Fig. 1: MUSHER lncRNA acts as a positive regulator of seed dormancy and DOG1 expression.

a Schematic representation of the DOG1-MUSHER genomic region of chromosome 5 in A. thaliana. The direction of transcription is indicated with arrowheads. Grey rectangles represent coding exons and introns with thick black lines. The green arrow represents the newly annotated MUSHER transcriptional unit. Numbers along the x-axis on the top indicate genome coordinates (TAIR10). Triangles represent T-DNA insertions of two T-DNA lines (msh-1 and msh-2). The green vertical lines show the positions of multiple guides for msh_dCas9-1 and msh_dCas9-2 dCas9 lines. The green horizontal line corresponds to the region deleted using Cas9 (∆msh). b Primer walking RT-qPCR analysis across the DOG1 locus. The position of amplicons corresponds to the DOG1 locus representation above. Expression levels in the hen2 mutant were normalised to WT and UBC21. Data are presented as mean values of four biological replicates +/− SD. c Positions of MUSHER transcript ends found in WT seeds using 3’ and 5’ RACE-seq, coloured in dark and light green, respectively. d Expression profiles of DOG1 and MUSHER transcripts during seed maturation in the WT. Expression levels were normalised to UBC21 mRNA and 16 DAP. The data are shown as the average of four biological replicates for DOG1 and MUSHER 11DAP, five for MUSHER 16DAP, and six for MUSHER 8,14DAP ± SD. e DOG1 relative expression levels, musher mutants relative to WT in freshly harvested seeds, normalised to UBC21. *p < 0.05, two-tailed Student’s t-test. Data are the mean of five biological replicates, with error bars indicating the standard deviation. f Freshly harvested seeds of WT and different musher mutants were scored for germination. Germination was defined as radical protrusion and counted 4 d after sowing. Data are the mean of six biological replicates +/− SD (n = 6, *p < 0.05, two-tailed t-test). g DOG1 expression profile during silique development in WT and msh_dCas9-1 plants. Expression levels were normalised to UBC21 and shown at the indicated number of days after pollination (DAP); Data are presented as mean values of three biological replicates for WT and four for msh_dCas9-1 + /− SD. h Subcellular localisation of selected transcripts in maturing WT Arabidopsis seeds. Each transcript was tested in 5 biological replicates represented as separate bars. Black horizontal lines represent the mean of chromatin and nucleoplasmic fractions contribution of the total amount of indicated transcript.