"Integrative analysis of the human cis-antisense gene pairs, miRNAs and their transcription regulation patterns".
Oleg V. Grinchuk 1, Piroon Jenjaroenpun 1, Yuriy L. Orlov 2, Jiangtao Zhou1, and Vladimir A. Kuznetsov 1, @.
1 Bioinformatics Institute, 30 Biopolis Street #07-01
and
2 Genome Institute of Singapore, 60 Biopolis Street #02-01,
Singapore 138672, Singapore
@ To whom correspondence should be addressed. Tel: +65
6478 8288;
Email: vladimirk@bii.a-star.edu.sg
Received July 10, 2009. Revised October 2, 2009. Accepted October 9, 2009.
Cis-antisense gene pairs (CASGPs) can transcribe mRNAs from
an opposite strand of a given locus. To classify and understand diverse
CASGP phenomena in the human we compiled a genome-wide catalog of CASGPs
and integrated these sequences with microarray, SAGE and miRNA data. Using
the concept of overlapping regions and clustering of SA transcripts by
chromosome coordinates, we identified up to 9000 overlapping antisense
loci. Four thousand three hundred and seventy-four of these CASGPs form
1759 complex gene architectures. We found that 35% (6347/18160) of RefSeq
genes are overlapped with the antisense transcripts. About 30% of Affymetrix
U133 microarray initial sequences map transcripts of 35% CASGPs and reveal
mostly concordant expression in CASGPs. We found strong significant overrepresentation
of human miRNA genes in loci of CASGPs. We developed a data-driven model
of cross-talk between co-expressed CASGPs and DICER1-mediated miRNA pathway
in normal spermatogenesis and in severe teratozoospermia. Specifically,
we revealed complex SA structural–functional gene module composing the
protein-coding genes, WDR6, DALRD3, NDUFAF3 and ncRNA precursors, mir-425
and mir-191, which could provide downregulation of ncRNA pathway via direct
targeting DICER1 and basonuclin 2 transcripts by mir-425 and mir-191 in
normal spermatogenesis, but this mechanism is switched off in severe teratozoospermia.
The database is available from http://globalisland.bii.a-star.edu.sg/jiangtao/sas/index3.php?link
INTRODUCTION:
A cis-antisense gene pair (CASGP) is a pair of genes mapped to opposite strands of the same locus and therefore transcribed in opposite directions. Corresponding pairs of cis-antisense transcripts are mRNAs that are at least partially complementary to each other. Cis-antisense mRNAs naturally transcribed from CASGP are known as naturally occurrding sense–antisense (SA) RNAs. Such SA transcripts (SAT) have been observed in prokaryotes, fungi, plants and animals (1–4). The overlapping of protein coding genes is a common event in prokaryotic genomes (5,6). On the other hand, up to 32% of yeast genes (3) and up to 25% of mammalian genes (1) have been estimated to form SATs. The number of CASGPs in the human genome remains the subject of considerable debate. Conservative RefSeq-based estimates and earliest counts ranged from 100 to 500 (7,8) to more than 2500 (9,10). Sophistication of computational antisense discovery tools [e.g. Serial Analysis of Gene Expression (SAGE) (4,11), cap analysis of gene expression (12) led to the ongoing growth of the cDNA and EST databases. As a result, new estimates of CASGPs have been raised by an order of magnitude to 4000–6000 (4). However, reliability, specificity and sensitivity of the computational estimates have not been well defined. In particular, Yelin et al. (10) have identified 2667 human transcripts, of which more than 1600 are predicted to be true SAT. More recently, analysis of many fully sequenced mouse cDNAs has predicted the existence of as many as 2500 distinct mammalian SATs (13). The widespread occurrence of natural SATs implies an evolutionarily advantage for this type of genome arrangement.
Natural SAT have already been found to function at several levels of molecular eukaryotic gene regulation including alternative initiation, splicing, termination (14), translational regulation (15), RNA stability, trafficking, apoptosis (4,16), genomic imprinting (17), antisense mediated silencing (18) as well as in development processes, such as X-inactivation (19) and eye development (20). Case studies showed that changes in CASGPs transcription could be implicated in pathological processes such as some cancer and neurology diseases (18,21).
The transcripts of CASGPs can share any exonic sequence, regardless of whether the exonic sequence is UTR (untranslated region) or protein-coding. However, many attributes of CASGPs (e.g. alternative transcription start sites, etc.), transcript isoforms and splice variants of the human genome still have not been well classified and studied. The reason for this is the absence of a uniform algorithm, which would integrate validated and predicted CASGPs. In particular, some groups identified SAT pairs from known mRNAs (22), other groups used predicted gene models or UniGene clusters (4). The reliability of predicted SA pairs was not validated by the sequences of well-characterized expression platforms.
In the mammalian genomes, CASGPs can be organized in complex SA gene architectures, in which at least one gene could share loci with two or more antisense partners (1,2,23). The study of these architectures could substantially contribute to our understanding of gene co-evolution and their association with genetic diseases. However, the complex SA structures in humans have not been systematically collected and studied. The publicly available search tools of SAT pairs, for example NATsDB [(4), last release on 7 September 2006] does not report the complex SA gene architectures and misses the gene pairs belonging to such natural SAT groups. For instance, only one gene pair of the complex SA architecture is reported by NATsDB; other pair(s) of such complex SA gene cluster were not reported and their graphic display is incorrect.
Eukaryotes produce various types of small RNAs, or small non-coding RNAs (sncRNAs) of 19–28 nt in length. sncRNAs can induce gene silencing through specific base pairing with the target molecules. Two relatively well-defined classes of small RNAs are involved in RNA silencing: short interfering RNAs (siRNAs) and micro-RNAs (miRNAs) (24). siRNAs and miRNAs are also involved in a wide range of functions such as cell growth and apoptosis, development, neuronal plasticity and remodeling. In cells, the long precursors of siRNAs are generated from long double-stranded RNAs, while miRNAs are generated from long single-strand hairpin-forming precursors.
Theoretically, both ncRNA precursors could be generated from the gene(s) of a CASGP. In case of siRNAs, such a possibility has been demonstrated in several case studies. The pioneer study reported about the protein-coding CASGP represented by SRO5 and P5CDH genes in Arabidopsis (25). Overlapped transcripts of SRO5 and P5CDH genes can generate endogenous siRNAs, which participate in regulation of salt tolerance. Additional evidences were found in a recent report (26): on one hand, after injection of sense and antisense transcripts in Xenopus oocytes, processing of SA transcripts into siRNAs (SAT-siRNA) was documented. On the other hand, a possibility of a switch from antisense-oriented to sense-oriented SAT-siRNAs was shown in zebrafish embryonic development.
A fine biological regulatory circuitry involving SAT-siRNAs was recently demonstrated via mechanism that has been termed ‘small RNA-induced gene activation’ (or RNAa) (27,28). RNAa targeting of a CASGP could direct the transcription activation of genes in such SA pair. It was shown that suppression of the p21 antisense non-coding RNA Bx332409 with siRNA leads to a significant suppression of this antisense transcript which correlated with significant increase in expression of p21 sense mRNA (28). However, in a case study of a non-coding–protein-coding SAT pair in human cells, an association of SAT expression regulation and Dicer-mediated pathway was not confirmed (29).
Systematic analysis of occurrence of precursors of miRNAs in transcripts of CASGPs and relationships of regulatory pathways of miRNAs genes embedded in CASGP loci has not yet been carried out. Recent findings of a large number of unique natural SATs and miRNAs in transcriptomes of different cell types of eukaryotic organisms and discovery of interconnections in regulatory network directed by natural SAT and ncRNA precursors (1,2,4,5,8,10–13,17,18,25–28,30) necessitate their comprehensive collection, accurate mapping on the genomes and appropriate analysis.
In this work, we report and imply an integrative method for computational identification of CASGPs and their complex architectures that:
(i) uses RefSeq, mRNA and EST tracks;
(ii) imposes stringent quality control filters on EST-to-genome
mapping;
(iii) provides mapping of Affymetrix U133A and U133B original target
sequences and SAGE tags on the genes of SAT pairs.
Finally, we analyse the co-localizations of SA genes with miRNA precursors.
Ultimately, the method allows identification and characterization of 9000
reliable SAT pairs found in the human genome including 1759 complex SA
gene architectures resulting in uniformly organized collection of CASGPs
stored in our new United SA Gene Pairs Pipeline (USAGP) DB. We report
about our finding of 128 miRNA-containing SA genes and describe a complex
transcription gene module of miRNA and SA genes co-localized on a same
chromosome territory. We predict the role of the miRNA-SA gene modules
in feed-back regulation of gene expression processes in development, oncogenesis
and tumor-suppression activity, and finally, we present a model of cross-talk
between co-expressed SATs and Dicer1-mediated miRNAs in silencing pathway
in normal spermatogenesis and severe teratozoospermia.
...
Figure 3. Intergroup redundancy removal procedure through step by step mapping–remapping of six groups of overlapping SAT pairs.
Figure 3. Intergroup redundancy removal procedure through step by step mapping–remapping of six groups of overlapping SAT pairs.
The group numeration at the same time corresponds to the each step (and level) of intergroup remapping procedure. Dashed arrows: if the outer level IDs perfectly matched (Supplementary Figure S1) onto the inner level IDs, they were removed from the outer level (e.g. if both IDs of a RefSeq/mRNA SAT pair from level 2 perfectly matched onto both IDs of a RefSeq/RefSeq SAT pair from level 1, they were removed from level 2).
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1. Frenster JH, and Hovsepian JA, (Dec. 2008a)
"Models of
successive levels of resolution during individual gene transcription".
2. Frenster JH, and Hovsepian JA, (Dec. 2008b)
"Micro
RNAs and adult neoplasms of embryonic type".
1. Each cell retains all of its embryonic genes for a lifetime.
2. Controls for embryonic genes are often absent in adults.
3. Uncontrolled embryonic genes can replicate wildly.
4. Replicating genes participate in intra-cellular competition.
5. The basis for gene competition is selective transcription.
6. MicroRNAs can reprogram embryomic transcription.
7. Gene reprogramming can produce normal phenotypes.
8. Normal phenotypes can by-pass chromosomal lesions.
9. MicroRNA therapy may need to be permanent.
10. Transplantation of microRNAs could be preferred.
1. Pathways within cell genomes involve a flow of information.
2. Information can flow by direct contact or by third parties.
3. Direct contact within whole genomes is difficult to regulate.
4. DNA-DNA direct contects are influenced by agents.
5. Nuclear agents include hydrophilic ionic and hydrophobic conforming ligands.
6. Third parties within genomes involve RNAs and proteins.
7. RNAs and proteins are easy to regulate or reverse.
8. Information can be shared, lost, or transformed.
9. Local information can be permanently lost during system entropy.
10. System information can be hidden during isolation.
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