Classical Conditioning Alters Short Noncoding RNA Expression in Drosophila The Harvard ...

332 Pages·2015·10.78 MB·English

by  Silas Maniatis

Classical 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.
Permanent link
http://nrs.harvard.edu/urn-3:HUL.InstRepos:17467392
Terms of Use
This article was downloaded from Harvard University’s DASH repository, and is made available
under the terms and conditions applicable to Other Posted Material, as set forth at http://
nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Share Your Story
The Harvard community has made this article openly available.
Please share how this access benefits you. Submit a story .
Accessibility
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