**2. Non-coding RNAs: biosynthesis and classification**

Non-coding RNAs are transcribed RNA molecules that are not translated into proteins. They play a remarkable variety of biological functions by engaging target transcripts through sequence-specific interactions. They regulate many biological processes, including gene expression (transcription, RNA processing and translation), protect genomes from foreign nucleic acids and can guide DNA synthesis or genome rearrangement [21]. In general, ncRNAs are classified according to size or function. According to size, ncRNAs are classified as (1) small or short ncRNA: <200 nucleotides in their mature forms (e.g. miRNA, PIWIinteracting RNA [piRNA], small nuclear RNA [snRNA], small nucleolar RNA [snoRNA] and endogenous small interfering RNA [siRNA]) and (2) long ncRNA: >200 nucleotides long (e.g. lncRNA). According to function, ncRNAs are classified as (1) housekeeping or translation-related ncRNAs: they are constitutively expressed and crucial for normal cellular function and viability and include tRNA, rRNA and snoRNA and (2) regulatory ncRNAs and include miRNA, lncRNA, siRNA and piRNA [22, 23]. The biogenesis of these various types of ncRNAs has been discussed extensively [23–26]. This chapter focuses particularly on the involvement of miRNA and lncRNA in ruminant mammary gland development and lactation.

#### **2.1. MicroRNAs**

but also provides a model for basic cellular processes (proliferation, differentiation, survival and death) [1], which may have important implications for productivity (milk yield) and disease status (e.g. breast cancer, mastitis, etc.). The endocrine regulation and physiological processes as well as the signalling pathways involved in these processes are fairly understood [1, 2]. Facilitated by the release of the whole genome sequences of cattle, sheep and goat [3–6] as well as availability of single nucleotide polymorphism (SNP) genotyping chips [7–11], the genetic mechanisms of ruminant lactation have been extensively explored (**Figure 1**). As a consequence, many quantitative trait loci (QTL) and genetic markers for lactationrelated traits (for instance, milk yield, milk components, lactation persistency, etc.) have been detected and catalogued in the animal QTL database (http://www.animalgenome.org/

Transcriptomics research by both microarray and RNA sequencing methods has allowed for a better understanding of the genes and regulatory networks of complex traits in animals [12], such as the biosynthesis of major milk components (reviewed in Refs. [13, 14]). Emerging studies now suggest that non-coding RNAs (ncRNAs) are key regulators of mammary gland development and lactation processes [15–17]. The results from the ENCODE (ENCyclopedia of DNA elements) project [18, 19] indicate that only a small portion of the genome, about

**Figure 1.** Growing research by year in the field of cattle genomics and transcriptomics (including non-coding RNA) from

cgi-bin/QTLdb/index).

56 Current Topics in Lactation

January 2000 to August 2016.

MiRNAs are an abundant class of short ncRNAs of about 22 nucleotides long. They regulate a variety of cellular processes through post-transcriptional repression of gene expression. MiRNAs consequently control the activities of about 60% of all protein-coding genes and participate in the regulation of almost every cellular process investigated in mammals [25]. Mature miRNAs are generated from a series of biochemical events beginning in the nucleus and culminating in the cytoplasm [24, 27, 28]. Briefly, these events occur in several main steps as follows: (1) nuclear processing of primary miRNA transcripts (pri-miRNAs) into precursor miRNAs (pre-miRNAs) by the DiGeorge Syndrome Critical Region Gene 8 (DGCR8)/Drosha complex, (2) cytoplasmic processing of pre-miRNAs into imperfectly paired miRNA duplexes by dicer, and (3) preferential incorporation of one strand (the 'guide' miRNA strand) onto the RNAinduced silencing complex (RISC) [25]. Most miRNA genes located in introns of protein-coding genes share the promoter of the host gene [29]. MiRNAs often have multiple transcription start sites and regulate gene expression through inhibition of translation initiation or elongation, cotranslational protein degradation and premature termination of translation [25, 30].

Since the discovery of the first miRNA, lin-4, in 1993 [31] and aided by deep sequencing technologies and developments in bioinformatics processing of deep sequence data, thousands of miRNAs have been detected in humans, mouse, farm animal species and plants and deposited in the miRNA data base (**Table 1**). Due to the crucial regulatory roles of miRNAs in many biological processes across species, they are being considered as candidate biomarkers of various human diseases, such as autoimmune [32], metabolic [33] and cardiovascular diseases [34], and various types of cancers [35–37].


\*Data source: MiRBase release 21 (http://www.mirbase.org/[38], and NONCODE database (www.noncode.org, Noncode 2016 [39]).

**Table 1.** Number of detected miRNAs and lncRNAs in farm animal species, mouse and human\* .

#### **2.2. Long non-coding RNAs**

Long non-coding RNAs are a diverse collection of non-coding RNAs with emerging regulatory roles in many biological processes in every branch of life [26, 40–42]. LncRNA transcripts are >200 nucleotides long and constitute the largest portion of the mammalian non-coding RNA transcriptome [40]. LncRNA closely resembles mRNA than other classes of ncRNA in terms of their biogenesis pathways and form. Most lncRNAs are transcribed by the activities of RNA polymerase II, have a 5′ terminal methylguanosine cap and are often spliced and polyadenylated [41]. Some non-polyadenylated lncRNAs arise through alternative pathways probably expressed from RNA polymerase III promoters [43, 44] or arise during splicing and small nucleolar RNA production [45]. Furthermore, some lncRNAs are regulated in different ways at different stages of their biogenesis, maturation and decay [26]. Thousands of genes encoding lncRNAs have been identified in mammalian genomes (including livestock species), birds and plants studied so far and deposited in the NONECODE database (www.nonecode. org [39], **Table 1**).
