Abstract

Non-coding RNAs (ncRNAs) are increasingly being implicated in diverse functional roles. Majority of these ncRNAs have their origin in the repetitive elements of genome. Significantly, increase in genomic complexity has been correlated with increase in repetitive content of the genome. Primate-specific Alu repeats, belonging to SINE class of repeats, is the most abundant repeat class inhabiting the human genome. Of the many possible functional roles of Alu repeats, they have been shown to modulate human transcriptome by virtue of harboring diverse array of functional RNA pol II TFBS, cryptic splice-site-mediated Alu exonization and as probable miRNA targets. Retro-transposition of Alu harboring TFBS has shaped up gene-specific regulatory networks. Alu exonized transcripts are raw material for dsRNA-mediated A–I editing leading to nuclear retention of transcripts and change in miRNA target. miRNA targets within Alu may titrate the effective miRNA or transcript concentration, thus acting as ‘miRNA sponge’. Differential levels of Alu RNA during different conditions of stress also await clear functional understanding. These have contributed toward evolution of complex regulatory repertoire leading to the evolution of primate-specific functions. Recent reports of co-localization of pol II and pol III binding sites near the gene and elsewhere in the genome, increase the possibility of dynamic co-ordination between both pol II and pol III determining the ultimate transcriptional outcome. Dynamic and functional Alu repeats seem to be centrally placed to modulate the transcriptional landscape of human genome.

Non-coding DNA and Genome Complexity

The proponents of molecular biology have always gone to great lengths to emphasize on what we know today as the Central Dogma. It precisely holds that for any biological expression to occur at the genotypic or phenotypic level, a gene must transcribe into an mRNA and this must then go on to make a protein. This has usually been interpreted to mean that genetic information flows from DNA to proteins via RNA, where RNA is a mere intermediate in the information flow cascade. However, only 2% of our genes functionally code for proteins; ∼50–75% of human genome is transcribed, and 98% of the transcripts do not get translated [1, 2]. Further, genomic complexity neither correlates with chromosome number (popularly known as K value paradox), genome size (C value paradox) or the number of genes (N value paradox) [3]. This was confirmed after human genome sequencing which revealed that humans have only approximately 21 500 protein-coding genes which are comparable to lesser complex genomes of Drosophila and Arabidopsis [4]. In contrast, non-coding component of the genome has increased substantially with genome complexity. These regions have assumed importance in recent times as they seem to hold a large number of cues that might explain the observed complexity in humans [5, 6]. This makes us sit back and question some of our good old notions of biology.

ncRNAs have no apparent ORF for translation, and it include ‘housekeeping’ ncRNAs [such as transfer RNAs (tRNAs), ribosomal RNAs (rRNAs) and small nuclear RNAs (snRNAs)], small/short ncRNAs (miRNA and piRNA), repetitive element-associated ncRNAs (Alu RNA) and long ncRNAs (lncRNAs). ncRNAs are transcribed by different RNA polymerase complexes, e.g. Alu is transcribed by pol III whereas miRNAs, snoRNAs, some snRNAs and some scRNAs are mostly transcribed by pol II [7–9]. A large class of ncRNAs like 7SL [10], 7SK [11] and miRNAs [12] are highly conserved across species and are being implicated in many genome functions [13]. Although conserved cis-regulatory elements in human genome like proximal promoter elements, enhancers, silencers and insulators comprise a larger fraction of the regulatory repertoire [14]. ncRNA can assume dynamic secondary structures and affect various cellular functions like splicing, editing, silencing through antisense, methylation and nuclear retention of transcripts [15–17]. They can also be a source of miRNA biogenesis and miRNA target. These RNAs govern diverse aspects of development, cellular differentiation and external/internal stimuli driven spatio-temporal expression [18–22]. They are also known to regulate transcription factors (TFs) as co-activators or co-repressors [23–26]. Different tiers of regulation across phyla from bacteria to humans can be regulated through variety of ncRNAs like cis/trans antisense RNA and transcribed pseudogene (Table 1). The ability of some RNAs to function as ncRNA or mRNA/protein depending on the cellular state has added a new dimension to the already rich repertoire of functions transacted by RNAs (Table 2). Different class of ncRNAs has also been correlated with many disease states like cancer, neurological and congenital diseases [27] as enumerated in Table 3.

Table 1:

Expanding functional landscape of ncRNAs

Organism ncRNA Function 
Xenopus laevis Xlsirt Anchors RNAs in vegetal cortex for normal embryonic development [28]. 
Bacteria Antisense RNA Affect bacterial virulence through its 3′-UTR, 5′-UTR, cis- and trans-antisense RNAs [29]. 
Drosophila melanogaster Hsr-ω Involved in intranuclear trafficking and availability of hnRNPs [30]. 
Sat III-induced ncRNAs Nuclear stress granules (nSGs), harbors Sat III repeat-induced ncRNAs, HSF1, splicing factors and hnRNPs which modulate chromatin structure [31]. 
Humans NRON Represses NFAT, a TF essential for T-cell receptor-mediated immune response [25]. 
Transcribed pseudogenes Act as decoy for functional mRNAs, involved in sequestering TFs and miRNAs [32]. 
Organism ncRNA Function 
Xenopus laevis Xlsirt Anchors RNAs in vegetal cortex for normal embryonic development [28]. 
Bacteria Antisense RNA Affect bacterial virulence through its 3′-UTR, 5′-UTR, cis- and trans-antisense RNAs [29]. 
Drosophila melanogaster Hsr-ω Involved in intranuclear trafficking and availability of hnRNPs [30]. 
Sat III-induced ncRNAs Nuclear stress granules (nSGs), harbors Sat III repeat-induced ncRNAs, HSF1, splicing factors and hnRNPs which modulate chromatin structure [31]. 
Humans NRON Represses NFAT, a TF essential for T-cell receptor-mediated immune response [25]. 
Transcribed pseudogenes Act as decoy for functional mRNAs, involved in sequestering TFs and miRNAs [32]. 

ncRNAs have been part of the transcriptome regulatory network across diverse species right from bacteria to humans. Different type of regulatory RNAs like ncRNA, antisense RNA and transcribed pseudogenes affect different functions from development to decoy for TFs or miRNAs.

Table 2:

RNA with dual functions

Organism Function
 
 ncRNA Protein/mRNA 
Bacteria SgrS 3′ region mediates mRNA degradation and translational repression, during sugar phosphate stress [33]. SgrT (43 aa polypeptide) 5′ region inhibits glucose transport [34]. 
Drosophila melanogaster oskar during oogenesis is required for development [35]. Functions as mRNA during embryonic stage [35]. 
Human SRA co-activates nuclear receptors like estrogen, progesterone and androgen [36]. SRAP interacts with SRA to inhibit myogenic differentiation through MyoD [37]. 
p53 and Mdm2 functional interplay regulates p53 synthesis [38]. p53 and Mdm2 also regulate p53 ubiquitination by proteasome [38]. 
Organism Function
 
 ncRNA Protein/mRNA 
Bacteria SgrS 3′ region mediates mRNA degradation and translational repression, during sugar phosphate stress [33]. SgrT (43 aa polypeptide) 5′ region inhibits glucose transport [34]. 
Drosophila melanogaster oskar during oogenesis is required for development [35]. Functions as mRNA during embryonic stage [35]. 
Human SRA co-activates nuclear receptors like estrogen, progesterone and androgen [36]. SRAP interacts with SRA to inhibit myogenic differentiation through MyoD [37]. 
p53 and Mdm2 functional interplay regulates p53 synthesis [38]. p53 and Mdm2 also regulate p53 ubiquitination by proteasome [38]. 

ncRNAs also display duality of function. In addition to its role as ncRNA, they also modulate cellular functions either as protein or mRNA.

Table 3:

Dark side of ncRNA

Disease ncRNAs 
In cancer  
    Breast cancer SRA [39], H19 [40], BC200 [41], miR-155 [42] and miR-21 [43]. 
    Prostate cancer DD3 [44] and PCGEM1 [45]. 
    Colon cancer OCC1 [46]. 
In neurological disorders  
    Alzheimer's BC200 [47]. 
    Schizophrenia and bipolar disorder DISC2 [48]. 
    Spinocerebellar ataxia type 8 SCA8 (KLHL1 antisense) [49]. 
In congenital syndromes  
    Autistic disorder RAY1/ ST7 [50]. 
    Prader–Willi syndrome ZNF127AS [51] and IPW [52]. 
    DiGeorge syndrome DGCR5 [53]. 
    Psoriasis PRINS [54]. 
Disease ncRNAs 
In cancer  
    Breast cancer SRA [39], H19 [40], BC200 [41], miR-155 [42] and miR-21 [43]. 
    Prostate cancer DD3 [44] and PCGEM1 [45]. 
    Colon cancer OCC1 [46]. 
In neurological disorders  
    Alzheimer's BC200 [47]. 
    Schizophrenia and bipolar disorder DISC2 [48]. 
    Spinocerebellar ataxia type 8 SCA8 (KLHL1 antisense) [49]. 
In congenital syndromes  
    Autistic disorder RAY1/ ST7 [50]. 
    Prader–Willi syndrome ZNF127AS [51] and IPW [52]. 
    DiGeorge syndrome DGCR5 [53]. 
    Psoriasis PRINS [54]. 

Differential levels of ncRNA have also been functionally correlated with disease states like cancer, neurological disorders, congenital syndromes and developmental disorders.

RNA Regulation: Economic and Efficient choice

Cells can benefit by using RNA molecules for regulation in diverse ways.

  • Through its increased regulatory repertoire: The repertoire of RNA is enriched through its multi-dimensional roles based on its location in 5′UTR, 3′UTR, intronic and intergenic regions. RNAs also have unique ability to fold in three-dimensional space and hybridize in a sequence-specific manner to other nucleic acids. dsRNAs act as substrates for AI editing and can form sense–antisense pairs.

  • Through transient expression: Regulation by RNA is more transient as they can be sequestered based on requirement or titrated through mechanisms such as ‘miRNA sponges’ and sense–antisense pairs.

  • At post-transcriptional level: Regulatory RNA can also act at post-transcriptional level, through miRNAs and nonsense-mediated decay (NMD).

  • In different cellular compartments: These RNAs can mediate differential transport of molecules thereby modulating downstream functions.

Some of these functions like increased regulatory repertoire by RNA mediated regulation are exclusive to RNA whereas other functions are also shared by the proteins. Thus it is remarkable that RNA in a cell has more than one way to achieve a single objective and provides choice for more than one way of differential regulation [29]. RNAs are proposed to modulate networks that process discrete information packets to yield continuously varying responses, thus functioning like digital-to-analog converters, allowing the expansion of complexity in biological systems, well beyond the scope of purely protein-based regulatory networks [55]. One class of ncRNA that almost reflects all the characteristic features displayed by ncRNA community as a whole, are primate-specific Alu repeats. As highlighted in Figure 1, transposons constitute ∼45% of the human sequences and are comprised of SINE elements, LINEs, long terminal repeats (LTRs), DNA transposons, MIR repeats, etc. Among the SINEs, Alus are the predominant fraction and occupy nearly 11% of the human genome. Moreover, compared to other repeats, Alu transcripts are also the most abundant. Alu density is enriched in intragenic regions (12.5%) compared to intergenic density of 9.6% and nearly 75% of the genes having at least one or more Alu insertion. Within genes, Alus occupy 12.8% intronic and 1.6% exonic regions. Alu repeats were also observed to be significantly enriched in genes belonging to specific functional categories like metabolism, transport and signaling whereas they are sparse in genes coding structural proteins and information pathways [56].

Figure 1:

Preferential transcription of Alu contrary to its genomic coverage. Broadly, genome is made up of ∼45% TEs and ∼55% non-repetitive elements. Transposons are constituted mainly of Alus, LINEs, LTR, DNA transposons and MIR repeats. Approximate percentage contribution of each class has been mentioned in the schematic diagram. Interestingly, of the total repeat content, Alus occupy ∼22% of genomic space but contribute ∼35% of the transcriptomic pool of repetitive elements.

Figure 1:

Preferential transcription of Alu contrary to its genomic coverage. Broadly, genome is made up of ∼45% TEs and ∼55% non-repetitive elements. Transposons are constituted mainly of Alus, LINEs, LTR, DNA transposons and MIR repeats. Approximate percentage contribution of each class has been mentioned in the schematic diagram. Interestingly, of the total repeat content, Alus occupy ∼22% of genomic space but contribute ∼35% of the transcriptomic pool of repetitive elements.

Genomic organization of Alus

The consensus sequence of Alu is comprised of ∼280 bp, formed by head to tail dimerisation of two similar but non-identical monomers joined by variable length of functional poly-A and a polymorphic poly-A tail at the 3′ end [57]. The two monomers are called as FRAM and FLAM, indicate the fossil right arm monomer and fossil left arm monomer, respectively. These monomers, in turn, have arisen from a 7SL RNA progenitor through a deletion of 141 nt [57]. The FLAM contains functional bipartite RNA pol III promoter comprising of Box A and B elements [58]. It also contains binding sites for many RNA pol II TFs. A schematic representation of consensus Alu sequence in Figure 2 summarizes the structure of Alu. Alu harbors a total of 23 cryptic splice sites in both 5′ and 3′ orientation. Unlike other pol III promoters, B-box promoter element is capable of initiating weak transcription on its own and also acts as a transcriptional enhancer [59].

Figure 2:

Basic structure of an Alu repeat showing different sequence features. Alu repeat is characterized by the FLAM and FRAM being joined by poly-A and having a poly-A tail at the 3′ end. In addition to Alu being transcribed by nearby pol III promoter of a LINE element, it also harbors RNA pol III binding site in its B-box and a terminator sequence at variable distance from FRAM. The functional repertoire is enhanced by the presence of RNA pol II binding sites within Alu like that for RARE, Vit. D, p53, etc. Asterisks show Transcription Factor Binding Sites (TFBS).

Figure 2:

Basic structure of an Alu repeat showing different sequence features. Alu repeat is characterized by the FLAM and FRAM being joined by poly-A and having a poly-A tail at the 3′ end. In addition to Alu being transcribed by nearby pol III promoter of a LINE element, it also harbors RNA pol III binding site in its B-box and a terminator sequence at variable distance from FRAM. The functional repertoire is enhanced by the presence of RNA pol II binding sites within Alu like that for RARE, Vit. D, p53, etc. Asterisks show Transcription Factor Binding Sites (TFBS).

The fusion of two 7SL RNAs happened ∼65 million years ago through retro-transposition via a single-stranded RNA intermediate transcribed by internal RNA pol III promoter [60]. Importantly, Alus are the only active SINE in the human genome capable of retro-transposition via younger Alu subfamilies. According to recent estimates, Alus retropose with a rate of approximately 1 integration every 20 births [61]. During expansion of Alu, it is speculated that the master gene acquired mutations which gave rise to Alu subfamilies, Alu J (oldest), Alu S (intermediate) and Alu Y (young), which share ∼80–85% similarity among them but can be identified independently by nucleotides at diagnostic positions [62]. Maximum amplification rate was observed about 30 million years ago, the time when Alu Sx subfamily appeared, leading to their highest representation in the genome [63]. A phylogenetic tree of Alu subfamilies is depicted in Figure 3 based on repeats catalogued in Repbase (15.07 release). Of the 1.18 × 106 copies of Alu (hg18) in the human genome, the ancestral form, i.e. fossil arm monomer (FAM) has 52 097 copies, Alu J has 310 562 copies, Alu S has 682 137 copies and the youngest retro-transpositionally active Alu Y has 142 089 copies (to calculate the exact number of copies, alternate genome assemblies and unplaced contigs have been excluded from the analysis). The percentage contribution of Alu in human genome is higher among closely related primates (Table 4). Depending on the location of Alu in the context of gene-like intergenic, exon, intron, promoter enhancer region or untranslated regions, it can affect functions at various hierarchies. When present in the:

  • Intergenic regions, Alus can mediate nonhomologous recombination leading to genome shuffling, affect nucleosome positioning/exclusion and chromatin remodeling [64–66], and alter methylation status/imprinting [67–69]. Alu nucleosomes are proposed to serve as ‘anchors’ in organizing the chromatin in human cells. Alus are generally hypermethylated whereas during disease states like cancer and Alzheimer, they are hypomethylated. Methylation status of Alu has the potential to be used as prognostic marker to evaluate progression of cancer.

  • Promoter region, may serve as TFBS or enhancers/repressor and thus regulating gene expression [70–73]. Alus have been the hub of host of TFs (discussed in detail in ‘Alus and TFBS’ section). Depending upon the position of Alu in the promoter region, they either act as enhancer or repressor.

  • Intronic region, affect transcriptional activity, produce alternate splice variants through its exonization and A–I edited transcripts [74–79]. Alu exons derived from intronic splicing of Alu are proposed to be one of the basic ingredients for evolution as they are known to regulate the human genes and vary in expression across different tissues.

  • Exons, can disrupt reading frame leading to truncated protein or loss of gene activity (pseudogene formation), genetic disorders or evolution of new functions [80–82]. Exons derived from Alu elements have also been associated with disease states in 40 cases. Alu insertion leads to human-specific transcript variants as well as disease like ‘Menkes’ due to insertion induced mutation.

  • UTR can affect alternate transcript isoforms in a tissue-specific manner and provide binding site for miRNAs [56, 78, 83, 84]. Alternative transcripts mediated by Alu exonization also show lineage-specific evolutionary events during primate evolution.

Additionally, intra- and inter-chromosomal recombination events mediated by Alu retro-transposition or Alu–Alu non-homologous recombination leads to chromosomal alteration, deletions and duplications (Table 5) and in some cases has resulted in genetic diseases [95, 96]. A summary of the regulatory role of Alu by virtue of its presence at different levels of genomic regulation namely, DNA, RNA and protein level has been summarized in Figure 4. The dynamic hub of pol II TFBS and pol III promoter within Alus make it an interesting player which modulates both pol II and pol III mediated transcriptional responses. Through recombination events, Alus are dispersed in a non-random manner in the human genome. By virtue of these characteristics, Alus harboring regulatory sites could have shaped novel regulatory networks [56, 72]. The possible non-coding regulatory networks in which Alus could be involved extensively are discussed below.

Figure 3:

The phylogenetic tree of various subfamilies of Alu. Briefly, the 34 Alu subfamilies consensus sequence were obtained from Repbase (15.07 release) and were aligned in multiple-sequence alignment mode using MUltiple Sequence Comparison by Log-Expectation (MUSCLE). It was analyzed using RepeatMasker (version 3.2.7, January 2009). As per count of Alu subfamilies from hg18, FAM is ∼4.4%, Alu J is 26.2%, Alu S is 57.5% and Alu Y is ∼12% of the total number of Alus. The intensity of the outer color band is indicative of the Alu subfamily age, newer the subfamily, darker is the color.

Figure 3:

The phylogenetic tree of various subfamilies of Alu. Briefly, the 34 Alu subfamilies consensus sequence were obtained from Repbase (15.07 release) and were aligned in multiple-sequence alignment mode using MUltiple Sequence Comparison by Log-Expectation (MUSCLE). It was analyzed using RepeatMasker (version 3.2.7, January 2009). As per count of Alu subfamilies from hg18, FAM is ∼4.4%, Alu J is 26.2%, Alu S is 57.5% and Alu Y is ∼12% of the total number of Alus. The intensity of the outer color band is indicative of the Alu subfamily age, newer the subfamily, darker is the color.

Figure 4:

Regulatory repertoire of Alu shaped by its preferential and non-random genomic distribution. All the four-tiers of regulation, namely, genome rearrangement/shuffling by Alu mediated non-homologous recombination, expression by chromatin modulation through Alu nucleosome, transcriptome through Alu-mediated transcript diversity and finally at the proteome level by Alu exons. All of this contributes and co-ordinate with each other to modulate human genomic diversity.

Figure 4:

Regulatory repertoire of Alu shaped by its preferential and non-random genomic distribution. All the four-tiers of regulation, namely, genome rearrangement/shuffling by Alu mediated non-homologous recombination, expression by chromatin modulation through Alu nucleosome, transcriptome through Alu-mediated transcript diversity and finally at the proteome level by Alu exons. All of this contributes and co-ordinate with each other to modulate human genomic diversity.

Table 4:

Genomic coverage of Alu in other primates

Primates Genome size (bps) Number of Alu (copies) Genomic coverage of Alu (bps) Percentage of genome 
Human (hg18) 3.08 × 109 11 93 407 3.10 × 108 10.08 
Chimpanzee (panTro2) 3.35 × 109 11 96 587 3.05 × 108 9.09 
Rhesus (rheMac2) 3.01 × 109 11 38 432 2.89 × 108 9.6 
Primates Genome size (bps) Number of Alu (copies) Genomic coverage of Alu (bps) Percentage of genome 
Human (hg18) 3.08 × 109 11 93 407 3.10 × 108 10.08 
Chimpanzee (panTro2) 3.35 × 109 11 96 587 3.05 × 108 9.09 
Rhesus (rheMac2) 3.01 × 109 11 38 432 2.89 × 108 9.6 

A brief summary of the percentage contribution of Alu repeats in closely related primate genomes. The percentage of Alu is higher in humans and importantly younger Alu subfamilies are still undergoing retro-transposition. The version used for calculations are mentioned in the bracket.

Table 5:

Alu shapes genomic landscape

Alu Function 
Alu insertion polymorphism Human population diversity marker [85–87]. 
Population identification [88]. 
DNA fingerprinting [89]. 
Alu recombination DNA methylation status [69]. 
Canonical polyadenylation signal [90]. 
Positioning of nucleosomes in chromatin [64, 91]. 
Segmental duplication and CNV [92]. 
Sequence-specific interaction with nuclear proteins [93]. 
Alu methylation Tissue-specific pattern of methylation. Especially, it seems to have shaped methylome of human cerebellum [94]. 
Alu Function 
Alu insertion polymorphism Human population diversity marker [85–87]. 
Population identification [88]. 
DNA fingerprinting [89]. 
Alu recombination DNA methylation status [69]. 
Canonical polyadenylation signal [90]. 
Positioning of nucleosomes in chromatin [64, 91]. 
Segmental duplication and CNV [92]. 
Sequence-specific interaction with nuclear proteins [93]. 
Alu methylation Tissue-specific pattern of methylation. Especially, it seems to have shaped methylome of human cerebellum [94]. 

Alu elements drive genomic rearrangements by virtue of insertion and recombination events. For example recombination-mediated DNA methylation imparts tissue-specific pattern of methylation which may shape the transcriptom in particular tissues.

Alus and TFBS

Older Alu subfamilies, with passage of time, have accumulated mutations that have led to creation of TFBS for many nuclear receptors. This has resulted in widespread distribution of RNA pol II regulatory sites from these RNA pol III dependant elements [72]. The plasticity or specificity has also been contributed by different Alu subfamilies, with an important role of variable midA-stretch joining the two monomers. Evidences have demonstrated that Alu elements have evolved to harbor hormone response elements (HRE) that overlap with the internal promoter of the Alu. These several hexameric sequences are related to a consensus binding site, AGGTCA, recognizable by members of the nuclear receptor super-family of ligand-activated TFs, including receptors for retinoic acid (RAR) [97], thyroid hormone (T3) (THR) [98], vitamin D (VDR) [99], progesterone [71], estrogen [100], gluco-corticoid [101], growth hormone [102], steroids and a number of orphan receptors. It has been recently shown that ∼90% of retinoic acid response element (RARE) sites reside within Alu and 95.5% of these are present within Alu S subfamily [103]. Many of the putative binding sites within Alu correspond to TFs associated with early markers of development processes [104]. Alus also harbor many orphan receptors like liver X receptors (LXRs) or PPARα, which are members of nuclear receptor super-family that play important role in functions like lipid homeostasis and regulation of pro-inflammatory genes, respectively [105, 106].

In recent times, there have been interesting reports highlighting the role of TFBS within Alu in modulating immune response. Human cathelicidin antimicrobial peptide (CAMP) gene is the direct target of Vitamin D receptor and is strongly up-regulated in response to 1, 25-dihydroxyvitamin D3. The binding site (BS) for vitamin D receptor is present within the Alu repeat present upstream of the gene [99]. This BS shows primate-specific purifying selection of the exapted Alu exclusively in human and primate lineages. Evolutionary selection to place the CAMP gene under regulation of vitamin D pathway in primates potentiates the innate immune response and may counter the anti-inflammatory properties of vitamin D [107]. It also helps to emphasize the point that presence of TFBS within Alu is a non-random phenomenon as it provides functional advantage to the host and is under positive selection. Exaptation of Alu as an additional promoter for human neuronal apoptosis inhibitory protein (NAIP) gene has led to a novel protein isoform of NAIP [108]. Recent studies have also highlighted that p53 binding site within Alu are generated by methylation and de-amination of CpGs on the genomic scale [70, 109]. It has been recently shown that an ankyrin-repeat containing protein p200 from an endoparasite Ehrlichia chaffeensis translocates to host nucleus and binds to Alu Sx elements in promoter and intronic regions of genes. These are enriched in biological processes such as transcription, apoptosis, ATPase activity and structural proteins associated with the nucleus and membrane-bound organelles [110]. Similarly, origin of NF-kB BS from Alu families in the proximal promoter region of the IFN-λ1 regulatory region also emphasize the role of Alu in innate immune response, in response to bacterial lipo poly-saccharide (LPS) membrane [111].

Transcribed Alu: Alu RNA

During normal cellular state, Alu RNAs accumulate at very low levels 103–104 molecules/cell but there is transient increase of nearly 20-fold in the levels of Alu RNA during stress, viral infection and cancer [112, 113]. Such pol III transcribed Alu RNA are referred to as ‘free Alu RNAs’. Alu is also transcribed as part of pol II transcription units of protein- and non-protein coding RNAs, termed as ‘embedded Alu RNAs’. This is a by-product of integration of Alu into various locations in the human genome [114]. As mentioned earlier, transcribed Alus contribute nearly 35% (Figure 1) of transcribed repetitive elements but an estimation of exact proportion of Alus has not been determined yet. It is compounded by the fact that due to the repetitive nature of Alu, it is not possible to map the transcribed free Alu RNA although embedded Alu RNAs can be mapped back to their genomic location. Free Alu RNA and embedded Alu RNA are regulated by different set of transcription rules governing pol III and pol II promoters. Pol III promoter element in older subfamilies have accumulated mutations and hence have reduced capability of driving transcription but in the process have gained pol II regulatory signals. Whereas, though newer subfamilies have less of pol II regulatory sites, their pol III promoters are in functional state [72]. Thus, older subfamilies would be transcribed more as embedded Alu RNA and newer subfamily as free Alu RNA. Most of the embedded Alus are preferentially present in the 3′UTR region of transcripts. Both free and embedded Alus play an important role in transcription and translational modulation. Alu RNA modulates functions like protein translation, alternative splicing and A–I editing. During heat shock stress, elevated Alu RNA acts as transcriptional co-repressor though direct interaction with RNA pol II in the promoter regions of heat shock responsive genes [115]. Conversely, the level of Alu RNA is down-regulated in human macrophages infected with Leishmania, a parasitic protozoan, during its initial stage of infection. The down-regulation is achieved through degradation of TFIIIC110, which is as essential component of RNA pol III mediated transcription of Alu via B-box promoter element [116]. It is possible that one of the purposes of knocking down of ncRNA gene expression like Alu RNA and 7SL RNA in macrophages would be to alleviate ncRNA mediated transcriptional arrest in these cells so that the parasites can establish infection as well as proliferate. Alu RNAs assemble with cellular proteins into ribonucleoprotein complexes and can be processed into the smaller scAlu RNAs [117]. The potential of Alu RNA as translational modulator through PKR (protein Kinase activated by RNA)-dependent mechanism has also been shown [118].

It is also equally plausible that ncRNA transcription in general and Alu transcription in particular could be non-functional. The skepticism about the functionality of pervasive transcription, mainly contributed by ncRNA has led to studies trying to understand the veracity of this phenomenon. These studies conclude that most ‘dark matter’ transcripts have low abundance than the known exons and that the genome is not as pervasively transcribed as widely perceived [119]. There has also been vigorous debate about what constitutes evidence for functional role of thousands of ncRNA loci which are located outside the protein-coding genes [120]. Possibly, this can only be answered through cellular phenotypes arising out of disruption of non-coding loci.

Alu Exonization and A–I Editing

In addition to the transcriptional landscape shaped by TFBS within Alu, cellular milieu is also replete with other important genomic variables shaped by Alu which could contribute to transcriptome diversity. Alu consensus sequence harbor 9 potential 5′ and 14 potential 3′ splice site, with most of them being present in the minus strand [56, 114, 121, 122]. This could potentiate Alu exonization leading to creation of novel splice isoforms. This phenomenon termed as ‘Aluternative splicing’, exhibits tissue-specific expression patterns [78, 123]. An estimate says that ∼5.2% of all identified alternative exons is derived from Alu elements. Alu exonization also generates new exons with altered mRNA translational efficiency. This could have global effects in primates through altered regulation of protein production of molecules, such as master transcriptional regulators in specific lineages [124]. Two Alu RNAs when present in a head to tail orientation in exonized transcripts are also substrate for AI editing mediated by adenosine deaminase that specifically target double-stranded RNAs (ADAR) [125]. This process of RNA editing involves co-transcriptional or post-transcriptional modification of RNA, most prevalent being the hydrolytic deamination of adenosine (A) to inosine (I). >90% of all A–I editing occurs within Alus with ‘A’ at positions 27, 28, 136 and 162 being more susceptible to such editing, compared to others [126]. Primates have high level of such editing occurring in their genomes. This opens up the avenue for speculating that appearance of Alu in the primate lineage led to such conspicuous editing, paving the path for primate evolution [127]. Inosine pairs with cytosine, not uracil, thereby creating novel secondary structure of RNA. Enhanced editing levels of Alus have been observed in human brain. This may add to the known significant enrichment of edited transcripts in the brain and their involvement in regulation of brain-specific transcripts [128]. Association of edited Alus with neuronal functions hints at the possible contribution of AI edited transcripts in the development of higher brain function. Alu editing may serve as an alternate information mechanism based on the binary A/I code [129]. Knockdown experiments of ADAR1 have shown high editing levels in non-coding sites of Alu in hESCs. This reverses to global decrease in editing levels during differentiation, particularly into the neural lineage. This highlights the role of AI editing in development [130].

Alus and NATs

NATs are endogenous RNA molecules containing sequences that are complementary to other transcripts. NATs primarily belong to two subcategories, cis-NATs, which are transcribed from opposing DNA strands at the same genomic locus, and trans-NATs, which are transcribed from separate loci. NATs reflect pervasive nature of transcription from bacteria to human, which suggests that they are a fundamental component of gene regulation [131, 132]. In human, ∼22% of transcripts form cis senseantisense pairs [133] and trans-NATs comprise ∼4% of the transcriptional units [134]. The size of NATs is variable ranging from a few bases to several kilobases [135]. Sense–antisense transcripts tend to be co-expressed or inversely expressed more frequently than would be expected by chance [136–138]. The humongous levels of NAT are also facilitated by (i) short introns in evolutionarily conserved antisense genes [139] and (ii) transcription of pseudogenes [140].

Genome-wide computation study shows that transcriptional start sites (TSSs) of NATs are overtly present within transposable elements (TEs) with as many as 48 718 human gene antisense TSS within TEs [141]. Interestingly, such TSSs are mostly present within 3′ ends of the genes which are also most favored sites for Alu integration. Although it lacks experimental evidence yet it is tempting to speculate that relative excess of antisense transcripts initiated from TEs and their enrichment closer to the 3′ ends of TUs might be contributed, in part by Alus. This may yield cis-NATs with biologically significant regulatory activities as Alus harbor TFBS which may initiate transcription. Such circumstantial evidence awaits experimental validation at the genomic scale but evidence of human-specific antisense transcripts like RNF144A, SYNE2 and CAMCK4 induced by insertion of TEs is an indication in the right direction. Such NATs may have played a role in acquisition of human-specific traits [142]. Abundance of Alu in the human transcriptome especially in the 3′UTR of genes and Alus ability to promote transcription potentiates its involvement in NAT mediated regulation [141]. Besides, Alus as part of antisense transcriptome has already been reported. Antisense Alus belong to older subfamily and show significantly greater sequence divergence from their consensus sequence than Alus that do not co-locate with TSS of antisense transcripts [141].

NAT may mediate regulation through transcriptional interference, RNA masking, dsRNA-dependent mechanisms and RNA interference, and antisense-mediated methylation and mono-allelic expression. One way by which cis-NAT can mediate regulation could be through transcriptional interference due to collision between two bulky RNA pol II complexes transcribing from opposite strands [143]. The other mechanisms could involve RNA masking which may hinder processes that require proteinRNA interactions such as splicing, mRNA transport, polyadenylation, translation and degradation. For example, antisense transcript-mediated masking of splice-site in thyroid hormone receptor gene erbAα results in shift of balance between the two splice variants [144]; Wrap53, a NAT to p53, mediate p53 induction in response to DNA damage by targeting the 5′UTR of p53 mRNA [145]; regulation of XIST by an antisense lncRNA Tsix leading to X chromosome activation [146]. Alu element harboring TFBS can activate transcription from pol II promoter as well as downstream regions. Besides, Alus can also potentiate transcription of Alu RNA when present in antisense orientation as well as provide TFBS for antisense transcription. This could not only result in transcriptional interference but also sense–antisense transcript pairing in cis. Due to their ability to be exonized in antisense orientation in a gene, Alu exonized transcripts could be substrates for a transcribing sense Alu from opposite strand or in trans. Alu-mediated genomic rearrangements leading to formation of pseudogenes may also help to enhance its NAT-mediated regulation.

Alus and miRNAs

Alus seem to share many features with miRNAs, the most characterized of the ncRNAs. These ∼22 nt short ncRNAs that now seem to be essential for rapid and transient response to external or internal changes are involved in wide variety of functions like stress response, cell fate determination, morphogenesis regulation, synaptic plasticity, apoptosis, mRNA splicing, DNA methylation, circadian rhythms, angiogenesis control, cell cycle control, endocrinological regulation, immune-modulation and neuro-protection [147–161]. The regulatory potential and specificity are also enhanced by editing of miRNA sequence as well as in the target site of the miRNA [162–165]. miRNA majorly regulate at post-transcriptional level by affecting mRNA stability after binding to 3′UTR region of the transcripts or at the translational level [166–168]. Each miRNA on average targets nearly 200 mRNA sequences and overall ∼40% of human genes [169].

In recent times, there have been bioinformatics predictions for origin of mammalian miRNAs from genomic repeats as well as miRNAs targeting the repeat elements. miRNAs have been mostly characterized to have target sites in the 3′UTR region of the transcripts. Alu exonization and especially its enrichment in the 3′UTR regions (84%) and the similarity of secondary structure of Alu RNA with the miRNA precursor potentiate Alu RNA in biogenesis of miRNA. Besides, presence of potential miRNA target sites within Alu also increase the possibility of Alu involvement in miRNA-based regulatory networks [170]. AI editing of exonized transcripts adds another dimension as editing within miRNA or the target region of miRNA have been shown to increase the functional repertoire of miRNA. An in silico analysis revealed that around 30 human miRNAs which include paralogous miRNAs like miR-17-5p, miR-20a, miR-20b, miR-93, miR-106a and miR-106b, are implicated in cancer and show 5′ seed complementarity against a specific site in the Alu sequences, which is highly conserved [83, 171]. Interestingly, several reports indicate that mRNAs containing Alu in their 3′UTRs are as a class associated with growth and differentiation and are subject to translational regulation. It has also been reported that there has been co-evolution of Alu and miRNAs in the humans with probable feedback mechanism helping to maintain homeostatic state [172]. This dual relationship is postulated on the premise that duplication events involving Alu facilitated growth of miRNA cluster and the expression of miRNAs. On the other hand, the expressed miRNAs target free Alu RNAs to sequester the level of Alu RNA. The probability of cross-talk was increased further with the discovery that in addition to pol II, miRNAs can also be transcribed by pol III as in case of C19MC where miRNAs are transcribed as part of the poly-cistronic transcript along with Alu by RNA pol III [173, 174]. In the absence of rigorous experimental evidence, it is amply clear that most of the Alu–miRNA links are circumstantial and it may be by chance. Thus, one has to have more than usual stringent controls to understand the functionality of the probable cross-talk.

Compartmentalization of transcripts: Alu involvement?

Compartmentalization of transcripts, active or passive, allows cellular functions to be separated in specialized organelles and provides an additional level of gene regulation. The compartmentalization is closely related to the export kinetics of mRNA from the nucleus to the cytosol and the degradation rate of mRNA in the cytosol [175]. Defects in mRNA export can result in human diseases [176]. Recent study by Barthelson et al., wherein nuclear and cytoplasmic transcripts were studied separately observed that both the compartments contain different, yet overlapping populations of transcripts with nuclear RNA especially enriched in non-coding sequences comprising of 41% intergenic and 25% intronic sequences [177, 178]. Differential compartmentalization of alternate transcript isoforms across cell types has been observed with cytosolic fraction having more and longer introns in their pre-mRNAs, more functional RNA folds and unique exons in the 3′-regions whereas nucleus enriched isoforms are more significantly associated with NMD [179]. Sub-cellular distribution of small RNAs has revealed that majority of miRNAs are imported to the nucleus with almost equal distribution of miRNAs between nuclear and cytosolic fraction [180].

The segregation/targeting are facilitated by particular signature sequences associated with individual transcripts. These signatures could be residing in Alus. Although, it has not been studied but it would be worthwhile to explore the importance of 3′-UTR Alu integration, its potential to form secondary structures as embedded Alu RNA, subsequent A–I editing and retention of A–I edited transcripts. It has been reported that ∼75% of AI editing in transcriptome involves Alu repeats [181]. AI hyper-edited transcripts are preferentially retained in the nucleus in spatio-temporal manner in membrane-less, transient but discrete ribonucleoprotein structure called ‘paraspeckles’. NEAT1 RNA is essential, along with PSP1, p54 and other factors regulating AI editing to initiate the de-novo assembly of paraspeckles. Interestingly, NEAT1 RNA is not AI edited which is consistent with its function of sequestering AI edited transcripts [182, 183]. Retention of AI was thought to be a norm rather than exception, with variable retention pattern in different tissues reflecting the editing levels [184] until altered retention pattern in hESCs were observed in comparison to the differentiated cells [185].

Future perspectives: Alu elements as ‘ornaments’ of human genome

With the paucity of experimental platforms to query for the functionality of repetitive elements in the human genome, systematic genome-wide studies for actively looking into the role of Alu at the global scale has not been carried out. Most of the observations have been serendipitous. Genome-wide study platforms like microarray and tiling array are devoid of probes to quantify ncRNA transcripts because of the technical limitations related to specificity and conservation. But with the availability of next-generation sequencing (NGS) platforms, it has been possible to undertake genome-wide studies involving RNA-seq and ChIP-seq to understand the role of non-coding part of the genome at the RNA and DNA level, respectively. NGS would also be of great help to explore RNA–protein interactions through sequencing of RNA-immunoprecipitated (RIP) samples.

From its initial days, when it was summarily rejected as ‘genomic scrap’, polluting and congesting the human genome; it has now been accepted and explored as ‘genomic gems’. It is evident that Alus as non-coding components of the human genome could modulate transcriptome in diverse ways. This would range from providing TFBS, their specific regulation, exonization, editing and involvement in antisense to miRNA regulatory networks. Additionally, through recombination they could further distribute these regulatory sites. Do these networks cross-talk and where could they assume importance? Parallel and peripheral information point toward the fact that ncRNA may be part of functional redundancy repertoire as a built-in safeguard for maintaining accurate regulation of the genome. Most of the characterized functional role of Alu repeats has been reported under different condition of stress, thus it is likely that Alus offer an alternative ‘safe exit’ route for survival during stress. It has also been reported that heat shock response in mammalian cells is RNA mediated [186]. Alus may be the unifying thread during different stress conditions as exemplified by the instances enumerated below. As Alus seems to be a partner in miRNA mediated regulatory networks, it would be worthwhile to explore Alu–miRNA cross-talk. During stress a concomitant transient increase in the level of Alu RNA along with heat shock proteins (HSPs) takes place till 12 h and then declines. Identification of diverse array of functional TFBS within Alu raises the possibility of Alu also harboring heat shock factor (HSF) binding site which could induce expression of Alu RNA and also heat shock responsive genes directly. Alu RNA may mediate complementary binding and sequester the expression levels of sense transcripts. Though Alu antisense transcripts have been reported to be abundant in the human genome, there are very few reports probing functional relevance of these antisense transcripts. It is possible that these transcripts are transcribed in response to rapidly changing environmental stimuli. The other possibility could be through a direct cross talk between pol II and pol III machinery in stress as Alu RNA levels are elevated and they are also known to act as transcriptional co-repressor. It would be important to mention that Alu is not itself recognized by pol III machinery instead ‘B-Box’ promoter is recognized. The transcriptional machinery thus transcribes both ‘free’ and ‘embedded’ Alu RNAs. Recent observations show close association of pol III and many TFs, like c-Fos, c-Jun and c-Myc that are otherwise tightly associated with pol II genes [187]. The preferential association of human pol III complexes near functional pol II promoters indicates that TFIIIC mediated recruitment of TFIIIB is regulated in a locus-specific manner. As Alu ‘B-Box’ is recognized by the pol III machinery, specifically the TFIIIC complex to initiate its transcription, these elements might be involved in such cross-talk. On the other hand, pol II modulating pol III activity is exemplified by the presence of pol II epigenetic marks, like histone acetylation with pol III transcribed ncRNA genes. It has been observed that pol III bound and expressed ncRNA are marked by H3K4me1/2/3 modifications. The widespread co-occurrence of pol II and pol III perhaps reflect much larger role of this phenomenon in organizing the human genome into discrete functional domains and TE like Alu could play such ‘insulator roles’ [188, 189]. Alu has already been shown to confer such position-independent expression in transgenic mice.

Since Alus are predicted to be targets for miRNAs, they can affect the function of miRNA through titration of miRNA by providing alternate target sites. miRNA mediated post-transcriptional degradation of non-canonical transcript isoforms made in stress, may be one of the possible mechanisms by which such transcripts can be cleared from the cellular milieu when the gene product is not of immediate use [190]. Such transcripts can be earmarked by the presence of exonized Alu, which may act like ‘miRNA sponges’ to protect the genome against the increased levels of miRNA in any particular condition/stimuli. With evidence of pseudogene, like PTENP1, regulating the corresponding protein-coding mRNA by acting as a decoy for miRNA that bind to the common site in the 3′UTR region, such a function mediated by Alu is also possible [32, 191]. Localization of miRNA associated Argonaute to the stress granules (SG), which are temporarily formed foci for undecided transcripts during condition of stress, also highlights the close association between miRNA and stress [147, 192].

Stress response is also mediated through the activation of signaling pathways which lead to phosphorylation of target splicing factors and change their sub-cellular distribution, activity and/or association with multi-protein complexes. In response to stress, hnRNP A1, a nucleo-cytoplasmic shuttling protein that antagonizes serine/arginine-rich (SR) proteins during alternative splicing, accumulates in the cytoplasm localizing to the SGs. This accumulation causes an altered ratio of the antagonistic alternative splicing factors SF2/ASF and hnRNP A1 in the nucleus and consequently affects alternative splicing regulation. Similarly, cell stress induces phosphorylation of the splicing factor RBM4 (RNA-binding motif protein 4) and drives its cytoplasmic accumulation and targeting to SGs, via the MKK3/6–p38 signaling pathway, where it inhibits translation. Overall it seems that hnRNAp A1 and RBM4 are not the only exception but stress stimuli also influence the sub-cellular localization of several other RNA binding proteins, including the second step splicing factor hSlu7, required for correct 3′ splice-site choice [193, 194] and HuR (human antigen R) [195]. These splicing factors may act on cryptic splice sites within Alu and add it as part of the coding exon. This may determine the fate of the transcripts, e.g. its retention in nucleus and compartmentalization or differential sensitivity to stress response. A summary of the validated and proposed regulatory functions mediated by Alu is present in Figure 5.

Figure 5:

Transcriptomic diversity leading to genome plasticity and complexity being modulated and mediated by transcribed Alus. Arrows with continuous line represent known experimental/computational facts whereas dotted lines are plausible functions based on peripheral and parallel evidences. Asterisk on dsRNA represent shared functions. Alu repeats seem to be an integral component of genome regulatory repertoire particularly for maintaining cellular homeostasis during stress and also for variability/specificity in cellular functions.

Figure 5:

Transcriptomic diversity leading to genome plasticity and complexity being modulated and mediated by transcribed Alus. Arrows with continuous line represent known experimental/computational facts whereas dotted lines are plausible functions based on peripheral and parallel evidences. Asterisk on dsRNA represent shared functions. Alu repeats seem to be an integral component of genome regulatory repertoire particularly for maintaining cellular homeostasis during stress and also for variability/specificity in cellular functions.

Finally, the increased levels of Alu RNA leading to toxicity of the cell in case of age-related macular degeneration has opened a totally new domain of research because till date it was only known that Alu RNA levels were either elevated or down-regulated. It has been shown that staufen-1 (STAU1)-mediated mRNA decay requires binding of STAU1 to the 3′UTR of translationally active mRNA. This binding is facilitated by the presence of Alu in 3′UTR of mRNA and another Alu element in a cytoplasmic, polyadenylated lncRNA [196]. Although this is not a universal phenomena but it reflects the genome plasticity contributed by Alu which may vary in time and space, different tissues, stimuli and interacting partners. The functional role of transcribed Alus is just being realized whereby new quanta of information are being strung together to understand the Alu RNA mediated regulation. The variable levels of Alu RNA during infection further substantiate its functional involvement in physiological homeostasis. The role of Alu in governing the dynamics of host–pathogen interaction would be an interesting avenue to explore. Human complexity and plasticity is characterized by its ability to adjust to different conditions. To achieve this, there must be stand-by scenario available which provides flexibility. The abundance of TE like Alu may capacitate the human genome with such flexibility.

Key points

  • ncRNA may be part of functional redundancy repertoire as a built-in safeguard for maintaining accurate regulation of the genome.

  • Differential levels of Alu RNA during stress conditions like heat shock, cancer and infection potentiates it for functional role in host–pathogen interaction.

  • Possible direct cross-talk between pol II and pol III machinery during condition of stress which may be mediated by the presence of binding sites for both within Alu repeats.

  • Probable miRNA targets within Alu potentiate the role of Alu as ‘miRNA sponge’ in response to external/internal stimuli.

  • Alu exonised transcripts may determine the compartmentalization of transcripts across nucleo-cytosolic fractions.

FUNDING

Financial support to M.M. and R.P. from Council of Scientific and Industrial Research (CSIR) grant (NWP-0036) and Council of Scientific and Industrial Research-Senior Research Fellowship (CSIR-SRF to R.P.) is duly acknowledged.

Acknowledgement

Financial support to M.M. and R.P. from Council of Scientific and Industrial Research (CSIR) grant (NWP-0036) and Council of Scientific and Industrial Research-Senior Research Fellowship (CSIR-SRF to R.G.) is acknowledged. The authors would like to acknowledge Amit K. Mandal for valuable discussions.

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