Table Of ContentClassical Conditioning Alters Short Noncoding RNA
Expression in Drosophila
Citation
Maniatis, Silas dana. 2015. Classical Conditioning Alters Short Noncoding RNA Expression in
Drosophila. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
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http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467392
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Classical
Conditioning
Alters
Short
Noncoding
RNA
Expression
In
Drosophila
A
dissertation
presented
by
Silas
Dana
Maniatis
to
The
Department
of
Molecular
and
Cellular
Biology
in
partial
fulfillment
of
the
requirements
for
the
degree
of
Doctor
of
Philosophy
in
the
subject
of
Biochemistry
Harvard
University
Cambridge,
Massachusetts
April
2015
©
2015
–
Silas
Dana
Maniatis
All
rights
reserved
Professor
Sam
Kunes
Silas
Dana
Maniatis
Classical
Conditioning
Alters
Short
Noncoding
RNA
Expression
In
Drosophila
Abstract
MicroRNAs
(miRNAs)
and
other
classes
of
short
non-‐coding
RNAs
regulate
essential
processes
in
the
development
and
function
of
the
nervous
system.
Regulation
of
miRNAs
by
neural
activity
has
also
been
reported.
Recently,
instances
of
piwi
interacting
RNA
(piRNA)
and
endogenous
short
interfering
RNA
(esiRNA)
mediated
modulation
of
neural
physiology
have
been
reported.
To
better
understand
the
role
of
miRNAs
and
other
classes
of
short
non-‐coding
RNAs
in
long
term
memory
(LTM)
formation,
we
have
conducted
high
throughput
sequencing
on
15-‐35nt
RNAs
isolated
from
heads
of
Drosophila
that
have
been
subjected
to
aversive
olfactory
conditioning.
We
developed
genome
wide
profiles
of
miRNA,
piRNA,
and
esiRNA,
and
tested
for
differential
expression
following
conditioning.
We
find
that
5
miRNAs
exhibit
significant
regulation
in
the
conditioned
group.
We
identify
several
esiRNA
generating
loci
within
genes
required
for
olfactory
LTM
formation.
Our
data
reveal
that
an
intron
of
the
multiple
wing
hairs
(mwh)
gene
forms
secondary
structures
and
generates
esiRNAs
following
conditioning
from
regions
that
correspond
to
lysozyme
family
genes
located
within
the
mwh
intron.
We
find
that
piRNAs
are
produced
in
fly
heads,
and
that
a
small
set
of
piRNA
generating
loci
mapping
to
LTR
retrotransposons
are
significantly
down
regulated
following
conditioning.
In
addition
to
the
well
characterized
classes
of
short
non
coding
RNAs,
we
describe
a
set
of
transcripts
that
produce
large
numbers
of
reads
with
a
broad
size
distribution
from
the
sense
strand.
We
find
that
a
subset
of
these
are
regulated
following
treatment
and
contain
consensus
elements
that
may
be
involved
in
their
regulation.
We
investigate
expression
of
one
such
gene
with
dramatically
up-‐regulated
reads
following
treatment,
the
Drosophila
beta-‐site
APP-‐cleaving
enzyme
(dBACE),
and
find
that
increased
reads
reflect
increased
mRNA
levels.
Further,
we
find
that
the
target
of
dBACE
protein,
drosophila
β
amyloid
protein
precursor-‐like
(APPL),
is
subjected
to
increased
cleavage
following
conditioning,
and
that
dBACE
is
required
for
LTM
formation,
but
not
for
learning
or
STM.
iii
Table
of
contents
Abstract…………………………………………………………………………………………………..………….iii
Acknowledgements………………………………………………………………………………………..…..vii
Chapter
I:
Introduction…………………………………………………………………………………………1
Part
I:
Synaptic
Plasticity
In
Learning
And
Memory,
And
Olfactory
Memory
In
Drosophila……………………………………………………………………..………………......……..4
Synaptic
plasticity
is
the
physiological
basis
of
learning
and
memory……………………………………………………………………………...……..……4
Many
Molecular
and
Genetic
Tools
Are
Available
For
Studies
Of
Drosophila
Learning
And
Memory…………………………………………………10
Paradigms
For
Behavioral
Studies
In
Drosophila…………………………….12
Olfactory
Learning
and
Memory
in
Drosophila………………………………..16
Genetic
Analysis
of
Drosophila
Olfactory
Memory……………...…………...18
The
Neural
Circuitry
of
Drosophila
Olfactory
Memory…...........................22
Part
II:
Short
Noncoding
RNAs
In
Drosophila
Memory
Formation......................27
A
Variety
Of
Short,
Non-‐Protein
Coding
RNAs
Regulate
Gene
Expression
In
Animals..............................................................................................27
Biogenesis
And
Function
Of
siRNAs...................................................................30
Biogenesis
And
Function
Of
miRNAs.................................................................36
Biogenesis
And
Function
Of
piRNAs...................................................................44
miRNAs
In
Neurophysiology
And
Behavior....................................................50
esiRNAs,
piRNAs,
And
Novel
sRNAs
In
Neurophisiology..........................60
Part
III:
The
Beta
Secretase
Beta-‐Site
APP-‐Cleaving
Enzyme
(BACE)
In
Memory
Formation
and
Cognitive
Impairment...........................................................65
Proteolytic
Processing
Of
Amyloid
Precursor
Protein
(APP)
Family
Proteins
Is
Involved
In
Alzheimer’s
Disease
Pathology............................65
APPL
Is
The
Drosophila
Homologue
Of
APP,
And
Its
Processing
And
Functions
Are
Conserved.......................................................................................69
Proteolytic
APPL
Processing.................................................................................71
APPL
And
Its
Metabolites
Are
Involved
In
Neurodevelopment,
And
Regulate
Synaptic
Structure..................................................................................73
APPL
Processing
Regulates
Neuronal
Activity..............................................76
Processing
Of
APPL
And
Its
Homologues
Is
Regulated
By
Neuronal
Activity...........................................................................................................................78
Tight
Control
Of
APPL
Expression
And
Processing
Is
Required
For
Drosophila
LTM...........................................................................................................81
Literature
Cited.........................................................................................................................84
iv
Chapter
II:
Drosophila
Olfactory
Long
Term
Memory
Formation
Alters
Short
Non-‐
Protein
Coding
RNA
Profiles............................................................................................................103
Summary....................................................................................................................................104
Introduction..............................................................................................................................106
Results.........................................................................................................................................112
Section
I:
Analysis
of
microRNA
expression
in
the
Drosophila
head
during
long-‐term
memory
formation.............................................................112
microRNA
expression
in
the
Drosophila
head
during
LTM
formation......................................................................................................112
Target
analysis
for
microRNAs
regulated
during
LTM
formation......................................................................................................118
Gene
ontology
analysis
of
targeted
genes......................................121
Expression
of
non-‐canonical
microRNA
sequences
in
the
Drosophila
head
during
LTM
formation..........................................124
Offset
isomiR
analysis
for
individual
pre-‐microRNAs...............128
Analysis
of
untemplated
nucleotide
tailing...................................130
Analysis
of
microRNA
editing..............................................................132
Section
II:
Analysis
of
esiRNA
and
piRNA
expression
in
the
Drosophila
head
during
long-‐term
memory
formation..................................................135
Identification
of
esiRNA
producing
loci...........................................136
esiRNA
expression
profile
in
the
Drosophila
head......................137
Changes
in
esiRNA
expression
during
LTM
formation.............143
A
profile
of
piRNA
expression
in
the
Drosophila
head..............149
Changes
in
piRNA
expression
during
LTM
formation...............153
Discussion...................................................................................................................154
Literature
Cited.........................................................................................................177
Chapter
III:
Beta-‐Site
APP-‐Cleaving
Enzyme
Is
Required
For
Long
Term
Memory
In
Drosophila.................................................................................................................................................184
Summary....................................................................................................................................185
Introduction..............................................................................................................................186
Results.........................................................................................................................................191
sRNAs
are
produced
from
highly
expressed
transcripts........................191
All
three
experimental
treatments
induce
significant
changes
in
HECT
read
counts.................................................................................................................195
Proteases
are
enriched
in
the
set
of
HECT
genes
with
increased
read
counts
in
the
LTM
condition...............................................................................196
Intronless
genes
are
overrepresented
in
the
set
of
HECT
genes
with
significantly
increased
reads
in
the
LTM
condition..................................197
Transcripts
harboring
a
consensus
sequence
that
facilitates
nuclear
export
and
expression
of
intronless
mRNAs
are
overrepresented
amongst
regulated
HECT
genes.........................................................................200
v
dBACE
mRNA
is
upregulated
by
LTM
training,
and
by
spaced
sessions
of
the
US
alone...........................................................................................................203
dBACE
expression
is
rapidly
upregulated
following
LTM
training
and
remains
elevated
24
hours
post-‐training......................................................207
APPL
processing
is
stimulated
by
LTM
training
and
spaced
sessions
of
US
exposure................................................................................................................209
APPL
and
dBACE
are
required
for
aversive
and
appetitive
LTM........211
Knockdown
of
APPL
or
dBACE
in
the
adult
MB
disrupts
LTM.............214
APPL
and
dBACE
are
not
required
for
STM..................................................216
Discussion..................................................................................................................................218
Materials
and
Methods.........................................................................................................230
Literature
Cited.......................................................................................................................235
Summary
and
Conclusion..................................................................................................................240
sRNA
profiles
are
altered
by
classical
conditioning................................................241
Identification
of
HECT
sRNAs............................................................................................250
dBACE
is
upregulated
during
LTM
formation............................................................251
Concluding
Remarks.............................................................................................................253
Literature
Cited.......................................................................................................................255
Appendix...................................................................................................................................................260
Supplementary
figures.........................................................................................................261
Literature
cited........................................................................................................................320
vi
Acknowledgements
The
writing
of
this
dissertation
represents
the
final
act
in
a
significant
phase
of
my
intellectual
and
personal
life.
Though
its
completion
obviously
brings
me
great
satisfaction,
I
feel
I
will
not
be
able
fully
embrace
and
enjoy
this
milestone
without
first
acknowledging
the
critical
support
and
important
contributions
of
those
around
me.
I
take
the
first
step
toward
an
adequate
expression
of
my
gratitude
here,
but
I
do
so
knowing
that
these
words
of
thanks
cannot
really
suffice.
I
wish
to
thank
Sam
Kunes
for
his
guidance
and
support
during
my
years
in
his
lab.
He
provided
me
with
the
immense
latitude
I
needed
in
developing
the
work
described
in
this
dissertation.
He
continued
to
support
me
when
other
advisors
might
not
have,
and
showed
enormous
patience
and
faith
in
my
work.
His
daily
presence
in
the
fly
room
and
at
the
bench
made
for
a
unique
lab
environment,
and
fostered
exchanges
that
are
unlikely
to
have
occurred
in
an
office
setting.
I
am
unaware
of
many
other
examples
in
which
a
senior
faculty
member
actually
performed
experiments
themselves
that
are
incorporated
in
their
students’
dissertations.
His
many
hours
of
labor
in
setting
up
and
conducting
the
olfactory
classical
conditioning
experiments
included
in
this
dissertation
were
absolutely
vital
to
my
work.
I
also
need
to
thank
the
members
of
my
advisory
committee,
Craig
Hunter,
Venkatesh
Murthy,
and
Joshua
Sanes.
They
too
showed
enormous
patience
and
faith
in
my
work,
and
without
their
interventions
and
encouragement
at
key
points,
I
would
not
have
been
able
to
complete
my
graduate
studies.
vii
My
friends
and
colleagues
from
the
de
Bivort,
Francis,
Kunes,
Lichtman,
and
Maniatis
labs,
as
well
as
those
from
the
“Secret”
journal
club
have
my
gratitude
for
the
many
forms
of
advise
and
help
they
have
given
me.
I
thank
them
for
keeping
science
fun
and
my
interests
broad.
During
the
course
of
their
graduate
work,
many
doctoral
students
receive
significant
support
from
their
families.
I
realize
that
I
cannot
really
compare
the
value
of
my
version
of
such
a
thing
to
that
of
anyone
else’s,
but
were
there
an
objective
scale,
I
am
confident
that
mine
would
be
at
the
top
end.
First,
and
foremost,
my
wife
has
stood
behind
me
at
every
turn,
and
in
every
manner
possible.
A
catalog
of
the
ways
in
which
her
support
has
been
essential
to
me
as
a
person,
and
to
the
completion
of
this
work
would
be
far
to
long
to
print
here.
Also,
the
aggregate
effect
of
the
various
forms
of
her
support
has
been
far
greater
than
their
sum.
Again,
words
will
fail,
but
in
essence,
she
has
provided
me
with
loving
encouragement,
shepherded
me
through
difficulties,
made
sure
I
have
celebrated
my
successes,
and
helped
those
around
us
who
have
not
been
down
the
doctoral
path
to
understand
my
struggles.
She
has
been
my
cut
man,
my
advocate,
and
my
defender,
and
for
all
of
this
I
am
grateful
in
a
way
that
cannot
be
concisely
distilled,
and
so
I
will
not
try
to
do
so
here.
Most
critically
though,
while
being
the
most
loving
and
supportive
spouse
one
could
imagine,
she
has
done
so
while
simultaneously
pushing
me
to
keep
progressing.
This
has
undoubtedly
been
a
herculean
task,
but
she
has
managed
to
attack
it
with
positivity
and
love.
The
patience
and
endurance
of
her
loving
support
has
kept
me
going
through
times
when
nothing
else
would
have.
Her
remarkable
strength
has
allowed
her
to
do
all
of
viii
this
while
simultaneously
pursuing
her
own
career
in
architecture.
She
has
achieved
more
than
can
be
listed
here,
but
her
work
in
many
ways
reflects
the
person
she
is.
Her
decision
to
change
the
direction
and
pursue
architecture
in
itself
displays
her
bravery.
Her
elegance
finds
expression
in
many
of
her
projects,
her
drive
manifests
in
the
volume
and
level
of
her
work,
and
her
toughness
was
in
evidence
when
she
learned
to
weld
and
to
operate
heavy
machinery.
Her
accomplishments
and
motivation
are
my
inspiration.
I
will
spend
the
rest
of
my
life
making
sure
she
understands
how
much
I
value
all
that
she
has
done
for
me,
how
much
I
respect
her,
and
this
brief
section
of
my
dissertation
cannot
contain
the
years
of
gratitude
I
owe
her.
I
also
have
the
great
fortune
of
being
born
into
a
family
for
whom
the
philosophy
of
science
is
a
guiding
force.
I
am
the
son
of
a
man
once
described
to
me
by
an
accomplished
professor
of
biology
as
“A
scientist’s
scientist
in
the
same
way
that
Ted
Williams
was
a
baseball
player’s
baseball
player.”
At
the
time,
this
sounded
great,
but
I
had
not
yet
begun
my
graduate
career,
and
was
thus
not
well
enough
read
in
biology
to
appreciate
the
remark.
Reflecting
back
upon
the
comment
now,
I
realize
how
apt
it
was.
Much
as
Williams’
book
“The
Science
of
Hitting”
remains
a
foundational
text
for
developing
baseball
players,
“Molecular
Cloning”
has
allowed
generations
of
scientists
working
in
diverse
areas
of
biology
to
bring
the
power
of
molecular
biology
and
biochemistry
to
their
work,
and
I
have
had
the
pleasure
of
putting
this
text
to
use
in
my
own
work.
My
father’s
course
on
gene
regulation
was
required
for
all
students
in
my
program,
and
it
was
one
of
the
most
difficult
I
have
taken.
By
relying
solely
on
current
publications
for
course
materials,
he
and
his
co-‐
ix
Description:In addition to the well characterized classes of short non coding RNAs, we .. might not have, and showed enormous patience and faith in my work.
