Supplementary notes on Alu-transcript numbers, location, subfamilies and function
Supplementary Document 1:
The transcript repeat element: the human Alu sequence
as a component of gene networks influencing disease
Paula Moolhuijzen1*, Jerzy K Kulski1, 2, 3 *, David S Dunn1, David Schibeci1, Roberto
Barrero1, Takashi Gojobori4, Matthew Bellgard1
Plant and animal small RNA target genes
Small RNAs have been found in all plant and animal multicellular organisms investigated
so far. Plant and animal miRNAs are evolutionarily ancient small RNAs, 19–24
nucleotides in length, generated by cleavage from larger highly structured precursor
molecules. In both plants and animals, miRNAs regulate gene expression post
transcriptionally through interactions with their target mRNAs. These miRNAs, excised
from endogenously encoded hairpin RNAs, negatively regulate endogenous target genes
by cleavage or translational inhibition of their mRNA (Millar and Waterhouse 2005).
To date, plant miRNAs share much higher complementarities to their target genes (zero
to three mismatches) than animal miRNAs to their target genes, although in both cases,
high to perfect complementarities between the target mRNA and the 5′-half of the
miRNA is required (Doench and Sharp 2004, Parizotto et al. 2004).
The current knowledge on animal miRNA-binding sites is limited and biased because
their discovery was primarily based upon computer predictions using databases
composed of only 3′- UTR sequences (Enright et al. 2003, Lewis et al. 2003, Xie et al.
1
Supplementary notes on Alu-transcript numbers, location, subfamilies and function
2003). In comparison, plant miRNA-binding sites are found almost exclusively within the
open-reading frames of the target genes (Millar and Waterhouse 2005, Xie et al. 2003).
Supplementary notes on Alu-cDNA numbers, location, subfamilies and function
Alu-cDNA Functions
The functional categories of the Alu-cDNA collected from the H-Inv human cDNA
database were analysed by Gene Ontology (GO), which classifies the known functions of
gene products into at least 5,175 categories according to biological processes, cellular
components and molecular functions (Ashburner et al. 2000). . GO analysis was
performed for cDNA with a NCBI gene identifier and CDS to determine the different
metabolic and regulatory pathways that are possibly associated with the cDNA-Alu in
normal and cancerous tissues. Over 8,000 cDNA-Alu from the H-Inv database were
classified into nearly 3000 of the GO functional categories, and then assigned into 35 GO
Slim categories for the Homo sapiens taxa identifier 9636
(http://www.geneontology.org/GO.downloads.shtml#ont). Over 70% of the cDNA-Alu
loci grouped into binding and catalytic activity molecular functions (Supplementary
Figure 3), and more than 80% were involved in biological processes, such as
physiological process, nucleotide metabolism, cell communication and transport. Less
than 3% of the cDNA and cDNA-Alu loci were designated “molecular function
unknown” or “biological process unknown”. The molecular function, biological process
and cellular component of loci unique to tissue traits for the full cDNA and cDNA-Alu
subsets showed no cDNA-Alu affect on the GO slim category proportions. The
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Supplementary notes on Alu-transcript numbers, location, subfamilies and function
proportion of loci unique to tissue traits for a few molecular functions did however vary
between unique normal and disease traits for all cDNA and cDNA-Alu. The molecular
functions of both cDNA and cDNA-Alu were associated mainly with binding (40%) and
catalytic activity (20%). The number of loci products involved in the function of nucleic
acid binding increased nearly two-fold for the disease loci, whereas loci associations with
signal transducer activity and transporter activity decreased in the cancerous trait three
and two fold, respectively. These shifts in function were significant by a Fisher’s exact
test at P<0.0001 (Supplementary Figure 3).
In the GO analysis of approximately half of the cDNA-Alu loci, there was no difference
between cDNA and cDNA-Alu for the biological and cellular processes or molecular
function. However, theproportion of categories for function, process and cellular
components of the expressed loci changed when the normal and cancerous trait of the
tissues were compared., with the number of cDNA and cDNA-Alu subsets increased for
nucleic acid binding and decreased for signal transducer and transporters activity in the
cancerous trait. In general, the majority of the cDNA isoforms containing full-length Alu
in the coding regions of the transcripts translated into hypothetical proteins rather than
known proteins. As the function of these cDNA-Alus is not known or is labelled
‘hypothetical’, it is proposed that they are either untranslated or destined for rapid
degradation or they serve a binding role against excessive retrotranspositional activity
within the genome.
3
Supplementary notes on Alu-transcript numbers, location, subfamilies and function
Alu Families and Subfamilies
When cDNA-Alu transcripts Alu elements were categorized into their particular families
and subfamilies, the contribution of the Alu families in cDNA was similar to the
proportion of Alu families within the genome; AluS 54%, AluJ 26%, AluY 10%,
Monomeric 8% and Alu 2% (Price et al. 2004) (Supplementary Figure 2B). The most
frequent Alu subtype was AluSx. No family bias was detected between the cancerous and
normal tissue sets.
Alu Locations Within the cDNA
The relative position and size of Alu insertions within the cDNA sequences was
determined for 17,819 of 17,861 distinct cDNA sequences with an annotated CDS that
contained at least one Alu. The Alus positions were partitioned into three groups
according to the regions in which they were found (1) overlapping the coding sequence
(CDS), (2) exclusively in the 5’ UTR, or (3) exclusively in the 3’ UTR. Supplementary
Figure 1 shows the number of fragments and distinct transcripts in normal or cancerous
tissues, and the transcripts with known functions are indicated in red.
The transcript Alu content was measured based on the proportion of transcripts
containing an Alu fragment represented within the CDS, 5’UTR or 3’UTR or represented
by sequence length (base pairs) to the total sum of sequence represented in these three
regions.
Alu sequence quantification within the cDNA
Greater than 28,000 Alu fragmented or full-length elements were found in the 17,000
cDNA-Alu sequences surveyed, at an average of 1.6 Alu per sequence. The Alu elements
4
Supplementary notes on Alu-transcript numbers, location, subfamilies and function
ranged in size from10 nucleotides in length to full-sized. All these Alu fragments were
within the RepeatMasker recommended RM score to be outside the threshold for a false
positive match (http://www.repeatmasker.org/). Supplementary Figure 2A shows the
number of Alu fragments plotted as a % of the consensus Alu sequence length for Alu
subfamilies, and for transcripts represented in cancerous and normal tissues, with known
and unknown (hypothetical) functions. The plots show that the number of cDNA-Alu in
normal tissues is clearly higher than in cancerous tissues. In the cDNA-Alu plot, minor
peaks represent a slightly increased number of the monomeric form of the Alu structure
(110-130 bp) and major peaks above 90% of the Alu consensus full length sequence
represent a greatly increased number of the dimeric form of the Alu structure (>250 bp).
This trend of minor and major peaks was similar for the cDNA-Alu in both the normal
and diseased tissues.
While multiple fragments of Alu elements can overlap the CDS within a single transcript,
the largest number of Alu contained in the CDS was 4 copies in one particular transcript
sequence. Full-length dimeric Alu (>280 bp) fragments overlapping the CDS that
represent half of the transcripts potentially have cryptic splicing sites provided by the Alu
sequences.
Alu-cDNA: small non-protein-coding RNA
There are several classes of small non-protein-coding RNA (npcRNA) that play
important roles in cellular metabolism including mRNA decoding, RNA processing and
mRNA stability. Indeed, altered expression of some of these npcRNAs has been
5
Supplementary notes on Alu-transcript numbers, location, subfamilies and function
associated with cancer, neurodegenerative diseases such as Alzheimer's disease, as well
as various types of mental retardation and psychiatric disorders (Xie et al. 2008).
It has been demonstrated that both free and embedded Alu RNAs play a major role in
post transcriptional regulation of gene expression, for example by affecting protein
translation, alternative splicing and mRNA stability (Hasler et al. 2007).
As transposable element evolution, frequencies and mechanisms for insertion differ
further opportunity remains to investigate other classes of retrotransposable elements.
Adenosine to inosine (A-to-I) RNA editing
DNA methylation and differences in histone modifications are the best-studied epigenetic
control mechanisms involved in cancer development (Feinberg and Vogelstein 1983,
Feinberg et al. 2006). Recently RNA silencing mechanisms, such as RNA interference
and microRNA regulation were also linked to cancer (Lu et al. 2005; Feinberg et al.
2006). Adenosine to inosine (A-to-I) RNA editing is a site-specific modification in stem–
loop structures within precursor mRNAs, catalyzed by members of the double stranded
RNA (dsRNA)- specific ADAR (adenosine deaminase acting on RNA) family (Bass
2002). ADAR-mediated RNA editing is essential for the normal development of both
invertebrates (Palladino et al. 2000) and vertebrates (Higuchi et al. 2000; Wang et al.
2000; Hartner et al.2004). The splicing and translational machineries recognize inosine
(I) as guanosine (G). These A-to-I editing events occur in noncoding repetitive
sequences, mostly Alu elements, and tend to undergo multi-editing in tight clusters. In
addition, RNA editing was shown to be involved in the regulation of nuclear retention
and in miRNAs biogenesis (Prasanth et al. 2005; Blow et al. 2006; Yang et al. 2006). Paz
6
Supplementary notes on Alu-transcript numbers, location, subfamilies and function
et al. (2007) found reduced editing levels at Alu sequences in a human brain tumor.
Alterations in editing and alternative splicing of serotonin receptor transcripts were found
to also correlate with a decrease in enzymatic activity of the editing enzyme adenosine
deaminase acting on RNA (ADAR) 2, as deduced from the analysis of ADAR2 selfediting. These results suggest a role for RNA editing in tumor progression (Maas et al.
2001). In the vast majority of edited RNAs, A-to-I substitutions are clustered within
transcribed sense or antisense Alu sequences. Edited bases are primarily associated with
retained introns, extended UTRs, or with transcripts that have no known corresponding
gene. Therefore, Alu-associated RNA editing may be a mechanism for marking
nonstandard transcripts to bypass the translational machinery (Kim et al. 2004).
7
Supplementary notes on Alu-transcript numbers, location, subfamilies and function
Supplementary Table 1 Subset of full-length Alu-transcript spanning CDS isoforms function exaimation.
accession
gene_id
loci
cancer
gene_id
gene_symbol
location
AK022432
GID:950
HIX0018713
normal
GID:950
SCARB2
4q21.1
AK025390
GID:25926
HIX0014090
normal
GID:25926
NOL11
AK026942
GID:411
HIX0018251
cancer
GID:411
ARSB
17q24.2
5p11q13
AK024421
GID:56996
HIX0006933
normal
GID:56996
SLC12A9
7q22
AK024421
GID:56996
HIX0006933
normal
GID:56996
SLC12A9
7q22
AK024494
GID:56996
HIX0006933
normal
GID:56996
SLC12A9
7q22
AK024494
GID:56996
HIX0006933
normal
GID:56996
SLC12A9
7q22
AK054840
GID:152641
HIX0007805
normal
GID:152641
FLJ30277
4q35.1
AK055885
GID:1
HIX0015537
normal
GID:1
A1BG
19q13.4
AK091237
GID:134266
HIX0005298
cancer
GID:134266
GRPEL2
5q33.1
AK092255
GID:169611
HIX0035002
cancer
GID:169611
OLFML2A
9q33.3
AK094948
GID:286207
HIX0008404
normal
GID:286207
C9orf117
9q34.11
gene_description
scavenger receptor
class B, member 2
nucleolar protein
11
repeat
isoformProtein
FLAM_C
hypothetical
arylsulfatase B
solute carrier
family 12
(potassium/chloride
transporters),
member 9
solute carrier
family 12
(potassium/chloride
transporters),
member 9
solute carrier
family 12
(potassium/chloride
transporters),
member 9
solute carrier
family 12
(potassium/chloride
transporters),
member 9
hypothetical
protein FLJ30277
alpha-1-B
glycoprotein
GrpE-like 2,
mitochondrial (E.
coli)
olfactomedin-like
2A
chromosome 9
open reading frame
AluSx
hypothetical
FLAM_A
AluSx
AluJb
AluSx
AluSx
AluSq
hypothetical
AluSq
FLAM_A
hypothetical
AluSq
hypothetical
AluSx
hypothetical
8
Supplementary notes on Alu-transcript numbers, location, subfamilies and function
AK000385
GID:8635
HIX0017958
normal
GID:8635
RNASET2
6q27
AK098245
GID:5549
HIX0001490
normal
GID:5549
PRELP
1q32
BC011823
GID:55652
HIX0010579
disease
GID:55652
FLJ20489
12q13.11
BC010030
GID:202020
HIX0004117
disease
GID:202020
FLJ39653
4p15.32
AL162039
GID:92597
HIX0004271
normal
GID:92597
MOBKL1A
4q13.3
AF010144
AK129645
GID:27308
GID:83546
HIX0028517
HIX0024449
normal
normal
GID:27308
GID:83546
AD7C-NTP
RTBDN
1p36
19p12
AK124186
GID:196743
HIX0009334
normal
GID:196743
PAOX
10q26.3
AK125252
GID:2523
HIX0010735
normal
GID:2523
FUT1
19q13.3
AK125657
GID:142679
HIX0002652
disease
GID:142679
DUSP19
2q32.1
AK126660
GID:59271
HIX0027783
normal
GID:59271
C21orf63
21q22.11
AK127614
GID:9462
HIX0023758
normal
GID:9462
RASAL2
AF218028
GID:81607
HIX0001228
normal
GID:81607
PVRL4
1q24
1q22q23.2
BC009467
GID:158960
HIX0056219
disease
GID:158960
LOC158960
Xq28
BC024593
GID:11165
HIX0033170
disease
GID:11165
NUDT3
6p21.2
BC037327
GID:22873
HIX0011407
normal
GID:22873
DZIP1
13q32.1
BC018643
GID:83716
HIX0020171
disease
GID:83716
CRISPLD2
16q24.1
117
ribonuclease T2
proline/argininerich end leucinerich repeat protein
hypothetical
protein FLJ20489
hypothetical
protein FLJ39653
MOB1, Mps One
Binder kinase
activator-like 1A
(yeast)
neuronal thread
protein AD7c-NTP
retbindin
polyamine oxidase
(exo-N4-amino)
fucosyltransferase
1 (galactoside 2alpha-Lfucosyltransferase,
H blood group)
dual specificity
phosphatase 19
chromosome 21
open reading frame
63
RAS protein
activator like 2
poliovirus receptorrelated 4
hypothetical
protein BC009467
nudix (nucleoside
diphosphate linked
moiety X)-type
motif 3
DAZ interacting
protein 1
cysteine-rich
secretory protein
LCCL domain
AluSq
hypoth
FLAM_C
hypoth
AluSx
hypoth
AluSg
hypoth
AluSx
hypoth
AluSc
AluSx
hypoth
hypoth
AluSx
AluSx
hypoth
AluSq
hypoth
AluSq
hypoth
AluSx
FLAM_A
hypoth
FLAM_A
hypoth
AluSg
hypoth
FLAM_C
AluSx
hypoth
9
Supplementary notes on Alu-transcript numbers, location, subfamilies and function
BC044913
GID:92597
HIX0004271
normal
GID:92597
MOBKL1A
4q13.3
BC060883
CR627453
GID:400581
GID:57653
HIX0039163
HIX0008209
normal
normal
GID:400581
GID:57653
LOC400581
KIAA1529
17p11.2
9q22.33
containing 2
MOB1, Mps One
Binder kinase
activator-like 1A
(yeast)
GRB2-related
adaptor protein-like
KIAA1529
AluSx
hypoth
AluSx
AluYb8
hypoth
hypoth
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Supplementary notes on Alu-transcript numbers, location, subfamilies and function
Acknowledgment
The H-Inv human cDNA data was analysed mostly by Paula Moolhuijzen as part of her
PhD requirements.
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Supplementary notes on Alu-transcript numbers, location, subfamilies and function
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