Characterization and analysis of the expression pattern of microRNAs in the grapevine Vitis vinifera

Motivation: MicroRNAs are small (19-24 nt) noncoding RNAs that play an important role in the regulation of multiple cell events, inhibiting gene expression at the posttranscriptional level by binding target mRNAs that are subsequently degraded or sequestered from translation. Plant microRNA genes are typically transcribed by Pol II to yield polyadenylated primary miRNAs (pri-miRNA). These undergo nuclear cleavage to produce to a stem loop intermediate (pre-miRNA) with specific thermodinamic features. Further processing yields a miRNA:miRNA* duplex with 2 nt 3’ overhangs that enters a cytoplasmic ribonucleprotein complex which mediates interaction with target mRNAs. Systematic analyses of micro RNAs and their expression patterns have been performed in only a few plant model species. The availability of the complete genome sequence of the grapevine (Vitis vinifera), has already permitted genome-wide predictions of microRNAs by purely computational methods. Here we present a comprehensive analysis of expression of both mature microRNAs and their primary transcripts in the grapevine using oligonucleotide arrays and next generation sequencing technologies. Methods: We integrate tanscriptome information derived from high-throughput Illumina SOLEXA and ABI SOLiD sequence tags derived from both polyA+ transcripts and isolated small RNAs with oligonucleotide array data. We are thus able to detect both mature microRNAs and to establish whether genomic loci corresponding to the pre-miRNA are expressed in various tissues. Results: Using “next generation” sequencing technologies and oligonucleotide arrays, we are able to demonstrate tissue specificity of expression of many microRNA genes and their precursor sequences. In many cases, the unambiguous alignment of sequence tags derived from polyA+ RNA to the genomic sequence allow provisional mapping of primary microRNA transcripts. It is hoped that the approach outlined here will ultimately provide insights into the regulation of processing of primary microRNAs and precursor microRNAs as well as facilitating identification of sequence elements involved in the regulation of transcription of microRNA genes.