Physcomitrella miRNAs

Novel miRNAs from Physcomitrella patens have been identified using cloning and computational approaches. Fractionated sRNAs were cloned and sequenced randomly. Sequences were subjected to different filtering steps to examine the origin of the sRNAs. BLAST searches against Physcomitrella genomic and EST databases were performed and sequences with 100% identity to the sRNAs were clustered and assembled using the Paracel Transcript Assembler. The parameters for clustering threshold, overlap length and overlap identity were 100 nt, 80 nt and 85%, respectively.

The generated contig sequences were analyzed using RNAshapes to predict the secondary structure of miRNA precursors. To do this, sequences spanning the putative miRNA site were trimmed in three different combinations upstream and downstream of the putative miRNA sequence: (1) 150 bp upstream and 50 bp downstream, (2) 50 bp upstream and 150 bp downstream, and (3) 150 bp upstream and 150 bp downstream of the miRNA sequence. Those EST sequences which were able to form a hairpin-like structure are available here (Fasta format).

MicroRNA-specific target genes were predicted for the Physcomitrella transcriptome using the RNAhybrid algorithm on both sequence orientations. The target prediction parameters were according to Schwab et al. (2005): no mismatch at positions 10 and 11, no more than one mismatch at positions 2-12, no more than two consecutive mismatches downstream of position 13, and at least 72% of free energy compared to a perfectly complementary target. The nucleotide sequences of putative targets were used for BLASTX searches against the UniProt and TrEMBL databases in order to get preliminary gene annotation. EST sequences of the target genes can be found here (Fasta format).

Contact: Wolfgang Frank

Citation: Fattash I., Voß B., Reski R., Hess W. R. and Frank W. (2007) Evidence for the rapid expansion of microRNA-mediated regulation in early land plant evolution. BMC Plant Biology 7:13