to each sample at a 1:1 ratio. Each sample was vortexed for ~30 seconds and

37
then centrifuged on a desktop microcentrifuge for 2 minutes at 15339xg. The top aqueous
layer was removed and ethanol precipitated.
Ethanol Precipitation
Each 100µl RNA sample had 400µl of 100% ethanol, 10µl of 3M Na-acetate, pH
5.3, and 2µl of Glyco-blue (Ambion) added to it. Larger samples contained the same
ingredients at the same ratios. The RNA was then left to precipitate overnight in a -70˚C
freezer. RNA was recovered by centrifugation at 15339xg for 25 minutes, which pelleted
the RNA out of solution. Ethanol was removed via aspiration and the pellet was then
washed in ice cold 70% ethanol and re-spun at 15339xg for 5 minutes. Ethanol was again
removed and the pellet was dried in a lyophilizer for 15 min at 23˚C.
Phosphatase Treatment of RNA
In order to add a radioactive phosphate to RNA, the 5` phosphate must first be
removed. We used Calf Intestinal alkaline Phosphatase (CIP) from NEB for this purpose.
Dried pellets (as described above) were re-suspended in 43µl of cold ddH2O. 5µl of 10x
Buffer 3 (NEB) and 2µl of 10 U/1µl CIP (NEB) were added to each sample. The samples
were then incubated at 37˚C. After one hour, 50µl of ddH2O was added to each sample to
aid in recovery of the RNA. The resulting 100µl samples were Acid-Phenol Chloroform
and then chloroform extracted as described above (RNA extraction of Pools). Then the
samples were ethanol precipitated overnight in preparation for labeling with 32
P.
32
P Labeling of CIP-treated RNA
Each dried pellet of CIP-treated RNA was re-suspended in 7µl of ddH2O. Next
1µl of 10X T4 PNK Buffer (NEB) and 1µl 10 U/µl T4 PNK (NEB) was added to each

38
sample. Finally 1µl of gamma 32
P labeled ATP (PerkinElmer) at a concentration of
10mCi/ml (6000 Ci/mmol) was added. Each sample was then incubated for 1 hour at
37˚C. Then 10µl of Formamide loading dye (49ml formamide, 1ml 0.5M EDTA, 0.013g
Bromophenol blue, 0.013g Xylene cyanol) was added to each sample and boiled at 95˚C
for 2 minutes to denature the RNA. Samples were then either loaded immediately on a
gel or stored in a rad-safe box at -20˚C.
Gel running of 32
P labeled RNA
32
P labeled RNA was loaded on a 15% polyacrylamide-urea denaturing gel in 1X
TBE (0.09M Tris-base, 0.09M Boric acid, 0.01M EDTA). Each gel was pre-run for 1 and
a ½ hours at 2000V/40W prior to loading. RNA that had been stored overnight in the -
20˚C freezer was first boiled at 95˚C for 2 minutes. Then 5µl of each sample was loaded
per lane. 10µl of an RNA ladder, made using the Decade Marker kit from Ambion (p/n
AM7778) was also loaded. The gel was run for ~2 ½ hours at 2000V/40W. The resulting
gel was exposed on a phosphor imager plate and imaged using a Typhoon
PhosphorImager (GE Health Care) and ImageQuant software.
Immuno-blotting
Proteins of fractions and lysates were denatured in Sample Buffer (2x version:
0.125mM Tris-Cl, pH 6.8; 20% (w/v) Glycerol; 4% (w/v) SDS; 10% (w/v) 2-
mercaptoethanol; and 0.05% (w/v) bromophenol blue) at a 1:1 ratio and run on a 10%
polyacrylamide SDS-PAGE mini-gel with Tris-Glycine SDS Running Buffer (0.38M
Glycine, 0.05M Tris, 0.2% (w/v) SDS) for ~1 hour at 100V (Laemmli 1970). Each gel
also contained a 1 cm 7% stacking region (This type of gel will henceforth be called a
10/7 SDS-PAGE Gel). The gel was then trimmed and the proteins were transferred to a

39
polyvinylidene difluoride (PVDF) membrane. To activate the PVDF membrane it was
incubated in 20% methanol for 4 minutes and then Transfer Buffer (0.19M Glycine,
0.025M Tris-Base, 10% methanol) for 5 minutes. Transfer took place in a Trans-Blot cell
(Biorad) at 70 volts, 1241mAmps for 1 hour at 4˚C. After the proteins were transferred to
the PVDF membrane, it was placed on an orbital shaker in a 1:10 dilution of
TBS:Western Blocking solution (Roche Diagnostics) for 1 hour (TBS is 0.05M Tris-Cl,
pH 8 and 0.15M NaCl). The membrane was then placed into a 50ml conical tube, into
which was added 4ml of TBS-T, 0.2ml of the diluted blocking solution, and 4µl of rabbit
anti-PIWIL1 antibody (Abcam, ab12337) (TBS-T is TBS as described previously plus
0.5ml of Tween-20 per 1 L). The tube was then placed on a tube rotator in a 4˚C
refrigerator room overnight. The next day the membrane was removed from the tube and
washed for 5 minutes, 4-6 times, in TBS-T on an orbital shaker. Next the membrane was
washed in a 2˚ solution containing 30ml of TBS-T and 6µl of HRP-conjugated goat anti-
rabbit antibody (BioRad) for 1 hour. The membrane was then washed again for 5
minutes, 4-6 times, in TBS-T, before being incubated in SuperSignal West Pico
Chemiluminescence solution (Thermo Scientific) for 5 minutes. The chemiluminescence
was then visualized using X-ray film and a film processor.
Silver stain
In order to see the protein complexity of my fractions, I ran proteins, denatured using
sample buffer, on a 10/7 SDS-PAGE gel. Silver staining enables detection of all proteins
in a sample even at extremely low concentrations. The SilverSNAP stain Kit II from
Thermo Scientific was used as follows. After running the gel at 100V/40w for
approximately one hour in SDS Running buffer, the gel was removed from the running

40
apparatus and placed in a tray on an orbital shaker. The gel was then washed for 5
minutes, twice, with ddH2O. Next the gel was washed for 15 minutes, twice, with 25ml of
Fixing Solution (6:3:1, ddH2O:EtOH:Acetic Acid). After removing the gel from Fixing
solution, it was washed for 5 minutes, twice, in 25 ml of 20% EtOH. Next the gel was
washed for 5 minutes, twice, with ddH2O before being washed in 50ml of Sensitizer
Working Solution (50µl of Sensitizer in 50ml of ddH2O) for exactly 1 minute. The gel
was then washed for exactly 1 minute, twice, with ddH2O before being placed into 50ml
of Staining solution (0.5ml Enhancer in 50ml of Stain). After approximately 30 minutes
the gel was removed from Stain solution and washed for 20 seconds, twice, with ddH2O.
Next the gel was placed in Developer solution (0.5ml Enhancer in 50ml of Developer)
and left to wash until bands reached desired intensity (typically around 1 ½ minutes).
Staining was then stopped using two washes of 5% acetic acid solution and the resulting
gel was photographed.

41
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