Analyzing the genomic context of LncRNAs can help predict their functional role. According to the relationship between LncRNAs and their associated protein-coding genes, LncRNAs detected by Arraystar Microarray are characterized as natural antisense, intronic antisense, bidirectional, exon sense overlapping, intron-sense overlapping and intergenic. Based on these specialized classifications, we can perform LncRNA subgroup analyses such as antisense LncRNA analysis, LincRNA analysis, Hox Loci LncRNA analysis, T-UCR analysis and enhancer-like LncRNA analysis, all of which will help identify the putative functional relationship between LncRNAs and their associated protein-coding genes.
I. Natural Antisense LncRNAs
Natural antisense LncRNAs are RNA molecules that are transcribed from the antisense strand and overlap in part with well-defined spliced sense or intronless sense RNAs. Antisense LncRNAs have a tendency to undergo fewer splicing events and typically show lower abundance than sense transcripts (He, Vogelstein et al. 2008). The basal expression levels of natural antisense LncRNAs and sense mRNAs in different tissues and cell lines can be either positively or negatively regulated (Katayama, Tomaru et al. 2005; Okada, Tashiro et al. 2008). Antisense LncRNAs are frequently functional and use diverse transcriptional and post-transcriptional gene regulatory mechanisms to carry out a wide variety of biological roles. According to the different portions of corresponding sense coding genes that antisense LncRNAs overlap with, natural antisense LncRNAs-sense mRNA pairing types can be characterized as follows: Divergent or head to head (5'-5' ) overlap, convergent or tail to tail (3'-3') overlap and fully overlapping.
II. Intronic Antisense LncRNAs
Intronic antisense LncRNAs are RNA molecules that are transcribed from the antisense strand without sharing overlapping exons. Intronic antisense LncRNAs are enriched in the introns of genes and are related to regulation of transcription. Most of the Intronic antisense LncRNAs have the same tissue expression patterns as the corresponding coding genes, and may stabilize protein-coding transcripts or regulate their alternative splicing (Nakaya, Amaral et al. 2007). Intronic antisense LncRNAs are correlated with the degree of tumor differentiation in prostate cancer, which is fine tuning of gene expression in eukaryotes (Reis, Nakaya et al. 2004).
III. Bidirectional LncRNAs
A Bidirectional LncRNA is oriented head to head with a protein-coding gene within 1,000 bp. A Bidirectional LncRNA transcript exhibits a similar expression pattern to its protein-coding counterpart which suggests that they may be subject to share regulatory pressures. However, the discordant expression relationships between bidirectional LncRNAs and protein coding gene pairs have also been found, challenging the assertion that LncRNA transcription occurs solely to "open" chromatin to promote the expression of neighboring coding genes (Chakalova, Debrand et al. 2005; Struhl 2007; Mercer, Dinger et al. 2008)
IV. Exon-sense Overlapping LncRNAs
These LncRNAs can be considered transcript variants of protein-coding mRNAs, as they overlap with a known annotated gene on the same genomic strand. Most of these LncRNAs overlap 3' UTRs and are transcribed from alternative promoters within 3' UTR exons. These 3' UTR variants are usually unspliced. In contrast, LncRNAs that overlap with 5' UTRs or coding exons are usually spliced (Carninci, Kasukawa et al. 2005).
V. Intron-sense Overlapping LncRNAs
These LncRNAs overlap with the intron of annotated coding genes on the same genomic strand.
VI. LincRNAs (large intergenic noncoding RNAs) Defined by Khalil et al.
The human genome encodes 3,289 large intergenic noncoding RNAs (LincRNAs) that are clearly conserved across mammals and thus functional (Khalil, Guttman et al. 2009). LincRNAs are named according to their 3'-protein-coding genes nearby. Gene expression patterns have implicated these LincRNAs in diverse biological processes, including cell-cycle regulation, immune surveillance and embryonic stem cell pluripotency. LincRNAs collaborate with chromatin modifying proteins (PRC2, CoREST and SCMX) to regulate gene expression at specific loci. In particular, PRC2 and LincRNA complex might have the role of a transcriptional repressor by directing silencing to specific loci.
VII. LncRNAs with Enhancer-like (LncRNA-a) Function Characterized by Ulf Andersson Ørom Research Group
LncRNAs with enhancer-like function (LncRNA-a) are identified using GENCODE annotations (Harrow, Denoeud et al. 2006) of human genes. The consideration of selecting LncRNAs with enhancer-like functions exclude transcripts mapping to the exons and introns of annotated protein coding genes, the natural antisense transcripts, overlapping the protein coding genes and all known transcripts. 3,019 enhancer like LncRNAs expressed from 2,286 unique loci of the human genome are identified after filtering out all the LncRNAs overlapping with protein coding genes and their promoters, antisense LncRNAs, as well as known LncRNAs. LncRNAs with enhancer like function and the LincRNAs show that about 13% of LncRNAs with enhancer-like function overlap with human LincRNAs identified by Khalil et al. The average size of these non-coding transcripts is about 800 nt with a range from 100 nts to 900 nts. They display a simpler transcription unit than that of protein-coding genes. Nearly 50% of these LncRNAs contain a single intron in the primary transcripts. The majority of them show evidence of polyadenylation. They are expressed and respond to cellular differentiating signals and function to enhance gene expression. It is temping to speculate that many of these LncRNAs and their promoters may correspond to mammalian enhancers or polycomb/trithrax response elements (PRE/TRE). In such a scenario, binding of polycomb or trithorax proteins to proximal promoters on these LncRNAs will regulate their expression which in turn will impact the expression of the protein-coding genes at a distance.
VIII. Hox Loci LncRNAs (Hox ncRNAs)
Rinn et al. characterized the transcriptional landscape of the four human Hox loci and identified a total of 407 discrete transcribed regions in the four Hox loci (Rinn, Kertesz et al. 2007). By using current genome annotations, they partition them into known Hox gene exons (101), introns (75) and intergenic transcripts (231). They found that most of these intergenic transcripts did not show any coding potential in all six transcriptional frames. These intergenic transcripts are referred as Hox ncRNAs. Like canonical Hox genes, Hox ncRNAs also systematically vary their expression along developmental axes of the body in a manner coordinated with the physical location on the chromosome. Systematic comparison of the expression pattern of every ncRNA with its immediate 5' and 3' Hox gene neighbor showed that the vast majority of ncRNAs (90%) are coordinately induced with their 3' Hox genes, while only 10% of instances are ncRNA expression anticorrelated with 3' Hox gene expression (Rinn, Kertesz et al. 2007). LncRNAs in the Hox loci become systematically dysregulated during breast cancer progression(Gupta, Shah et al. 2010). A distinct set of Hox LncRNAs are sometimes overexpressed in primary breast tumors, and very frequently overexpressed in metastases. Notably, a LncRNA termed HOTAIR is increased in expression level in primary breast tumors and metastases, and HOTAIR expression level in primary tumors is a powerful predictor of eventual metastasis and death.
IX. Ultraconserved Regions Encoding LncRNAs (T-UCRs)
The UCRs are a subset of conserved sequences that are located in both intra- and intergenic regions. They are absolutely conserved (100%) between orthologous regions of the human, rat and mouse genomes (Bejerano, Pheasant et al. 2004). The UCRs are frequently located at fragile sites and genomic regions involved in cancers. A large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs (T-UCRs) whose expression is altered in human cancers. Genome-wide profiling revealed that T-UCRs have distinct signature in human leukemia and carcinomas. The expression of T-UCRs may be regulated by microRNAs abnormally expressed in human chronic lymphooytic leukemia (Calin, Liu et al. 2007)
X. LncRNAs from NRED
The Non-coding RNA Expression Database (NRED) provides gene expression information for thousands of long ncRNAs in human and mouse. The database contains both microarray and in situ hybridization data. Using publicly available data from the Genomics Institute of the Novartis Research Foundation (GNF), Dinger et al. identified 1287 human organ- and cell specific expression LncRNAs, which are LncRNAs from NRED.
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