Novel micro-RNAs and intermediates of micro-RNA biogenesis from ...

8 Mali Talmor-Neiman et al.Figure 6. Northern blot analysis of candidate miRNAs.Total RNA was extracted from isolated 8–10-day-old protonema (P) and 45-day-old gametophores (G). Protonemal total RNA was also used to preparelow-molecular-weight RNA (C). Samples of total RNA (100 lg) and lowmolecular weight RNA (equivalent to 500 lg total RNA) were separated on adenaturing polyacrylamide gel, blotted and probed with oligonucleotideprobes complementary to indicated miRNA sequence. The tRNA and 5S rRNAbands were visualized by ethidium bromide staining of polyacrylamide gels,and served as loading controls.complementary to them according to the empirical parametersas formulated by Schwab et al. (2005) and described inexperimental procedures. In addition, gaps were not allowed,and the G:U non-canonical pair was treated as amismatch. This analysis identified only one potential targetfor miR1218 (pphf2k14) that was predicted to encode a NAM/ATAF/CUC (NAC) domain-containing protein (Table 2).To identify additional targets, a similar search wasperformed against the P. patens raw genomic sequencesdeposited at the NCBI trace archive data base (http://www.ncbi.nlm.nih.gov/Traces/trace.cgi?). This search identifiednumerous genomic sequences with complementarityto all the newly identified miRNAs according to the aboverules (data not shown). However, to determine their orientationand thus their relevance as miRNA targets, redundantsequences were first clustered by using the CLUSTALWsequence alignment tool (Thompson et al., 1994). Then aBLASTX search against the NCBI protein database (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi?) was performed toidentify significant similarities to known proteins in the database. This search identified 12 sequences with similarities toknown proteins, which could serve as feasible targets fornine out of the 19 identified miRNAs (Table 2). In nine ofthese genes the miRNA complementary site was foundwithin a predicted open reading frame (ORF). Five of thesegenes, which were predicted to be targeted by eithermiR1215 or miR1217, encoded a protein with a putativekinase domain. These genes showed homology to a proteinkinase (756726571), to phytochrome-like kinases(830632667, 816301061, 815676221), and to a disease resistance-likeprotein (692445811). The remaining targets appearedto encode proteins with homology to an F-BOXprotein, a B-box zinc finger, NAC-domain transcriptionfactors, a retrotransposon nucleocapsid protein, an ABCtransporterand a protein with strong homology to Arabidopsiscytokinin response 1 (CRE1)/wooden leg (WOL)/Arabidopsishistidine kinase 4 (AHK4) cytokinin receptor(Table 2).It was suggested that intermediates of Arabidopsis shortpre-miR163 processing might function as miRNAs (Kuriharaand Watanabe, 2004). To test this possibility, Pp_82, Pp_112and Pp_115 target prediction was performed as describedabove, against the P. patens EST and genomic databases.This search identified one potential target for Pp_115 thatshowed a significant similarity to an unnamed Arabidopsisprotein that contains a conserved domain of unknownfunction (gnl|CDD|26301) termed DUF647 (Table 2).Experimental validation of predicted P. patens miRNAtargetsMost higher plant miRNAs control gene expression posttranscriptionallyby targeting cognate mRNAs for degradation(Llave et al., 2002; Palatnik et al., 2003; Tang et al.,2003). To verify whether newly identified P. patens miRNAscan mediate the cleavage of their predicted targets in vivo,we isolated mRNAs from 10-day-old protonema and 45-dayoldgametophores and performed an RNA-ligase mediated5¢-rapid amplification of cDNA ends (5¢-RACE) (Llave et al.,2002) on five target genes that were predicted to be targetedby an miRNA (Table 2): two genes were representatives oftargets of weakly expressed miRNAs (756726571 targeted bymiR1215 and 816301061 targeted by miR1217) and three ofª 2006 The AuthorsJournal compilation ª 2006 Blackwell Publishing Ltd, The Plant Journal, (2006), doi: 10.1111/j.1365-313X.2006.02768.x