Journal: Nucleic Acids Research

Article Title: Principles of miRNA/miRNA* function in plant MIRNA processing

doi: 10.1093/nar/gkae458

Figure Lengend Snippet: Sequentially processed MIRNAs generate several small RNA duplexes with different structural requirements. ( A ) Scheme of a sequential MIRNA . The first DCL1 cut is highlighted with a black arrow. Small RNAs derived from sequential precursors. MiRNA is shown in red, distal miRNA duplex (miRNA.2) is depicted in light blue and proximal miRNA duplex (miRNA.1) is indicated in dark grey (right). ( B ) Top panel: logarithm in base 10 of number of reads of small RNA duplex (n.d.: not detected). See for the number of reads detected for each duplex. Bottom panel: dot plot representing |Δ G | calculated for each small RNA duplex from evolutionary conserved sequentially processed MIRNAs . The colour code of the small RNA duplexes is shown in (A). ( C ) Structural analysis of MIR319a . Denaturing 8% (w/v) polyacrylamide gels shown: OH−: alkaline hydrolysis; V1: RNAse V1 in decreasing concentrations; H2O: incubation with water (control). Blue lines indicate the double-stranded bases obtained after V1 digestion as determined from polyacrylamide gels. The arrows correlate the position of DCL1 cuts in the precursor (right) with the experimental determination of the secondary structure (left). See for the original image of the polyacrylamide gel. ( D, F and H ) Small RNA blots of transgenic lines expressing different precursors from the 35S promoter. Each sample corresponds to 15 pooled inflorescences from independent primary transgenic plants. Numbers above small RNA blots correspond to miRNA levels quantified relative to the wt precursor. The EB staining of each gel is shown below. Top panels show a schematic representation of MIR319a including the wt and modified sequences of (D) miR319.2 (miR319a.2GU and miR319a.2AU) (F) MIR319a-MIL where a black arrow indicates how the central internal loop in miRNA.1 duplex was moved closer to DCL1 second cut (F); and MIR319a-3MM wt and MIR319a-5MM (H). These depictions represent the mfold secondary structure prediction for each precursor (see Appendix II). Black letters specify the closed mismatches, and the numbers indicate their position counting from the 5′end of the miRNA or miRNA.2. MiRNA is shown in red and miRNA* in grey, miRNA.2 is shown in light blue, and miRNA.2* is shown in grey. |Δ G | is shown above each small RNA sequence. ( E , G and I ) RT-qPCR of pri-miRNA determined in three biological replicates are shown as triangles in different shades of grey. Lines represent the mean value for each MIRNA . No statistically significant differences in E, G and I. See for the statistical analysis conducted in all the samples.

Article Snippet: Radioactively labelled products of in vitro transcription were partially digested using T1 RNAse (Fermentas, denaturing conditions), V1 RNase (Ambion, native conditions) and S1 nuclease (Fermentas, native conditions) as described before ( ).

Techniques: Derivative Assay, Incubation, Control, Transgenic Assay, Expressing, Staining, Modification, Sequencing, Quantitative RT-PCR