13.1: Introduction

Large-scale analyses in the 1990s using expressed sequence tags have estimated a total of 35,000 - 100,000 genes encoded by the human genome. However, the complete sequencing of human genome has surprisingly revealed that the numbers of protein-coding genes are likely to be ∼20,000 – 25,000 [12]. While this represents <2% of the total genome sequence, whole genome and transcriptome sequencing and tiling resolution genomic microarrays suggests that over >90% of the genome is still actively transcribed [8], largely as non-protein- coding RNAs (ncRNAs). Although initial speculation has been that these are non-functional transcriptional noise inherent in the transcription machinery, there has been rising evidence suggesting the important role these ncRNAs play in cellular processes and manifestation/progression of diseases. Hence these findings challenged the canonical view of RNA serving only as the intermediate between DNA and protein.

ncRNA classifications

The increasing focus on ncRNA in recent years along with the advancements in sequencing technologies (i.e. Roche 454, Illumina/Solexa, and SOLiD; refer to [16] for a more details on these methods) has led to an explosion in the identification of diverse groups of ncRNAs. Although there has not yet been a consistent nomenclature, ncRNAs can be grouped into two major classes based on transcript size: small ncRNAs (<200 nucleotides) and long ncRNAs (lncRNAs) (≥200 nucleotides) (Table 13.1 ) [6, 8, 13, 20, 24]. Among these, the role of small ncRNAs microRNA (miRNA) and small interfering RNA (siRNA) in RNA silencing have been the most well-documented in recent history. As such, much of the discussion in the remainder of this chapter will be focused on the roles of these small ncRNAs. But first, we will briefly describe the other diverse set of ncRNAs.

Table 13.1: ncRNA classifications (based on [6, 8, 13, 20, 24])

Name	Abbreviation	Function
Ribosomal RNA Transfer RNA Small nucleolar RNA Small Cajal body-specific RNA Small nuclear RNA Guide RNA	Housekeeping RNAs rRNA tRNA snoRNA (∼60-220 nt) scaRNA snRNA (∼60-300 nt) gRNA	translation translation rRNA modification splicesome modification RNA splicing RNA editing
MicroRNA Small interfering RNA Piwi interacting RNA Tiny transcription initiation RNA Promoter-associated short RNA Transcription start site antisense RNA Termini-associated short RNA Antisense termini associated short RNA Retrotransposon-derived RNA 3’UTR-derived RNA x-ncRNA Small NF90-associated RNA Unusually small RNA Vault RNA Human Y RNA	Small ncRNAs (<200 nt) miRNA (∼19-24 nt) siRNA (∼21-22 nt) piRNA (∼26-31 nt) tiRNA (∼17-18 nt) PASR (∼22-200 nt) TSSa-RNA (∼20-90 nt) TASR aTASR RE-RNA uaRNA x-ncRNA snaR usRNA vtRNA hY RNA	RNA silencing RNA silencing Transposon silencing, epigenetic regulation Transcriptional regulation? unknown Transcriptional maintainence? not clear not clear not clear not clear not clear not clear not clear not clear not clear
Large intergenic ncRNA Transcribed ultraconserved regions Pseudogenes Promoter upstream transcripts Telomeric repeat-containing RNA GAA-repeat containing RNA Enhancer RNA Long intronic ncRNA Antisense RNA Promoter-associated long RNA Stable excised intron RNA Long stress-induced non-coding transcripts	Long ncRNAs (≥200 nt) lincRNA T-UCR none PROMPT TERRA GRC-RNA eRNA none aRNA PALR none LSINCT	Epigenetics regulation miRNA regulation? miRNA regulation? Transcriptional activation? telomeric heterochromatin main- tenance not clear not clear not clear not clear not clear not clear not clear

Small ncRNA

For the past decades, there have been a number of well-studied small non-coding RNA species. All of these species are either involved in RNA translation (transfer RNA (tRNA)) or RNA modification and processing (small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA)). In particular, snoRNA (grouped into two broad classes: C/D Box and H/ACA Box, involved in methylation and pseudouridylation, respectively) are localized in the nucleous and participates in rRNA processing and modification. Another group of small ncRNAs are snRNAs that interact with other proteins and with each other to form splicesomes for RNA splicing. Remarkably, these snRNAs are modified (methylation and pseudouridylation) by another set of small ncRNAs - small Cajal body-specific RNAs (scaRNAs), which are similar to snoRNA (in sequence, structure, and function) and are localized in the Cajal body in the nucleus. Yet in another class of small ncRNAs, guide RNAs (gRNAs) have been shown predominately in trypanosomatids to be involved in RNA editing. Many other classes have also been recently proposed (see Table 13.1) although their functional roles remain to be determined. Perhaps the most widely studied ncRNA in the recent years are microRNAs (miRNAs), involved in gene silencing and responsible to the regulation of more than 60% protein-coding genes [6]. Given the extensive work that has been focused on RNAi and wide range of RNAi-based applications that have emerged in the past years, the next section (RNA Interference) will be entirely devoted to this topic.

Long ncRNA

Long ncRNAs (lncRNAs) make up the largest portion of ncRNAs [6]. However the emphasis placed on the study of long ncRNA has only been realized in the recent years. As a result, the terminology for this family of ncRNAs are still in its infancy and oftentimes inconsistent in the literature. This is also in part complicated by cases where some lncRNAs can also serve as transcripts for the generation of short RNAs. In light of these confusions, as discussed in the previous chapter, lncRNA have been arbitrarily defined as ncRNAs with size greater than 200 nts (based on the cut-off in RNA purification protocols) and can be broadly categorized into: sense, antisense, bidirectional, intronic, or intergenic [19]. For example, one particular class of lncRNA called long intergenic ncRNA (lincRNA) are found exclusively in the intergenic region and possesses chromatin modifications indicative of active transcription (e.g. H3K4me3 at the transcriptional start site and H3K36me3 throughout the gene region) [8].

Despite the recent rise of interest in lncRNAs, the discovery of the first lncRNAs (XIST and H19), based on searching cDNA libraries, dated back to the 1980s and 1990s before the discovery of miRNAs [3, 4]. Later studies demonstrated the association of lncRNAs with polycomb group proteins, suggesting potential roles of lncRNAs in epigenetic gene silencing/activation [19]. Another lncRNA, HOX Antisense Intergenic RNA (HOTAIR), was recently found to be highly upregulated in metastatic breast tumors [11]. The association of HOTAIR with the polycomb complex again supports a potential unified role of lncRNAs in chromatin remodeling/epigenetic regulation (in either a cis-regulatory (XIST and H19), or trans-regulatory (e.g. HOTAIR) fashion) and disease etiology.

Recent studies have also identified HULC and pseudogene (transcript resembling real genes but contains mutations that prevent their translation into functional proteins) PTENP1 that may function as a decoy in binding to miRNAs to reduce the overall effectiveness of miRNAs [18, 25]. Other potential roles of lncRNAs remains to be explored. Nevertheless, it is becoming clear that lncRNAs are less likely to be the result of transcriptional noise, but may rather serve critical role in the control of cellular processes.