Jan

24

The Latest RNA-Seq Application – Degradome Sequencing

Filed Under Information, Sequencing Protocols 

Degradome sequencing for identification of miRNA targets in plants

MicroRNAs (miRNAs) are endogenous regulators of a broad range of physiological processes and act by either degrading mRNA or blocking its translation. Mature miRNAs function within large complexes to negatively regulate specific target mRNAs. Plant miRNAs generally interact with their targets through perfect or near-perfect complementarity and direct mRNA target degradation.

In plants, miRNAs not only post-transcriptionally regulate their own targets but also interact with each other in regulatory networks to affect many aspects of development, such as growth, development and responses to biotic and abiotic stresses. Hundreds of miRNAs have been identified in higher plants by direct cloning or more recently by next-gen sequencing. To determine the function of these miRNAs we must first identify their targets.

Originally, plant miRNA targets have been studied via computational prediction, which is based on either perfect or near-perfect sequence complementarity between miRNA and the target mRNA or sequence conservation among different species. However, target prediction is very challenging, especially when a high level of mismatches exists in miRNA:target pairing.

Recently, a new method called degradome sequencing, which combines high-throughput RNA sequencing with bioinformatic tools, has-been successfully established to screen for miRNA targets in plants. Using degradome sequencing, many of the previously validated and predicted targets of miRNAs have been verified indicating that it is an efficient strategy to identify smRNA targets on a large scale in plants.

Degradome sequencing reveals miRNA targets by globally identifying the remnants of small RNA-directed target cleavage by sequencing the 5′ ends of uncapped RNAs. Sequencing reads are mapped to mRNAs and the 5′ terminal nucleotide of miRNA-cleaved mRNA fragments corresponds to the nucleotide that is complementary to the 10th nucleotide of the miRNA. Therefore, the cleaved RNA targets have distinct peaks in the degradome sequence reads at the predicted cleavage site relative to other regions of the transcript. Confirmed miRNA targets are presented in the form of target plots (t-plots).

t-plots

Oilseed rape (Brassica napus) is one of the most important crops in China, Europe and other Asian countries with publicly available expressed sequence tags (ESTs) and genomic survey sequence (GSS) databases. However, unlike Arabidopsis and other plants, much less is known about its miRNAs and their targets. Now, researchers at the Chinese Academy of Agricultural Sciences, Beijing have used Illumina high-throughput sequencing analysis of small RNAs as well as degradome sequencing of B. napus to identify novel miRNAs and miRNA targets1.

Forty-one conserved B. napus miRNAs and 62candidate novel B. napus-specific miRNAs were identified through small RNA sequencing and further verified by real-time RT-PCR. A total of 33 non-redundant target ESTs for 25conserved miRNAs, and 19 non-redundant target ESTs for 17 B. napus-specific miRNAs were identified through degradome sequencing and verified by RNA ligase-mediated 5’RACE mapping. This study describes large scale sequencing and identification of B. napus miRNAs and their potential targets, providing the foundation for further characterization of miRNA function in the regulation of diverse physiological processes in B. napus.

miRNAs also play important regulatory roles in development and stress response in plants. Wild soybean (Glycine soja) has undergone long-term natural selection and may have evolved special mechanisms to survive stress conditions as a result. However, little information about miRNAs especially miRNAs responsive to aluminum (Al) stress is available in wild soybean.

Now, researchers at the South China Agricultural University, Guangzhou have sequenced two small RNA libraries and two degradomelibraries constructed from the roots of Al-treated and Al-free G. soja seedlings2. Among all the identified miRNAs, the expressions of 30 miRNAs were responsive to Al stress. Through degradome sequencing, 86 genes were identified as targets of the known miRNAs and five genes were found to be the targets of the novel miRNAs obtained in this study. Gene ontology (GO) annotations of target transcripts indicated that 52 target genes cleaved by conserved miRNA families might play roles in the regulation of transcription. Additionally, some genes known to be responsive to stress, were found to be cleaved under Al stress conditions. These findings provide valuable information to understand the function of miRNAs in Al tolerance.

Cucumber (Cucumis sativus) is among the most important greenhouse species in the world, but only a limited number of miRNAs from cucumber have been identified and the experimental validation of the related miRNA targets is still lacking. In this study, a team led by researchers at Zhejiang University, Hangzhou constructed two independent small RNA libraries from cucumber leaves and roots, respectively, and sequenced them with the high-throughput Illumina system3. Based on sequence similarity and hairpin structure prediction, a total of 29 known miRNA families and 2 novel miRNA families containing a total of 64 miRNA were identified.

With the recently developed ‘high throughput degradome sequencing’ approach, 21 target mRNAs of known miRNAs were identified for the first time in cucumber. These targets were associated with development, reactive oxygen species scavenging, signaling transduction and transcriptional regulation. This study provides an overview of miRNA expression profile and interaction between miRNA and target, which will help further understanding of the important roles of miRNAs in cucumber plants.

  1. Xu MY, Dong Y, Zhang QX, Zhang L, Luo YZ, Sun J, Fan YL, Wang L. (2012) Identification of miRNAs and their targets from Brassica napus by high-throughput sequencing and degradome analysis. BMC Genomics 13:421. [article]
  2. Qiao-Ying Z, Cun-Yi Y, Qi-Bin M, Xiu-Ping L, Wen-Wen D, Hai N. (2012) Identification of wild soybean miRNAs and their target genes responsive to aluminum stress. BMC Plant Biol 12(1), 182. [article]
  3. Mao W, Li Z, Xia X, Li Y, Yu J. (2012) A Combined Approach of High-Throughput Sequencing and Degradome Analysis Reveals Tissue Specific Expression of MicroRNAs and Their Targets in Cucumber. PLoS One 7(3), e33040. [article]

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