Conventionally unconventional: Anecdote of small RNAs discoveries

Past decade has witnessed an incredible increase in a number of small RNAs. As the name indicates, small RNAs are RNA transcripts of small (approximately 21-24 nucleotide) length [1-8]. These small RNA transcripts regulate various biological processes ranging from a response to biotic/abiotic stress to the determination of tissue specificity [1-8]. Non-coding RNAs are basically classified based on their biogenesis protocol and mode of function. Presently, two classes of noncoding RNAs are well characterized. First is micro RNA (miRNA) and the second one is small interfering RNA (siRNA). siRNAs are further categorized as trans-acting siRNAs (ta-siRNAs), natural antisense siRNAs (nat-siRNAs), repeat-associated siRNAs (ra-siRNAs) or heterochromatic siRNAs (hc-siRNAs) and long siRNAs (l-siRNA) [8, 9]. Through the scope of this journal, I have attempted to consolidate the storyline of small RNA discovery. However, for a detailed insight of experimental setup, materials used, initial findings and challenges faced readers are recommended to go through the original research work.

            The story of small RNAs started in the year 1990 when Napoli et al. reported the occurrence of variegated or white flowers upon exogenous supplementation of chalcone synthase gene in petunia petals [10]. Chalcone synthase is the key enzyme that catalyzes a pathway (phenylpropanoid pathway) which leads to the production of anthocyanin in petunia petals. In their experiments which are originally aimed to identify the rate-limiting step of the pathway, authors observed that in transgenic plants (having chalcone synthase over-expression cassette) did not improve upon the intensity of petal color (purple in wide type) rather appeared white or variegated.

            The molecular aspect of this unconventional result was deciphered by Fire et al. in the year 1998 [11]. Fire and Mello were working on Caenorhabditis elegans to identify the regulatory activity of unc22 encoding mRNA on ‘muscle twitching’. Unc22 gene in C. elegans codes for a myofilament which causes characteristic muscle twitching phenotype under low levels of unc22 protein. From their multiple experiments, authors were able to report that exogenous supply of double-stranded RNA (dsRNA) can lead to silencing of the endogenous gene. This unusual silencing was termed as RNA interference (abbreviated as RNAi). Nowadays RNAi is a potent tool widely utilized in functional genomics and biotechnology for targeted gene silencing. It should be noted that siRNAs (small interfering RNAs) are the effector molecules of RNAi.

            With advancements in time and technology, it was realized that there are several other categories of small RNAs which causes homology-dependent silencing of genes. Micro RNA (miRNA) is one such class. miRNA was first discovered by Lee et al. in the year 1993 [12]. The first discovered miRNA was lin4 which was identified in C. elegans. This small RNA causes inhibition of lin14 mRNA. The second miRNA discovered was let7 which was again discovered in C. elegans [12]. Both of these miRNAs regulate developmental transitions.

After these discoveries, several small RNA has been identified from different domains of life using direct cloning methods and characterized to play significant roles [1-7]. Today, researchers apply next-generation sequencing technologies to perform wide-scale sequencing of these small RNAs [1-7].

In the past half decade, the bioinformatics-based discovery of small RNAs, particularly miRNAs, has gained immense popularity. This method is preferred by researchers owing to its throughput, fast nature, and low cost. As a fact, miRNAs are evolutionarily conserved among related species and hence share sequence and structural homologies. These conserved properties (conservation across species, the secondary structure of precursor RNA and miRNA/miRNA* position in the precursor) can be readily picked up by using a computational tool.  However, identification of a novel (not known so far) small RNAs still a computational challenge.  One drawback of computational discovery method is that many non-small RNA sequences are also identified along with small RNAs and secondly, this method has no scope in organisms where the genome is not sequenced.

In miRBase, a central repository of miRNAs, a record of astonishingly 28,645 miRNAs is present (miRBase v21, http://www.mirbase.org/). It is now accepted beyond doubts that the expression of many genes is governed by small RNA species. Henceforth, it is commonsensical to understand the conventionally unconventional mechanisms of genic regulation pertaining to plant growth, development and stress tolerance and for improving useful agronomic traits in crops.

Authors: Ankur Bhardwaj, Ritu Pandey, G. Joshi

References:

1. Bhardwaj AR, Joshi G, Kukreja B, Malik V, Arora P, Pandey R, Shukla RN, Bankar KG, Katiyar-Agarwal S, Goel S, Jagannath A, Kumar A and Agarwal M. Global insights into high temperature and drought stress regulated genes by RNA-Seq in economically important oilseed crop Brassica juncea. (2015) BMC Plant Biology 15(9), DOI 10.1186/s12870-014-0405-1.
2. Bhardwaj AR, Joshi G, Pandey R, Goel S, Jagannath A, Kumar A, katiyar-Agarwal S and Agarwal M. A genome-wide perspective of miRNAome in response to high temperature, salinity and drought stresses in Brassica juncea (Czern) L. (2014) PLoS ONE 9(3): e92456.
3. Bhardwaj AR, Pandey R, Agarwal M, Katiyar-Agarwal S (2012) Northern Blot Analysis for Expression Profiling of mRNAs and Small RNAs.In: Jin H. and Gassmann W. (Eds.) RNA Abundance Analysis: Methods and Protocols, Methods in Molecular Biology, vol. 883, DOI 10.1007/978-1-61779-839-9_2, Springer Science+Business Media New York 2012.
4. Kohli D, Joshi D, Deokar AA, Bhardwaj AR, Agarwal M, Katiyar-Agarwal S, Srinivasan R, Jain PK. Identification and characterization of wilt and salt stress-responsive microRNAs from chickpea by high-throughput sequencing. (2014) PLoS ONE 9(10): e108 851. ISSN: 1932-6203
5. Katiyar-Agarwal S, Jin H. Role of small RNAs in host-microbe interactions. (2010) Annual review of phytopathology 48: 225-246.
6. Lakhotia N, Joshi G, Bhardwaj AR, Katiyar-Agarwal S, Agarwal M and Kumar A. Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum) by high-throughput sequencing. (2014) BMC Plant Biology. 2014 Jan 7;14:6.
7. Pandey R, Joshi G, Bhardwaj AR, Agarwal M, KatiyaSr-Agarwal. A Comprehensive study on identification and expression profiling of microRNAs in Triticum aestivum during abiotic stress and development. (2014) PLoS ONE 9(4): e95800.
8. Sunkar R, Chinnusamy V, Zhu J, Zhu J-K. 2007. Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. (2007) Trends in plant science 12: 301-309.
9. Lelandais-Brière C, Sorin C, Declerck M, Benslimane A, Crespi M, Hartmann C. Small RNA diversity in plants and its impact in development. (2010) Current genomics 11: 14.
10. Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. (1990) The Plant Cell Online 2: 279-289.
11. Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE and Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. (1998) Nature 391, 806-811
12. Lee RC, Feinbaum RL, Ambros V. The elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. (1993) Cell 75: 843-854.