Figure 3

A novel population of sRNAs derived from the U2 snRNA. (A). Screenshot of the reads profile associated to the U2 snRNA in chromosome 5 (chr5: 696.774–696.999). The U2-5′ (in orange) and U2-3′ (in green) sRNA populations associated to this region are indicated. (B) Validation of the U2-3′ expression by low molecular weight Northern blotting. Total RNA was extracted from cell cultures grown under different light conditions (High Light (HL), Normal Light (NL), Low Light (LL), Dark (D) and iron starvation (−Fe)) and Northern blot was probed using an oligonucleotide recognizing the U2-3′ sRNAs and the U6 snRNA as reference loading control. M, microRNA Marker (New England Biolabs, USA). (C) Relative transcript levels of U2-3′ and U2-5′ sRNAs determined by stem-loop qPCR, in cells grown under the same growth conditions described above. The expression values U2-3′ and U2-5′ were normalized to the snoRNA. Error bars are relative to three independent experiments. (D) Relative transcript levels of the U2 snRNA by qRT-PCR in cells grown under iron starvation and different light conditions. Normalization was done relative to histone H4 mRNA, used as a reference gene. Error bars are relative to three independent experiments. The U2 snRNA expression was also analyzed by Northern blot by using an oligonucleotide recognizing a region of the U2 snRNA upstream the U2-5′ small ncRNA, as a probe. (E) U2 snRNA secondary structure as annotated in Rfam [82] and the distribution of U2-5′ and U2-3′ highlighted on it. (F) Analysis of the U2-3′small RNAs in wild-type and overexpressing snRNA U2 lines (OE1 and OE2) by Northern blotting from cells grown under normal light condition. A total of 20 μg of RNA was loaded per lane and sRNA products were detected as described above.