"Primate-Specific Endogenous Cis-Antisense Transcription in the Human 5q31 Protocadherin Gene Cluster".

Leonard Lipovich ¹, Ravi Raj Vanisri ¹, Say Li Kong ¹, Chin-Yo Lin ¹, and Edison T. Liu ¹

¹ Genome Institute of Singapore, 60 Biopolis Street #02-01, Singapore, 138672, Singapore

Leonard Lipovich   Email: LL@gis.a-star.edu.sg

NetworkEditor's Perspective: Antisense RNA synthesis regulates sense RNA expression.
Abstract:
Introduction:
Material and Methods:
Results:
   Table 1: Novel transcriptional units cis-antisense to human Protocadherin exons:
   Fig. 1: Distinct genomic footprint of a novel human transcriptional unit on the negative strand of PCDHb3:
   Fig. 2: Conservation of canonical signals and splice junctions on the PCDH antisense strand:
   Fig. 3: Genomic structure and transcriptional activity of targeted portions of the protocadherin gene cluster:
   Fig. 4: Antisense-strand splice site conservation between human PCDHy5 and human PCDHb15:
   Fig. 5: Summary of protocadherin sense and antisense expression in human, rhesus, and mouse:
   Fig. 6: Quantitated expression levels of sense-strand protocadherin transcripts:
   Table 2: Sense expression levels with and without endogenous cis-antisense:
Discussion:
   Fig. 7: Evolutionary map of mammalian PCDH sense and cis-antisense transcription:
Acknowledgments:
Supplementary Material:
References:
Additional References:
Further Topics:
Other Links:
Further Information and Feedback:

Protocadherins (PCDH), localized to synaptic junctions, contribute to the formation of neuronal networks during brain development; thus, it is speculated that protocadherins may play a role in evolution of neuronal complexity. While protocadherin genes are highly conserved in vertebrates, EST evidence from the locus suggests apparently species-specific cis-antisense transcripts. Novel cis-antisense transcripts, which partially overlap the PCDHa12 variable exon, PCDHb3 single-exon gene, and PCDHy5 unprocessed pseudogene in the human 5q31 PCDHa/b/g gene cluster and which are coexpressed with sense-strand transcripts in fetal and adult brain, were identified computationally and validated by gene-specific strand-specific reverse transcriptase PCR (SSRTPCR) and sequencing. Absence of antisense transcripts arising from equivalent genomic locations in mouse indicates that the antisense transcripts originated in the primates after the primate-rodent divergence. Furthermore, not all expected orthologues of human sense and antisense PCDH transcripts were detected in rhesus macaque brain, implying that protocadherin expression patterns differ between primate species. RT followed by quantitative real-time PCR (QPCR) analysis of the three genes in the brain of all three species, and of the PCDHb15 gene paralogous to PCDHy5 in human and rhesus, revealed that the presence of antisense transcripts was significantly associated with lower sense expression levels across all orthologues. This inverse relationship, along with the pattern of sense and antisense coexpression in the brain, is consistent with a regulatory role for the primate-specific PCDH cis-antisense transcripts, which may represent recent evolutionary inventions modulating the activity of this conserved gene cluster.

Protocadherins: Localization and Function

Protocadherins (PCDHs), the largest subgroup of the cadherin superfamily of calcium-dependent cell-cell
adhesion glycoproteins (Frank and Kemler 2002), are major structural and functional components of synapses
and are expressed on the surfaces of neurons at synaptic junctions (Noonan et al. 2003). Each PCDH displayed
on the surface of a given cell potentially facilitates homophilic adhesion formation with adjacent cells displaying
the identical PCDH (Vanhalst et al. 2001). Evidence for heterophilic interactions of Pcdh a and g proteins in the brain also exists (Murata et al. 2004). Individual neurons are capable of expressing distinct but overlapping
subsets of PCDHs, which may provide a combinatorial molecular code for neuron-to-neuron connections (Wang
et al. 2002). By determining which neurons interact with which other neurons in the vicinity, PCDHs likely
account for some of the combinatorial complexity in neuronal networks, affecting brain development and
possibly memory (Noonan et al. 2003) through neuronal morphogenesis, synaptic connection formation, and
synaptic transmission regulation (Frank and Kemler 2002).

Structure of the 5q31 Protocadherin Gene Cluster

The majority of PCDH genes are found in three adjacent clusters, mapping in human (Homo sapiens) to 5q31.
The three clusters—PCDHa, PCDHb, and PCDHg—occur sequentially to one another, with PCDHa closest to the centromere and PCDHg closest to the 5q telomere. Together, the three human 5q31 PCDH clusters span ~750 kb and contain putative regulatory elements upstream of each variable exon. For a detailed diagrammatic representation of PCDH gene cluster structure, see Wu et al. (2001).

The PCDHa cluster consists of a tandem array of multiple alternative first exons, followed by three
constitutive exons. Any one of the multiple alternative first exons can be spliced to the downstream
constant-exon cassette. The PCDHg cluster is organized in the same fashion. Each alternative PCDHa and
PCDHg first exon encodes an entire extracellular domain, the transmembrane segment, and a part of the
intracellular C-terminal domain. The PCDHb cluster, which maps between the PCDHa and PCDHg clusters,
consists exclusively of single-exon genes.

Most human PCDHa first exons, PCDHb genes, and PCDHg first exons have one-to-one mouse (Mus
musculus) orthologues. However, some are the products of duplications that postdated the human/mouse
divergence and lack true one-to-one orthologues in the mouse. Some other PCDH exons and genes are
functional in one species but pseudogenic in another (Vanhalst et al. 2001). PCDHa and PCDHg expression
regulation is in agreement with the “alternative promoter choice and cis-splicing” model. However, specific
mechanisms of PCDH expression remain uncharacterized (Wang et al. 2002).

Cis-Antisense: Definition, Incidence, and Significance

A cis-antisense gene pair is operationally defined as a pair of genes which reside on opposite strands in the
same locus in such a configuration that at least one exon of one gene overlaps at least one exon of the other.
Cis-antisense has been detected in prokaryotes (Vanhee-Brossolet and Vaquero 1998), Arabidopsis (Yamada
et al. 2003), and Drosophila (Misra et al. 2002). Up to 22% of human genes may participate in cis-antisense
pairs (Chen et al. 2004).

Cis-antisense is a gene expression regulatory mechanism which functions at both transcriptional and
post-transcriptional levels. At the transcriptional level, competitive transcriptional interference (Prescott and
Proudfoot 2002) and sense-strand silencing by antisense-mediated promoter methylation (Tufarelli et al. 2003)
have been demonstrated. Posttranscriptionally, cis-antisense transcripts can regulate alternative splicing
(Hastings et al. 1997), may be involved in RNA editing (Lavorgna et al. 2004), and have been shown to form
double-stranded RNA duplexes with their sense counterparts. Although the duplexes may be targeted for
degradation by cellular RNAses, translation attenuation through formation of stable undegraded RNA
duplexes may occur as well (Podlowski et al. 2002).

Confirmed functions of cis-antisense are diverse. Antisense transcripts are associated with autosomal
imprinting and X-inactivation (Shibata and Lee 2004) and may function by allele-specific silencing (Verona et
al. 2003). Transcript abundance ratios of certain key developmental regulators and their noncoding
cis-antisense partners are inversely correlated and change during cell differentiation, suggesting a function for
cis-antisense in modulating sense levels (Blin-Wakkach et al. 2001). Mutations in a noncoding cis-antisense
transcript are sufficient for pathogenesis of a neurodegenerative disorder whose mechanism depends on the
sense-encoded protein (Nemes et al. 2000). Downregulation of sense-encoded protein expression by an
endogenous trans-antisense transcript has been demonstrated in a mammalian cell line, although the antisense
in that case involved an interspersed repetitive element in trans (Stuart et al. 2000). Cis-antisense–mediated
downregulation of sense expression, albeit in an in vitro system, has been confirmed as well (Thenie et al.
2001).

Early samplings of the mammalian cis-antisense subtranscriptome suggested that only a minority of the
cis-antisense pairs (27% in mouse [Kiyosawa et al. 2003]) involve solely protein-coding genes. Noncoding
cis-antisense transcripts in the remainder of the dataset differ drastically from other types of regulatory RNAs,
i.e., microRNAs, which have received more attention in recent years. Unlike microRNAs, cis-antisense
noncoding RNAs are mRNA-like in all respects, except that they do not code for protein. Cis-antisense
noncoding RNAs are 5'-capped (Imamura et al. 2004; Kiyosawa et al. 2003), mostly canonically spliced (Chen
et al. 2004) and polyadenylated, longer than microRNAs and snRNAs, pol(II)-promoted, encoded by the same
locus as the target (Lavorgna et al. 2004), independent of cytoplasmic Dicer (Tran et al. 2004), and
complementary to coding targets both within and outside of 3' UTRs (Chen et al. 2004; Yelin et al. 2003).

Origins of New Genes and Primate-Specific Functions

The genomic basis of phenotypic distinctions between closely related species remains uncertain. Existing
explanations for the drastic differences in phenotypes between closely related species such as chimpanzees
and humans invoke regulatory element differences responsible for distinct expression profiles of homologous
genes (King and Wilson 1975) or lineage-specific phenotypes related to the loss of function of particular genes
during evolution (Olson and Varki 2003). However, it is conceivable that some phenotypic differences might be
due to a gain of function in one lineage, encoded by a gene absent in the other. In fact, origin of new genes,
which enable novel functions and contribute to genetic diversity, is recognized as a fundamental biological
process. In view of the obvious and pronounced differences in mental ability, immune response, and
reproductive biology between primates and nonprimates—as well as within primates—novel functions encoded
by primate-specific new genes are important to study. Nevertheless, the exact mechanisms giving rise to new
genes remain to be elucidated, although several case studies suggest that one pathway by which new genes are
created is the shuffling of existing coding-gene exons, which generates both coding and noncoding new genes
and is often facilitated by retrotransposition (Long et al. 2003). For example, the primate-specific genes
PMCHL1 and PMCHL2 have formed through a complex combination of cis-antisense transcription,
retrotransposition, novel splice site recruitment, and block duplication during primate evolution (Courseaux
and Nahon 2001). Nonconservation of multiple genes in mammalian antisense pairs (Shendure and Church
2002; Veeramachaneni et al. 2004) makes it plausible that certain cis-antisense transcripts have recent
evolutionary origins. If such transcripts exist at the PCDH gene cluster, they can represent attractive
functional candidates underlying the genomic basis of mammalian interspecies differences in neuronal and thus
behavioral complexity.

Although the structure of the human 5q31 PCDH gene cluster is known in exquisite detail, no mention of
endogenous cis-antisense transcription in this locus could be found in the literature. We report in silico
discovery and experimental validation of novel cis-antisense transcripts in the 5q31 PCDH gene cluster,
followed by a qualitative and quantitative multispecies analysis of sense and antisense transcription.

Materials and Methods:

Sequence Analysis

Identification of Human Cis-Antisense Transcription

Finished and HTGS-draft human genomic clones encompassing the 5q31 PCDH cluster (tiling path, 5cen to
5qter: AC005609.1, AC010223.6, AC025436.2, AC005754.1, AC074130.3, AC005752.1, AC005618.1,
AC005366.1) were visualized using Seqhelp software (Lee et al. 1998). All visual clusters of ESTs partially
overlapping sense-strand PCDH exons but having a discordant genomic footprint (distinct genomic locations of
transcription start and end sites, and of splice donor/acceptor sequences for spliced ESTs) relative to the sense
exons were noted. Orientation of representative ESTs from each cluster was determined by BL2SEQ
(Tatusova and Madden 1999) and Spidey (Wheelan et al. 2001) pairwise alignments to genomic sequence. For
plus/minus HSPs with 3' ESTs, strandedness was inverted, because by convention the first nucleotide of 3'
EST sequences in GenBank represents the 3' end of the corresponding transcript.

Identification of Sequences Orthologous to Human PCDH Regions

Putatively orthologous chimpanzee sequences were identified by a BLAT search (Kent 2002) of the November
2003 chimpanzee WGS assembly at the UCSC Genome Browser portal (Kent et al. 2002) with human queries.
Putatively orthologous rhesus monkey (Macaca mulatta) sequences were identified by a TraceDB
MegaBLAST BLASTN search (http://www.ncbi.nlm.nih.gov/blast/mmtrace.shtml ) of the “Macaca
mulatta—WGS” and “Macaca mulatta—other” databases with human queries. Mouse orthologues and best
homologues were defined as the gene hits with simultaneously highest BLASTN and BLASTP scores relative
to the given human query. All orthologues were verified by reciprocal BLAST against the appropriate human
databases, and nonhuman sequences whose top-scoring human matches differed from the original human query
were discarded. Precomputed global human/chimpanzee and human/mouse BLASTZ outputs (Schwartz et al.
2003), underlying the “chained BLASTZ alignments” track of the UCSC portal, were utilized to obtain pairwise
alignments of orthologous regions.

Experimental Protocols

Note: PCR conditions, along with all primer and probe sequences, are cited in the supplementary information
file.

Nonquantitative PCR (SSRTPCR)

Strand-Specific cDNA Synthesis

Human adult brain total RNA (Clontech; one donor; 43-year-old male Caucasian; no pathology noted), human
fetal brain total RNA (Clontech; pooled from 59 spontaneous abortuses, Caucasian, male and female, 20–33
weeks), Macaca mulatta adult brain total RNA (BioChain Institute, Inc.; one donor; no pathology), and mouse
pooled RNA from a male and a 12.5-day-pregnant female (a gift from Sai-Kiang Lim, GIS, Singapore) were
used for reverse transcription (RT) reactions to make strand-specific cDNAs.

Mouse pooled RNA was treated with DNase I before RT reaction. One microgram of total RNA was incubated
with 1 ml of DNase I (2 U/ml;Ambion,USA) at 37°C for 60 min and the reaction was inactivated at 95°C for 5
min.

Two different types of RT reactions were performed, with SuperScriptII reverse transcriptase and
ThermoScript reverse transcriptase, using gene-specific untagged primers and gene-specific tagged primers,
respectively. For human and rhesus PCDHb15, as well as human PCDHy5, ThermoScript was used to detect
transcription in both sense and antisense orientations, whereas for other amplicons solely SuperScriptII was
used.

For SuperScriptII reverse transcriptase (Invitrogen) reaction, 200 ng total RNA, 20 mM gene-specific primers,
and 10 mM dNTP (Invitrogen) were incubated at 65°C for 5 min and the tubes were immediately placed on the
ice. The contents were colleted by brief centrifugation. Then 5× first-strand buffer, 0.1 M DTT, 1 ml
RNaseOut, and 1 ml SuperScript 11 (200 U) were added to the tube for a final volume of 20 ml. RT was carried
out for 60 min at 42°C and the reverse transcriptase activity was inactivated at 70°C for 15 min.

For ThermoScript reverse transcriptase (Invitrogen) RT, 200 ng total RNA, 20 mM gene-specific tagged
primer, and 20 mM dNTP were incubated for 5 min at 70°C. The tubes were immediately placed on ice. Then 5×
cDNA synthesis buffer, 0.1 M DTT, 1 ml RNaseOut, and 1 ml TheromoScript reverse transcriptase (15 U/ml)
were added. The temperature was reduced for primer annealing for 2 min and then returned to 70°C for a
further 30 min. Reverse transcriptase activity was inactivated at 98°C for 15 min. Three negative controls
accompanied each RT reaction: exclusion of template, exclusion of enzyme, and both.

For ThermoScript reverse transcriptase RT, Exonuclease 1 (10 U; Amersham International) was added to 10 ml
of cDNA to degrade unincorporated primers upon completion of RT and incubated at 37°C for 45 min, followed
by inactivation at 98°C for 15 min.

Nested PCR Amplification

After optimization of amplicons on genomic DNA (details available upon request), 2 ml of cDNA was used in the
first-round PCR and 2 l of the first-round product was used in the second-round PCR. PCR products were
analyzed by 2% agarose gel electrophoresis.

Both genomic PCR products and cDNA PCR products were purified using a QIAquick Gel Extraction kit and
25 to 50 ng of purified PCR products was used for cycle sequencing reactions with 3.2 pmol of forward or
reverse primers and 4 ml of sequencing Premix (Big Dye terminator), to confirm all amplicon identities at the
sequence level.

Quantitative PCR (QPCR)

We quantified the sense expression level of PCDH using a real-time fluorescence detection method. Human
adult and fetal brain total RNAs (Clontech), Macaca mulatta adult brain total RNA (BioChain Institute, Inc.),
mouse adult brain total RNA (Clontech), and mouse embryonic brain total RNA (E17; Zyagen Laboratories;
catalog no. MR-201-E17) were used in a nested RT-PCR. Single-stranded cDNAs were generated using
Superscript 11 reverse transcriptase (Invitrogen) and random primers according to the manufacturer’s protocol
and then 1 ml of serial 100-, 50-, 25-, and 12.5-fold dilutions of cDNA, a 5 mM concentration of each primer, and
2 ml of LightCycler FastStart DNA Master PLUS SYBR Green1 (Roche Diagnostics Asia Pacific Pte. Ltd.)
were used in a 10-l total volume. The relative sense-RNA transcript abundance was obtained by calculating
the ratio of the fluorescent intensity (cross-point value). Each sample was normalized on the basis of its ß-actin
content. Real-time quantitative PCR experiments were performed with a Roche Lightcycler instrument.

The cycling conditions were as follows: 95°C for 10 min, 95°C for 10 s, annealing dependent on the primer
temperature, and 72°C for 10 s. Melting curve analysis was performed depending on the primer annealing
temperature using the Lightcycler software supplied with the instrument.

PCR products were visualized on a 2% agarose gel to confirm amplicon sizes prior to quantification.

Northern Blot Analysis

We used human adult and fetal multiple tissue Northern (MTN) blots from Clontech (catalog nos. 636818 and
636803) in an attempt to detect PCDH antisense-strand transcription. 5’-End-labeled 50-mer oligonucleotide
probes were used in hybridization. Thirty picomoles of antisense oligonucleotide, 7 ml of [g-³²P]ATP (6000
Ci/mmol; Amersham Biosciences Ltd.), and one tube of Ready-To-Go T4 PNK (Amersham Biosciences Ltd.)
were used in final volume of 50 ml. The reaction was incubated for 60 min at 37°C, then stopped by the addition
of 5 ml of 250 mM EDTA. The labeled probe was separated from unincorporated nucleotides through Sephadex
G25 Quick Spin Columns (Roche Diagnostics Asia Pacific Pte. Ltd.). Specific activity of the probe was
quantified by scintillation counting.

Membranes were prehybridized in ExpressHyb Solution at 42°C for 2 h. Denatured probes were added to
ExpressHyb Solution (0.73 × 10⁷ cpm/ml) and the membranes were hybridized overnight at 42°C. Membranes
were rinsed a few times in 2× SSC and 0.05% SDS and washed two times at room temperature, followed by two
washings in 0.1× SSC and 0.1% SDS at 42°C. Blots were exposed for 3 days against a PhosphorImaging
screen and visualized using a PhosphorImager System (Typhoon 9410; Amersham Biosciences). A human adult
MTN blot was stripped by hot water containing 0.5% SDS for 10 min. Then it was used for a control
hybridization with a human ß-actin probe. The blot was hybridized at 0.8 × 10⁷ cpm/ml ExpressHyb solution
overnight. After washing, PhosphorImager signal detection was performed as above.

Results:

In Silico Discovery of Putative Novel Cis-Antisense Transcripts in the Human PCDH Gene Cluster

During manual annotation of the 5q31 PCDH gene cluster, we encountered 12 novel transcriptional units (TUs)
supported by EST and/or flcDNA evidence (partial listing; Table 1). Their genomic locations partially
overlapped those of PCDH exons, but their genomic footprints (transcriptional unit boundaries and splice
junction locations) were distinct from those of the sense-strand PCDH exons which they overlapped. In all 12
cases, this distinction was due to the cis-antisense orientation of the novel transcripts relative to the PCDH
cluster.

Table 1: Characterization of flcDNA- and EST-supported novel transcriptional units cis-antisense to human
PCDH exons.
 

Human  PCDH  gene  or exon	Mouse  orthologue	Human  sense  strand  expression	Mouse  sense  strand  expression	Number and list  of human  antisense ESTs	Number  of mouse  antisense  ESTs	Number of  exons in the  human  antisense  transcript
PCDHa1 variable exon	PCDHa1	+	+	1 flc DNA BC019862	0	1
PCDHa9 variable exon	PCDHa7 variable exon	+	+	10 ESTs AI247431, AA437139, AI015406, AW236832, BF510424, AI632155, BE502295, AI968755, BG719192, AA927013	0	1
PCDHa11 variable exon	PCDHa11 variable exon	+	+	1 EST BI562026	0	1
PCDHa12 variable exon	no one-to-one orthologue	+	not applicable	2 ESTs AW134813, BG150315	0	1
PCDHa13 variable exon	PCDHa12 (Wu et al. 2001)	+	+	1 EST CN428072	0	2
PCDHac1 variable exon	PCDHac1 variable exon	+	+	1 EST AA772180	0	1
PCDHb1 single-exon gene	PCDHb1 single-exon gene	+	+	1 EST BX097466	0	2
PCDHb3 single-exon gene	PCDHb3 single-exon gene	+	+	1 flc DNA BC016751,  and 6 ESTs,  BG773164, AI825548,  AW183407, AW136319, AI633930, AI990795	0	3
PCDHb10 single-exon gene	PCDHb10 single-exon gene	+	+	1 EST BX113296,	0	2
PCDHb16 single-exon gene	PCDHb16 single-exon gene	+	+	4 ESTs AI684744, AI376824, AW079889, AA453283	0	1
PCDHy5 single-exon unprocessed b-class pseudogene	no one-to-one orthologue	-	not applicable	17 ESTs:  BE463846, AI623196, AW294155, AI969803, AI652903, BE672046, AI954650, AW025845, AI219898, AW593514, AW241532,  AI692192, AA437196, AA296664, AA757142, AA904724, AI911128.	not  applicable	3
PCDHy3 unprocessed g-class pseudogenic variable exon	PCDHgB8 variable exon	-	+	2 ESTs AW070271, BX281564	0	1

Three lines of evidence supported the antisense orientation of the novel TUs relative to PCDH genes. First,
their canonical polyadenylation signals (e.g., AATAAA) and canonical splice donor and acceptor sites (GT-AG)
resided on the strand opposite to that encoding the PCDH exons. Figure 1 highlights the 3' end of a
representative antisense transcript (anti-PCDHb3), including the antisense-strand polyadenylation signal and
the genomic footprint difference with sense, as visualized in SeqHelp (Lee et al. 1998). Second, the orientation
of these polyadenylation signals and splice sites universally conformed to submitter-indicated transcription
orientation of the ESTs and flcDNAs comprising the novel TUs; for example, AATAAA, or an associated
consensus variation, was found within the 50 bp nearest the submitter-indicated 3' end of the ESTs and
flcDNAs, suggesting that there was no artifactual reversal of transcript sequences in GenBank/dbEST. Finally,
BL2SEQ and Spidey pairwise alignments of ESTs and flcDNAs comprising the novel TUs against known
PCDH exons were invariably in the antisense (plus/minus) orientation.

Figure 1: Antisense-strand canonical polyadenylation signal and distinct genomic footprint of a novel human
transcriptional unit on the negative strand of PCDHb3.

For initial assessment of interspecies conservation of PCDH antisense transcription, we identified the true
orthologues or nearest homologues (Wu et al. 2001; Vanhalst et al. 2001) of the 12 human
antisense-overlapped PCDH exons in the mouse and manually curated all EST-to-genome alignments
corresponding to the mouse exons and adjacent genomic sequences. No evidence of antisense-strand
transcription was seen in the mouse, suggesting that antisense transcripts at these specific locations are not
conserved (Table 1).

Comparative Sequence Analysis of PCDH Cis-Antisense Transcripts

To further investigate the possibility that human PCDH cis-antisense transcripts are not evolutionarily
conserved, we focused on the three transcripts with the greatest extent of EST support: anti-PCDHa12,
anti-PCDHb3, and anti-PCDHy5—all of which are putatively noncoding. Anti-PCDHa12, anti-PCDHb3, and
anti-PCDHy5 encode the longest ORFs, sized at 121, 49, and 149 amino acids, respectively. The ORFs contain
no conserved domains and no similarities to any known proteins outside of low-complexity regions.

Since splice sites and polyadenylation signals are major contributors to transcript structure and boundary
definition, the absence of these sequence elements in a nonhuman species would indicate either a major
interspecies difference in antisense transcript structure or a lack of the antisense transcript. To determine the
extent to which these elements are conserved in mammalian genomic sequences and to estimate the time at
which they first arose in evolution, we searched for antisense-strand splice sites and polyadenylation signals at
orthologous genomic locations in one nonhuman great ape (chimpanzee), one old world primate (rhesus
macaque), and mouse. Results are summarized in Fig. 2.

Figure 2: Conservation of canonical polyadenylation signals and splice junctions on the PCDH antisense
strand.

Figure 2: Conservation of canonical polyadenylation signals and splice junctions on the PCDH antisense
strand.

Columns 3 and 5: mouse is at top, human is at bottom, and short alignments are excised out of a
substantially longer context of one-to-one sequence-level orthology represented by the pairwise sequence
alignment underlying the UCSC Chained BLASTZ Alignments track. Arrows indicate direction of transcription
of the human PCDH cis-antisense transcripts; boxes indicate consensus poly(A) signals and splice sites, all of
which are on the reverse strand of alignments shown. Column 4: “no info” denotes splice site located in
genomic sequence not currently covered in the Macaca mulatta division of Trace DB. 

All three cis-antisense transcripts were characterized by canonical polyadenylation signals in human. AATAAA
is the most frequent polyadenylation signal in mammals, while AGTAAA and CATAAA are acceptable variants
of the broader polyadenylation hexamer consensus, occurring at frequencies of 2.83% and 1.82%,
respectively, in the FANTOM2 mouse cDNA collection (Carninci et al. 2003). All signals were fully conserved
in chimpanzee and rhesus, with the exception of the rhesus genomic location equivalent to the human
anti-PCDHa12 AGTAAA polyadenylation signal, which contained a strongly noncanonical AGTACA (the C is
supported by a Q40 peak in TraceDB accession 331289929 and also remains in the January 2005 rhesus
genome assembly at UCSC). The full conservation in chimpanzee, however, implies that the whole-genome
shotgun assembly method used to derive the chimpanzee genomic sequence, while potentially less accurate
than the BAC/PAC tiling path method used to assemble the human genome (for an in-depth discussion see
Green 2002), was not problematic for this analysis.

In contrast, no antisense-strand polyadenylation signals were found in mouse. The AGTAAA near the 3' end
of anti-PCDHa12 localized to a human-specific insertion in the global human/mouse BLASTZ alignment.
Although the sequence containing the AATAAA of anti-PCDHb3 was found in the mouse, two of the six bases
differed between human and mouse due to single-nucleotide substitutions, thereby completely abolishing the
polyadenylation signal consensus in mouse. Similarly, three of the six bases of the CATAAA polyadenylation
signal utilized by human anti-PCDHy5 diverged from the polyadenylation signal consensus in mouse.

Although all human anti-PCDHa12 EST evidence indicates a single-exon transcript, the other two human
antisense transcripts were spliced, allowing an assessment of splice donor and acceptor conservation in
homologous nonhuman sequences. The splice donor/splice acceptor sequence of the intron used by all
anti-PCDHb3 transcripts except AI633930 was GTGCG-AG and completely conserved in chimpanzee. The
splice acceptor was conserved in rhesus as well, although the splice donor lacked genomic sequence coverage
at the time of analysis due to incompleteness of the rhesus trace archive. The anti-PCDHy5 transcript had two
introns, with alternative splicing within the second. The long second intron variant, represented most frequently
in anti-PCDHy5 ESTs, had a splice donor (GTGGC) and splice acceptor (AG) completely conserved at
orthologous locations in both chimpanzee and rhesus.

However, splice site conservation of PCDH antisense-strand transcriptional units did not extend to mouse. The
GTGCG splice donor and AG splice acceptor utilized by human anti-PCDHb3 did not exist in mouse due to two
single-base substitutions in the donor and one in the acceptor sequence. Two of the five bases of the human
anti-PCDHy5 splice donor were substituted in mouse with nonconsensus bases as well. Thus, multispecies
comparison of PCDH antisense transcript splice sites and polyadenylation signals within orthologous sequence
context demonstrates conservation of these sequence elements in the primate genomes we considered but not
in mouse.

Experimental Validation of Primate-Specific PCDH Cis-Antisense Transcripts Coexpressed with
Corresponding Sense Exons in Brain

To test the hypothesis that PCDH cis-antisense transcripts are primate-specific, and to validate the expression
of the sense and antisense transcripts in brain, we performed gene-specific, strand-specific RT followed by
nested PCR and sequencing in an attempt to detect the anti-PCDHa12, anti-PCDHb3, PCDHa12, and PCDHb3 transcripts in human, rhesus, and mouse and anti-PCDHy5 and PCDHy5 in human and rhesus. In addition, weperformed the same orientation-specific transcription assay on the mouse Pcdhb15 locus, which is in a one-to-two homologous relationship with the human PCDHb15 and PCDHy5 genes (Vanhalst et al. 2001), and
on the human and rhesus PCDH?15 genes, even though they did not have antisense-strand flcDNAs or ESTs.

Figure 3 summarizes the genomic structure of all PCDH loci in all species vis-à-vis the sense and antisense
transcript exon-intron structures. Exon-intron structures were determined by curation of EST-to-genome
alignments, prior to experimental validation. For human, antisense-specific RT primers were designed from
sequences which, based on flcDNA and EST evidence, were exonic with respect to the antisense but not the
sense transcripts. In addition, the orientation-specific nature of our single-primer RT reactions (see Materials
and Methods) assures the strand specificity of results.

Figure 3: Multispecies analysis of genomic structure and transcriptional activity of targeted portions of the
protocadherin gene cluster.
 

A. The human PCDHa12 variable exon and orthologous regions in rhesus and mouse.

A. The human PCDHa12 variable exon and orthologous regions in rhesus and mouse.
 

B. The human PCDHb3 single-exon gene and orthologous regions in rhesus and mouse.

B. The human PCDHb3 single-exon gene and orthologous regions in rhesus and mouse.

C. The human PCDHy5 single-exon b-class unprocessed pseudogene; the orthologous region in rhesus; and the mouse Pcdhb15 gene, whose two closest primate homologues are PCDHy5 and PCDHb15.

C. The human PCDHy5 single-exon b-class unprocessed pseudogene; the orthologous region in rhesus; and the mouse Pcdhb15 gene, whose two closest primate homologues are PCDHy5 and PCDHb15.

D. The human PCDHb15 single-exon gene; the orthologous region in rhesus; and the mouse Pcdhb15 gene, whose two closest primate homologues are PCDHy5 and PCDHb15.

D. The human PCDHb15 single-exon gene; the orthologous region in rhesus; and the mouse Pcdhb15 gene, whose two closest primate homologues are PCDHy5 and PCDHb15.

SSRTPCR only. QPCR amplicons are *not* shown. Left side of each module (genomic structure): thick black dashed horizontal lines separate species within a gene grouping. For genes where both genomic DNA and transcripts are shown, the transcripts are indicated by horizontal arrows pointing in the direction of transcription below genomic DNA. Solid arrows indicate transcripts, or portions of transcripts, documented by our sequenced RTPCR products and/or by public flcDNA/EST evidence. Dotted arrows indicate portions of transcripts which are inferred to exist based on sequence homologies, but which are outside of our sequenced RTPCR products and lack public flcDNA/EST support. Thin black solid horizontal lines are genomic DNA sequences (human except for PCDHb15, which is rhesus) and flcDNA sequences (mouse and human PCDHb15). Interspecies thick vertical lines demarcate genomically equivalent sequence positions, based on one-to-one orthology for all genes except mouse Pcdhb15 and its partners, in which case they are based on one-to-two homology to the primate genes. Primary RTPCR amplicons are shown as thin horizontal lines bounded by vertical lines. See supplementary information for accession numbers, sequence coordinates, and nested amplicon locations. Not to scale. Right side of each module (SSRTPCR results): all PCR products on gels are nested. Identities of all products were confirmed by sequencing. (All chromatograms are on file; data not shown.) “Antisense” is a nested PCR product obtained after gene-specific, strand-specific, single-primer RT with antisense-specific RT primer. “Sense” is a nested PCR product obtained after gene-specific, strand-specific, single-primer RT with sense-specific RT primer. Controls on gels, left to right, are as follows.

(1) Mock RT. +RT –primer. Outer and nested PCR as usual. Shows no contamination of RT and buffer with genomic DNA.
(2) Mock RT. –RT +primer. Outer and nested PCR as usual. Shows absence of genomic DNA in starting RNA sample.
(3) Mock RT. –RT –primer. Outer and nested PCR as usual. Shows no contamination of primer aliquots with genomic DNA.

Human and rhesus “antisense” lanes are followed by 1 and 2; “sense” lanes, by 1, 2, and 3.
Mouse “antisense” and “sense” lanes are both followed by 1, 2, and 3.
Templates: human—adult brain and fetal brain total RNA; rhesus—adult brain total RNA; mouse—pooled
whole-body adult and fetal total RNA. Additional details pertaining to this figure are given in the
supplementary information.

If antisense transcript sequence and structure are conserved in a nonhuman primate, then antisense transcript
boundaries and splice sites can be predicted from the human sequence. We refer to these aligned nonhuman
positions as positional equivalents of the human genomic structure elements. For rhesus, orthologous regions
were localized, and primers were designed within these positionally equivalent spans. The position
equivalencies are indicated by vertical lines in Fig. 3. For mouse, genomic sequence conservation appeared
limited to sense-strand exon boundaries. Therefore, mouse SSRTPCR amplicons covered solely portions of the
Pcdh exons which were positionally equivalent to antisense-covered portions of human PCDH exons.

SSRTPCR results are presented in Fig. 3 to the right of the genomic diagrams. Transcription in both directions
was detected in human adult and fetal brain for the unspliced PCDHa12 amplicon. Though the primers were
initially designed solely for antisense-specific SSRTPCR, the sense signal indicates that the sense-strand TSS
is located at least 63 bp upstream of its previously reported location at bp 16625 of AC005609.1. Transcription
in both directions was detected in adult rhesus brain as well. However, no antisense-specific signal was
detected in the mouse total-body fetal and adult sample. Therefore, the cis-antisense transcript overlapping the
PCDHa12 variable exon is primate-specific and is coexpressed with PCDHa12 in brain.

Similarly, transcription of PCDHb3 was observed in both directions in human adult and fetal brain total RNA
samples. The presence of the sense-strand signal suggesting that the PCDHb3 TSS is at least 136 bp upstream of its previously known location at bp 78155 of AC005754.1. In contrast with PCDHa12, no antisense transcription could be detected in the rhesus region equivalent to the 3’ terminal exon of the human
anti-PCDHb3 transcript. The detection of sense PCDHb3 transcript in the rhesus suggests that, as in human,
the rhesus PCDHb3 TSS is upstream (at least 341 bp) of the location predicted based on the human TSS
defined by the full-length cDNA AF217755. Consistent with our interpretation of the multispecies sequence
alignment showing primate specificity of the cis-antisense transcripts, no antisense transcription was seen in
the mouse equivalent of the antisense-overlapped portion of human PCDHb3 (Fig. 3B).

Human PCDHy5 is an unprocessed single-exon ß-class PCDH pseudogene, originating from an ancient tandem
duplication within the PCDHB cluster, and is known to be transcribed in both sense (Vanhalst et al. 2001) and
antisense (this study) orientations. We also show sense-strand transcription of PCDHy5 in human fetal and
adult brain. As expected for a PCDHB-class gene, the transcript is unspliced. Although numerous ESTs in this
locus suggest the existence of a spliced cis-antisense transcript, antisense-specific SSRTPCR shows a spliced
product of the appropriate size only in fetal brain, whereas an unspliced antisense transcript is found in adult
brain. The corresponding region of the rhesus PCDHy5 was not transcriptionally active in adult brain in either
orientation (Fig. 3C).

Multiple attempts to detect PCDHa12, PCDHb3, and PCDHy5 antisense strand transcription in human by
Northern blotting were unsuccessful with both Integrated DNA Technologies and standard T4 polynucleotide
kinase labeling protocols, using both Clontech and U.S. Biologicals fetal and adult multitissue blots. Since an
ACTB control Northern blot was successful, PCDH cis-antisense transcript levels are likely below the
threshold of detection by Northern analysis but detectable by SSRTPCR.

To determine whether the paralogous PCDHb15 and PCDHy5 transcriptional units, originating from a
duplication in the primate lineage after the primate-rodent divergence, both possess cis-antisense transcripts,
we applied our SSRTPCR to human PCDHb15 and to the orthologous rhesus sequence. Sense and antisense
transcripts were detected in both fetal and adult human brain. However, they were also detected in adult rhesus
brain, even though rhesus brain appeared to lack PCDHy5 transcription. Therefore, expression profile
differences in brain between two paralogous protocadherin genes, PCDHy5 and PCDHb15, may exist between
human and rhesus.

We detected PCDHb15 antisense transcripts using both SuperScript II and the much more stringent tagged
primer, exonuclease I-ThermoScript RT system. Sequence comparison of PCDHy5 and PCDHb15
demonstrates that antisense-strand canonical splice sites specifying anti-PCDHy5 major isoform intron 2, as
well as the antisense-strand canonical polyadenylation signal of anti-PCDHy5, are conserved on the antisense
strand of human PCDHb15 (Fig. 4). This conservation of key genomic structure elements of the cis-antisense
transcriptional units between paralogues is consistent with our detection of PCDHb15 cis-antisense
transcription. This suggests that cis-antisense arose at the ancestral PCDHb15 locus prior to the
PCDHb15-PCDHy5 gene duplication.

Figure 4: Antisense-strand splice site (GT-AG) and polyadenylation signal (_ATAAA) conservation between
human PCDHy5 and human PCDHb15.

Figure 4: Antisense-strand splice site (GT-AG) and polyadenylation signal (_ATAAA) conservation between
human PCDHy5 and human PCDHb15. All genomic coordinates are on AC005752.1. Genomic DNA is at top.
Transcribed sequence and transcription direction are below. GT...AG text indicates introns. Not to scale. The
splice site and polyadenylation signal conservation is observed in the following one-to-one paralogous pairwise
sequence alignment context: 47590–48988 vs 41921–43330, 85% identity, 1% in gaps; 49043–49434 vs
43386–43777, 73% identity; 49528–49760 vs 43881–44113, 71% identity.

As expected from lack of sequence conservation, no murine antisense transcription could be detected in
total-body pooled RNA in the homologous Pcdhb15 region (Fig. 3C). This supports the emergence of
cis-antisense transcription at this locus after the primate/rodent divergence. Our qualitative assessment of
sense- and antisense-strand transcription of PCDHa12, PCDHb3, and PCDHb15 in the brain in all three
species, as well as PCDHy5 in human and rhesus, is summarized in Fig. 5.

Figure 5: Summary of protocadherin sense and antisense expression in human, rhesus, and mouse.

Figure 5: Summary of protocadherin sense and antisense expression in human, rhesus, and mouse.

Cis-Antisense Transcription Is Associated with Lower Levels of PCDH mRNA in Quantitative Orthologue
Expression Comparisons

Our study is the first to report protocadherin sense expression quantitation in mammalian brains. To test for a
correlation between the level of a sense transcript and the presence of its cis-antisense partner in the PCDH
endogenous antisense system, we used a Roche LightCycler to quantitatively compare the expression levels of
each sense transcript (PCDHa12, b3, and b15) across the three species (Fig. 6), taking into account the
presence or absence of cis-antisense transcription. The presence of cis-antisense transcripts was visually
associated with lower sense expression levels.

Figure 6: Quantitated expression levels of sense-strand protocadherin transcripts, as a percentage of the
ß-actin transcript levels within the same samples.

Figure 6: Quantitated expression levels of sense-strand protocadherin transcripts, as a percentage of the
ß-actin transcript levels within the same samples.

To quantify the relationship between cis-antisense incidence and sense expression levels, we considered
separately each of the three sets of gene expression measurements from paralogous gene sets (Fig. 6). The
maximum observed quantitated expression level within each set was denoted as 100%. No expression was
denoted as 0%. For each paralogous gene, the species results were separated into two subsets: those samples
from species with SSRTPCR-confirmed cis-antisense transcripts and those without evidence of any
cis-antisense expression. Mean quantitated expression levels, as percentages of the maximum, were computed
for each subset (adult and fetal expression levels for the same gene in the same species were considered
different data points). For all three gene sets, the mean expression levels in the presence of cis-antisense were
two to three times lower than the levels in the absence of cis-antisense (Table 2). This shows a strong trend
toward lower sense transcript levels in the presence of a cis-antisense moiety.

Table 2: Percentage means of PCDH sense expression levels with and without endogenous cis-antisense
Orthologous/homologous gene group.

Table 2: Percentage means of PCDH sense expression levels with and without endogenous cis-antisense
Orthologous/homologous gene group.

The 10 expression level measurements in samples showing expression of cis-antisense transcripts were further
compared to the 7 measurements taken from loci and species where cis-antisense coexpression was not
detected. The difference between the lower PCDH sense expression levels in the presence of cis-antisense and
higher sense expression levels in the absence of cis-antisense was statistically significant (t-test for two
independent samples: p= 0.038). Taken together, these results suggest an inverse relationship between
levels of cognate sense and antisense transcripts through evolution: i.e., in the presence of a cis-antisense
transcript, the level of the sense transcript is reduced.

Putative Human-Specific PCDHy5 Expression in Brain

PCDHy5 has the highest number of antisense-strand ESTs (17) of any PCDH transcript. This robust
cis-antisense transcription may account for its low level relative to that of the three PCDH genes. In addition,
our repeated attempts to detect a PCDHy5 sense transcript in rhesus adult brain both qualitatively and
quantitatively were unsuccessful (data not shown). Therefore, PCDHy5 transcription in adult brain may take
place in human but not rhesus.

Identification and Experimental Validation of PCDH Cis-Antisense Transcripts in Human and Rhesus

Despite EST evidence for PCDH cis-antisense transcription, antisense transcripts at this locus had not been
validated by orientation-specific RTPCR prior to our study. We demonstrate that cis-antisense transcription in
the brain, associated in all cases with simultaneous sense-strand transcription, occurs at four human PCDH
exons and at two of the four rhesus orthologues of those exons but does not take place at the mouse
counterparts of any of these exons.

Partial Conservation Between Human and Rhesus and Lack of Conservation Between Primate and Mouse
Cis-Antisense Transcripts at Orthologous PCDH Exons

We show that the broad framework of protocadherin sequence diversity and divergence between mammalian
species is characterized by the birth of novel cis-antisense transcripts after the primate/rodent divergence (Fig.
7). Furthermore, we demonstrate that PCDHb3 cis-antisense transcription, as well as transcription from both
strands of the PCDHy5 pseudogene, occurs in human, but not rhesus, adult brain. In the case of PCDHb3, the
conservation of the cis-antisense splice sites and polyadenylation signals between human and rhesus suggests
that the gene birth predated the human-rhesus divergence, while expression pattern differences between
human and rhesus appeared after the divergence, although the possibility that the gene birth was due to a
human-specific sequence change cannot be formally excluded.

Figure 7: Evolutionary map of mammalian PCDHa12, PCDHb3, and PCDHb15/PCDHy5 sense and
cis-antisense transcription, presented as simplified gene trees.

Figure 7: Evolutionary map of mammalian PCDHa12, PCDHb3, and PCDHb15/PCDHy5 sense and
cis-antisense transcription, presented as simplified gene trees.

Filled circles indicate species divergences.
Filled squares indicate de novo birth of cis-antisense transcripts.
Open squares indicate the loss of cis-antisense expression in brain along an evolutionary lineage.
Filled triangle indicates a tandem gene duplication.
The origin of PCDHb15/PCDHy5 cis-antisense from a single ancestral copy is inferred from the
sequence conservation shown in Fig. 4. 

Although pseudogenes and pseudogenized exons account for just 5 of 58 (9%) of the PCDHb genes and
PCDHa/g variable exons in the human PCDH gene cluster, 2 of the 8 cis-antisense transcripts (25%) overlap
the PCDH pseudogenic elements. We propose an antisense-mediated exon turnover model that can explain the
association of cis-antisense transcripts and pseudogenes. This model starts with de novo birth of a robustly
transcribed cis-antisense TU at the genomic location of an existing sense-strand PCDH exon as a chance
event. By competitive transcriptional interference with the sense strand, and/or by posttranscriptional
facilitation of sense mRNA decay, the appearance of anti-PCDH transcripts during evolution could have
decreased or eliminated the translation of the corresponding PCDH proteins. This would be an alternative to
promoter mutations as an evolutionary stratagem to attenuate the expression of paralogous genes. The initial
function of such cis-antisense transcripts, in effect, is to convert a gene into a pseudogene. The utility of the
cis-antisense thus evolved is then to suppress the useless transcription of that pseudogene. In the resulting
absence of purifying selection on the PCDH sense transcripts, mutations that made their exons pseudogenic
would accumulate. Accordingly, any synaptic connection formation patterns specified by those proteins prior to
the birth of the antisense transcripts would disappear from the set of possible patterns.

Examination of the mouse Pcdh cluster using the UCSC Genome Browser revealed antisense to the Pcdhb12
gene and to five Pcdhg variable exons: a10, a11, a12, b7, and c3. The human orthologues/closest homologues
of these mouse Pcdh exons do not match any ESTs in the antisense orientation. EST-supported human
anti-PCDH transcripts are limited to the a and b PCDH clusters, whereas mouse demonstrates cis-antisense
transcription in the Pcdhg cluster (entirely unaffected by antisense in human) as well as at Pcdhb exons whose
human equivalents lack antisense. While protocadherin cis-antisense transcripts are thus not a primate-specific
phenomenon, cis-antisense transcription in the a and b portions of the region may have first appeared in
primates and rodents respectively after the two lineages diverged.

Cis-Antisense Is Associated with Lower Sense Expression in Orthologue Comparisons

Cis-antisense transcripts have been hypothesized to bind their sense counterparts upon coexpression in the
same cell, preventing translation of the protein-coding sense mRNA. Therefore, molar excess of an antisense
RNA might be predicted to decrease the effective copy number of the sense RNA in the cell, since only
antisense transcripts will remain after most of the sense has been sequestered into RNA duplexes targeted for
degradation. Competitive transcriptional inhibition of one member of a sense-antisense pair by another might
also be expected to cause the levels of the two to be inversely related. Together, these arguments comprise the
basis of the “sense-high, antisense-low” hypothesis, stipulating that, if antisense functions by downregulating
sense, then the levels of the two should be inversely related.

Our results were mostly consistent with this hypothesis. In all three orthologue comparisons (PCDHa12,
PCDHb3, and PCDHb15), expression level of the same orthologue relative to the intraspecies ACTB standard
was higher in mouse than in human (i.e., the Pcdha12/Actb ratio in mouse was higher than the PCDHa12/ACTB
ratio in human), allowing for the possibility of sense transcript depletion in human by the primate-specific
cis-antisense. In adult brain, the lowest PCDHb3 sense expression level was seen in human, consistent with the
hypothesis that human-specific PCDHb3 cis-antisense downregulates the sense. For PCDHb15, the mouse is
the only species lacking cis-antisense at that locus. Consistent with our sense-high, antisense-low hypothesis,
the mouse exhibited the highest sense expression level. In aggregate, percentage mean expression levels in
tissues without cis-antisense were approximately two- to threefold higher than in tissues expressing
cis-antisense (p = 0.038). Altogether, our combined qualitative and quantitative PCR evidence supports an
inverse correlation between sense expression levels and the presence of antisense transcripts in the
mammalian PCDH system.

Biological Significance of PCDH Cis-Antisense Transcription

Cis-antisense transcription covering variable or alternative exons within extended combinatorial gene clusters
may be a widespread phenomenon not limited to protocadherin genes. Extensive cis-antisense transcription
over the V segments of the mouse immunoglobulin heavy chain region has been recently demonstrated
(Bolland et al. 2004). In this report, we document novel cis-antisense TUs within the conserved PCDH gene
cluster in diverged mammalian lineages and show evidence for quantitative regulation of gene expression by
cis-antisense transcripts. We also demonstrate that an antisense-mediated regulatory mechanism arose at
specific exons after the primate-rodent divergence. Such a mechanism would be consistent with species-specific
evolutionary pressures on PCDH genes (Vanhalst et al. 2001), might provide a recent parallel to the
vertebrate-specific origin of the protocadherins (Frank and Kemler 2002), and suggests a role for cis-antisense
in the regulation of synaptic plasticity in human brain (Cheng et al. 2002). In view of the potential coexpression
of sense and antisense transcripts from this locus in primate brains, as well as the apparently recent
evolutionary origin of PCDH cis-antisense transcription, the antisense transcripts merit consideration as
factors contributing to the complexity of primate brains and behaviors. Our work identifies those cis-antisense
components that can be experimentally manipulated in functional studies using transgenic models.

Acknowledgments:

This work was funded in its entirety by the Agency for Science, Technology, and Research, Republic of Singapore, through Genome Institute of Singapore budget no. GIS/03-114102. We thank Zhang Tao for fruitful discussions of SSRTPCR artifacts and the ThermoScript protocol and Sanjay Gupta and Jane Thomsen for expert assistance with Northern blotting.

Electronic Supplementary Material  Electronic Supplementary material is available for this article at http://dx.doi.org/10.1007/s00239-005-0041-3 and accessible for authorised users.

This exciting new study by Leonard Lipovich, Ravi Raj Vanisri, Say Li Kong, Chin-Yo Lin, and Edison Liu
reveals brain-specific gene expression during fetal and adult life, unique to primates, with differences between humans and non-human higher primates. The focus of this gene expression is on antisense RNA synthesis, which is found to be inversely related to sense RNA expression. This inverse relationship is consistant with a regulatory role for the newly-synthesized antisense RNA species.

1. Coudert AE, Pibouin L, Vi-Fane B, Thomas BL, Macdougall M, Choudhury A, Robert B, Sharpe PT, Berda A, and Lezot F, "Expression and regulation of the Msx1 natural antisense transcript during development".

2. Katayama S, Tomaru Y, Kasukawa T, Waki K, Nakanishi M, Nakamura M, Nishida H, Yap CC, Suzuki M, Kawai J, Suzuki H, Carninci P, Hayashizaki Y,  Wells C, Frith M, Ravasi T, Pang KC, Hallinan J, Mattick J, Hume DA, Lipovich L, Batalov S, Engström PG, Mizuno Y, Faghihi MA, Sandelin A, Chalk AM, Mottagui-Tabar S, Liang Z, Lenhard B, and Wahlestedt C,
"Antisense Transcription in the Mammalian Transcriptome".

3. Sun M, Hurst LD, Carmichael GG, and Chen J, "Evidence for a preferential targeting of 3'-UTRs by cis-encoded natural antisense transcripts".

4. Chen J, Sun M, Kent WJ, Huang X, Xie H, Wang W, Zhou G, Shi RZ, and Rowley JD,
"Over 20% of human transcripts might form sense–antisense pairs".

5. Barclay C, Li AW, Geldenhuys L, Baguma-Nibasheka M, Porter GA, Veugelers PJ, Murphy PR, and Casson AG, "Basic Fibroblast Growth Factor (FGF-2) Overexpression Is a Risk Factor for Esophageal Cancer Recurrence and Reduced Survival, which Is Ameliorated by Coexpression of the FGF-2 Antisense Gene".

6. Hovsepian JA, and Frenster JH, "Sense and Antisense during RNA Initiation of the DNA Transcription Bubble".

7. Frenster JH, and Hovsepian JA, "Ultrastructure of Euchromatin Contact Points between the Closed Loops of Adjacent Interphase Chromosomes".

Links to RNA and Biological Causality:

Links to Euchromatin Activator RNA Reviews:
Links to Euchromatin Activator RNA Research:
Links to Ultrastructural Probes of DNase I-Sensitive Sites:
Links to RNA as a Therapeutic Agent:
Links to Hodgkin Lymphoma Immuno-Pathology:
Links to Activated T-Lymphocyte Immunotherapy:
Links to Medical Systems Biology:
Links to Selective Gene Transcription:
Links to RNA-Induced Epigenetics:
Links to RNA-Induced Embryogenesis:
Links to RNA and Biological Causality:
Links to Reprogramming and Neoplasia:

"Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".

For Further Information and Feedback:
Phone:  +1 650 367 6483
E-mail: frenster@euchromatin.net