**1. Introduction**

The transcripts whose sequences are complementary to those of mRNA have been reported in many genes regardless of species, from bacteria to mammals. Because protein is encoded by mRNA, whose sequence is the same as the sense strand of a gene, i.e., double-stranded DNA, the transcript has been called a natural antisense transcript (NAT or AS transcript) [1, 2]. The AS transcripts do not code for proteins or only short peptides and are classified as one class of noncoding RNA (ncRNA). Accumulating genome-wide transcriptome analyses have demonstrated that natural

antisense transcripts are transcribed from many eukaryotic genes [3]. HUGO proposed the nomenclature of human gene symbols for natural antisense transcripts *AS* (suffix) [4]. In this chapter, we use 'AS transcript' as a natural antisense transcript.

In contrast, classical types of ncRNA species, such as ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA), are well known and have definite functions in gene expression. These classical ncRNAs do not overlap mRNAs. Among other ncRNA species, microRNA (miRNA or miR), which is 20–23 nucleotides (nt) in the length, was found in nematodes and mammals. This very short ncRNA species, which is complementary to the 3′-untranslated region (3′UTR) of several mRNAs, inhibits translation and induces mRNA degradation [5]. Therefore, microRNA hybridizes with mRNA to regulate its functions.

Furthermore, long ncRNAs (lncRNAs), which are more than 200 nt long [6], were found. At first, their functions were unclear, but it has gradually been revealed that these lncRNAs are involved in gene expression [7]. To date, huge number of lncRNA sequences have been reported by RNA-seq analysis and deposited in public databases, such as LNCipedia 5 (human lncRNA transcripts) [8]. Nowadays, ncRNA species, including miRNA, and lncRNAs, are known as *regulatory RNAs* [9].

Many studies have demonstrated that the AS transcript, one class of lncRNA, is involved in various steps during gene expression [7]. When focusing on the AS transcript that overlaps with an mRNA, this type of AS transcript interacts with mRNA and plays an important role in gene expression, especially at post-transcriptional levels [1, 2]. Interestingly, most AS transcripts are transcribed at low levels [1, 2]. The analyses and application of AS transcripts are summarized by the reviews, for example, see [1, 2, 10].

During our functional analyses of AS transcripts, we found the mRNA-AS transcript interactions that regulate mRNA stability (described later). Although conventional methods, i.e., antisense and short interference RNA (siRNA) technologies [11, 12] were available for the analyses of AS transcript functions [13], we first used synthetic sense oligonucleotides that are complementary to the AS transcript. We found that sense oligonucleotides resulted in decreases in cytoplasmic mRNA levels, which may be applied to 'knockdown of mRNA.'

Here, our method to regulate mRNA levels based on the mRNA-AS transcript interactions is described, and the application of this technology to treat disease is discussed.
