The spectacular escalation in complexity in the plant genomes correlates well with the aberrant increase in the number of naturally occurring small RNAs, termed as microRNAs (miRNAs) and small interfering RNAs (siRNAs). MicroRNAs are an abundant class of 19-24 nucleotides which are small endogenous non-coding RNA that negatively regulate gene expression at the post-transcriptional level by directing the cleavage of mRNAs or interfering with translation [1–5]. As key regulators of diverse biological processes, this group of small RNAs act by base pairing to complementary target sites and mediating mRNA cleavage or translation repression . In addition, expression of these mature miRNAs varies between tissues and over time .
Biogenesis of miRNA occurs in the nucleus . First, miRNAs are single stranded RNA molecules encoded by nuclear genes which are processed into primary miRNA (pri-miRNA) transcripts by the action of RNA polymerase II . Then, these pri-miRNA are cropped into stem loop structures called precursor miRNAs (pre-miRNAs) with sequence of 70-150nt long through the action of RNase III [6, 7]. Pre-miRNAs are then transported into the cytoplasm through the nuclear transport receptor complex [6, 7]. Inside the cytoplasm, these pre-miRNAs with characteristics of stem loop secondary structures are further processed to generate mature miRNAs [7–9]. On the other hand, siRNAs are processed from long double stranded RNA (dsRNA) introduced exogenously into cells, formed by convergent transcription, extended hairpin structures or RNA-dependent RNA polymerization [7, 8].
RNA interference (RNAi) is a natural phenomenon of specific gene silencing in fungi, plants and animals . As RNAi has become a standard experimental tool in biological research, understanding their biogenesis is imperative in order to better understand the mechanism of gene silencing trigger by miRNAs and siRNAs . In plants, RNAi machinery are triggered by the assembling of these small RNAs into RNA-induced silencing complexes (RISC) [7, 10] which further guide the post-transcriptional gene silencing [11–15]. Although biogenesis of both mature miRNAs and siRNAs are different from each other, both of them depend on the Dicer for appropriate processing .
Although the mechanisms of RNAi initiated by miRNAs are about the same as siRNAs, however, miRNAs are said to play an essential role in plant growth, development and stress response [17, 18]. MicroRNA play a critical regulatory behaviour in root, leaf, flower and shoot development [18–22]. Recent findings have demonstrated that majority of the plant miRNAs are conserved. For instance, miR156, miR159, miR164 and miR172 regulate the LFY expression, flowering time and floral organ identity which are important characteristics in the normal plant growth and development [23–26]. MiR413 regulates the expression of FLOWERING LOCUS C (FLC) . Gene expression study has revealed that majority of the plant miRNA families in Populus trichocarpa are expressed at some level associated with cambium differentiation activities . Tissues specific expression of miRNAs (ptr-miR160, 164, 171, 473, 477, 478, 479, and 480) suggested the specific roles of these miRNAs families in xylem tissue .
In Arabidopsis, bioinformatic analysis revealed that highly conserved miRNA families encoded transcriptional factor as their predicted target . Several studies using model plant species have validated the roles of miRNAs in regulating the specific lignin pathway genes or the entire lignin biosynthetic pathway genes by controlling the binding between transcription factors with the AC elements present in the promoter of monolignol biosynthetic genes. Transcriptional factors like MYB, NAC, HD-Zip, SQUAMOSA PROMOTER BINDING PROTEIN-LIKE and many others are known to be differentially regulated in developing wood, tension wood, xylem, wood forming stem, developing stem and differentiating xylem [27–30]. Although conserved miRNAs families are present in all plant families, some of the isoforms might play a specific role with their expression being unique to that particular tissue of the species [17, 31–34].
High throughput sequencing approach is so far the most economic and accurate method for miRNA isolation compared to computational and cloning based approach [35, 36]. This is because cloning approach might exclude underexpressed miRNAs in a particular investigated tissue [35, 36]. This limitation could be overcome using deep sequencing strategies as the entire low abundance novel small RNA classes could be discovered [35, 36]. Identification of the entire set of small RNAs is the best strategy to better understand the mechanism of gene regulation and gene silencing in a complex organism [35–39]. In our study, Illumina GAII sequencer was employed to profile the relative expression of the small RNAs between samples with contrasting lignin content. Changes in the relative abundance of the expressed small RNAs between A. mangium secondary xylem with contrasting lignin content indicated the specific adaptation and behavior of the identified miRNAs or siRNAs.