1. INTRODUCTION
APOBEC1-mediated editing of amyloidogenic
RNA transcripts in microglia
MATERIALS & METHODS
RESULTS
DISCUSSION
ABSTRACT
Chittampalli Yashaswini (The Bronx High School of Science)
Dr. Nina Papavasiliou & Dr. Theodoros Sklaviadis (Rockefeller University)
ACKNOWLEDGEMENTS
RNA editing is a very important process that contributes to
adaptations and organismic complexity. This study focuses on
the RNA editing enzyme APOBEC1.
• APOBEC1 converts cytidine to uridine in AU-rich areas of the
3’ untranslated region (UTR).
• The 3’ UTR determines mRNA stability which can affect the
amount of that protein produced from the translated mRNA.
• Others in the lab have shown that APOBEC1 edits the 3’ UTR
in macrophages.
Microglia are immune cells of the brain derived from
macrophages.
• They trim synapses, which is important for learning.
• They play a role in Alzheimer’s disease (AD) though this role
is still quite ambiguous.
• Positive: engulf amyloid plaques
• Negative: cause neuroinflammation
The RNA transcripts studied here all have to do with amyloids.
• B2M can adopt the configuration of amyloids, and is most
likely a component of amyloids.
• The proteolysis of APP can lead to amyloid formation
• ADAM10 is the alpha secretase (cleaver) of APP.
Figure 1: General map of a gene.
Figure 2: Microglia are glial cells that
protect the brain from pathogens.
Figure 4: Amyloid plaques create barriers between
neurons, blocking communication.
WT
KO
Isolate microglia
Isolate microglia
Grow BV2 cells
Isolate RNA
Isolate RNA
Isolate RNA Make cDNA
APOBEC1
ADAM10
B2M
APP
Make cDNA
APP
Gel ElectrophoresisAmplify DNA/ cDNA
Isolate DNA
Transform into E. coli
using pSC vector
Amp
EcoR1 EcoR1Insert
pSC-B-amp/kan
4.3 kb
Kan
Clone
Digest
Insert
Gel Electrophoresis
Purify plasmid
Sequence
and Align
RESULTS
Microglia, the immune cells of the brain, play a crucial, though
controversial role in the central nervous system. Microglia trim
synapses, a process important for learning. However, defective
microglia can cause neurological disorders, such as Alzheimer’s
disease (AD). This study focuses on three genes of microglia: beta
2-microglobulin (B2M), amyloid precursor protein (APP), and a
disintegrin and metalloproteinase domain-containing protein 10
(ADAM10) . These genes are involved in the creation of amyloid
plaques—the hallmark AD. It was hypothesized that
Apolipoprotein B-editing enzyme, catalytic polypeptide-1
(APOBEC1)—a cytidine deaminase—edits the 3' UTRs of these
transcripts of microglia. By means of RNA and DNA isolation,
cDNA synthesis, PCR amplification, bacterial transformation,
DNA purification, gel electrophoresis and sequence analysis using
the MacVector program, we have concluded that APOBEC1 is
responsible for editing of APP in murine microglia from brain
tissue, and that cytidine-to-uridine (C to U) RNA editing occurs in
the 3’ UTRs of B2M, APP and ADAM10 in an immortalized,
murine, microglial cell line (BV2). It is likely that APOBEC1 is
responsible for the editing present in the BV2 cell line, because
the sites edited correspond with the editing preferences of
APOBEC1.
I would like to thank my mentors Dr. Nina Papavasiliou and Dr.
Theodoros Sklaviadis for their guidance in this research project,
Violeta Rayon for providing macrophage sequences, Ted Scovell
whose passion for science is insipiring, my father Dr. C. S.
Narayanan who helped me achieve a better understanding of the
biological concepts involved, and my research project advisor Dr.
Ramon Bonfil for all his help over the years.
Conclusions:
• In the murine microglia from brain tissue, the RNA from the
APOBEC1 KO mice has no editing at all; however, the RNA
from the WT mice does contain edits. This suggests that
APOBEC1 is required for C to U RNA editing to occur in
APP in murine microglia.
• In the BV2s, the DNA sequences were void of any edits, while
the RNA sequences contained several C to U RNA edits. This
suggests that RNA editing occurs in the 3’ UTRs of
ADAM10, B2M and APP in microglial cells.
• APOBEC1 is most likely responsible for this editing:
• It is present in BV2 cells
• It edits ADAM10, B2M and APP in macrophages
• All edits occur in 3’ UTR
• All observed edits are C to U
• All edited sites are flanked by A or U on both sides
Possible future experiments based on findings:
• Will editing still occur if APOBEC1 is knocked down in a BV2
cell line?
• Is there RNA editing of ADAM10 and B2M in murine
microglia as well? Would this editing be absent in the
APOBEC1 knockout microglia?
• What is the function of the edits found?
• Will there be RNA editing in a neuronal cell line?
MATERIALS & METHODS
B2M in BV2s undergoes RNA editing
Figure 5 & Table 1: The 1st sequence is genomic DNA of
B2M, which has no edits. B2M is edited at 10 sites. Site
516 is edited in 8.3% of BV2 clones and 50% of
macrophage clones. Site 580 is edited in 8.3% of BV2
clones and 18.8% of macrophage clones. Site 678 is
edited in 12.5% of clones and 43.8% of macrophage
clones. These 3 sites were considered to be significantly
edited because of the relatively high amount of BV2 and
macrophage clones that were edited.
Figure 5:
Table 1:
• The 3’ UTRs of B2M, APP and ADAM10 from BV2 cells all
have C to U RNA editing. Some of these edited sites are also
edited in a relatively high percentage of macrophages, which
we recognized as significantly edited.
• In the murine microglia from brain tissue, C to U editing only
occurs in the WT mouse; it does not occur in the APOBEC1
KO mouse.
Alzheimer’s is characterized
by amyloids (clumps of
misfolded proteins).
• Amyloids accumulate in
the hippocampus and
cortex.
• Amyloids create barriers
between neurons.
Figure 3: Microglia respond to factors that
pose harm to the Central Nervous System.
Table 2:
Figure 6:
APP in BV2s undergoes RNA editing
Figure 6 & Table 2: The 1st sequence is genomic DNA
of APP, which has no edits. APP is edited in 16
positions (only 10 are displayed in the accompanying
table). Six positions were considered significant. 2767 is
edited in 8.3% of BV2s and 16.7% of macrophages.
2894 is edited in 4.2% of BV2s and 16.7% of
macrophages. 2925 is edited in 8.3% of BV2s and 25%
of macrophages. 2952 is edited in 4.2% of BV2s and
20.8% of macrophages. 3014 is edited in 8.3% of BV2s
and 20.8% of macrophages. 3280 is edited in 8.3% of
BV2s and 37.5% of macrophages.
• Mouse BV2 cells were used because of their microglial properties.
• Isolate RNA from BV2 cells and from wild type (WT) and
APOBEC1 knockout (KO) murine microglia.
• Reverse transcription to make cDNA.
• PCR amplification of 3’ UTRs of APOBEC1, APP, ADAM10,
B2M.
• Gel electrophoresis to ensure cDNA amplification.
• Transformation followed by cloning.
• Digestion and gel electrophoresis to ensure that insert is in plasmid.
• Purify and sequence cDNA.
• Align sequences with MacVector to macrophage sequences.
• Look for C to U edits (evident as C to T mutations).
• Repeat to get genomic DNA of each gene.
BV2Macrophage
ADAM10 in BV2s undergoes RNA editing
Table 3:
Figure 7 & Table 3:The 1st sequence is genomic DNA of
ADAM10, which has no edits. ADAM10 is edited in 3 positions.
Each of the first 2 positions (2778 and 2936) are edited in 4.2%
of BV2 clones. The 3rd position (3076) is edited by 4.2% of BV2
clones, and by 12.5% of macrophage clones.
Figure 7:
Macrophage
BV2
BV2Macrophage
Table 2:
Figure 8 & Table 4: The 1st sequence is the cDNA sequence of APP from the microglia of APOBEC1 KO
mouse, which has no edits. 2968 was edited in 8.3% of WT clones and 4.2% of macrophage clones. 3265
was edited 4.2% of WT clones.
WT
KO
APOBEC1 is responsible for RNA editing of APP in
murine microglia from brain tissue
Figure 8: Table 4: