Methodologies for in vitro cloning of small RNAs and application for plant genome(s).

Eric J Devor,Lingyan Huang,Ibrokhim Y Abdurakhmonov,Abdusattor Abdukarimov

doi:10.1155/2009/915061

Abstract

The “RNA revolution” that started at the end of the 20th century with the discovery of post-transcriptional gene silencing and its mechanism via RNA interference (RNAi) placed tiny 21-24 nucleotide long noncoding RNAs (ncRNAs) in the forefront of biology as one of the most important regulatory elements in a host of physiologic processes. The discovery of new classes of ncRNAs including endogenous small interfering RNAs, microRNAs, and PIWI-interacting RNAs is a hallmark in the understanding of RNA-dependent gene regulation. New generation high-throughput sequencing technologies further accelerated the studies of this “tiny world” and provided their global characterization and validation in many biological systems with sequenced genomes. Nevertheless, for the many “yet-unsequenced” plant genomes, the discovery of small RNA world requires in vitro cloning from purified cellular RNAs. Thus, reproducible methods for in vitro small RNA cloning are of paramount importance and will remain so into the foreseeable future. In this paper, we present a description of existing small RNA cloning methods as well as next-generation sequencing methods that have accelerated this research along with a description of the application of one in vitro cloning method in an initial small RNA survey in the “still unsequenced” allotetraploid cotton genome.

Highlights

In the 1990s two independent discoveries opened up the previously unsuspected world of noncoding RNAs
The world of miRNAs has grown from the two original nematode “genes” to number more than one thousand loci in plants and animals and their role in regulating cellular processes has expanded to a point where virtually all normal and pathogenic cellular processes are affected at some point by one or more of these tiny entities
The performance obtained by the Roche 454 Life Science commercial system Genome Sequencer (GS-FLX) platform of 99.5% accuracy and average read lengths of over 250 bp resulting in outputs exceeding 200 000 reads with acceptable Phred values is ideal for searching genomes for new small RNAs and, such studies have already resulted in the discovery of the curious 21U RNA class of small RNA in C. elegans

Summary

Introduction

In the 1990s two independent discoveries opened up the previously unsuspected world of noncoding RNAs (ncRNAs). Over the past several years RNAi has become a powerful tool for understanding the role played by dozens of plant and animal genes in a wide range of cellular processes, both normal and pathogenic [14]. The world of miRNAs has grown from the two original nematode “genes” to number more than one thousand loci in plants and animals and their role in regulating cellular processes has expanded to a point where virtually all normal and pathogenic cellular processes are affected at some point by one or more of these tiny entities. The universe of small RNAs has expanded to include miRNAs but new classes including endogenous small interfering RNAs (siRNAs), 21U RNAs, and Piwi-interacting RNAs (piRNAs) [17]. We present a compilation of extant small RNA cloning methods, options for sequencing, and some of the small RNA results that we have obtained in the “still unsequenced” allotetraploid cotton genome

Small RNA Cloning Strategies

Cloning with Adenylated Linkers

Sequencing Strategies

Application

Findings

Conclusions