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. 2018 Dec 14;20(1):e46588. doi: 10.15252/embr.201846588

Figure 5. Rrm4 recognises UAUG in vivo .

Figure 5

  1. Logo representation of the most enriched sequence motif at Rrm4 binding sites. At each position, the height of the stack is proportional to the information content, while the relative height of each nucleotide within the stack represents its relative frequency at this position.
  2. Frequency of the Rrm4 motif UAUG around Rrm4 and Grp1 binding sites. Shown is the percentage of binding sites that harbour an UAUG starting at a given position in an 81‐nt window around the binding site summit.
  3. Box plot comparing relative binding strength at Rrm4 binding sites (signal‐over‐background, SOB; see Materials and Methods) with or without UAUG (n = 2,201 and n = 4,211, respectively; unpaired Student's t‐test). Box limits represent quartiles, centre lines denote 50th percentiles, and whiskers extend to most extreme values within 1.5× interquartile range.
  4. Schematic representation of the yeast three‐hybrid system: lexA operator (lexA op) sequences are bound by the LexA‐MS2 coat protein (CP) hybrid (grey), recruiting the MS2‐SELEX‐RNA hybrid (black and red, respectively) to the promoter region of the HIS3 reporter gene. Transcription is activated by binding of the third hybrid AD‐Rrm4‐Gfp (green) carrying a Gal4 activation domain (AD).
  5. RNA structure prediction of aptamer SELEX‐A1 with UAUG (red) or the mutated version mUAUG containing UCUC(A) (mutated bases in green).
  6. Colony growth on control and selection plates of yeast cells expressing protein and RNA hybrids indicated on the right. RNA binding is scored by growth on selection plates (SC ‐his +3‐AT, 3‐amino‐1,2,4‐triazole). mRRMx, Rrm4 variants harbouring mutations in RRM1, 2, 3 or 1 and 2.
  7. Percentage of Rrm4 bs containing the motif UAUG, shown for all bs and separated into transcript regions. The total number of binding sites is indicated on top.
  8. Relative contribution of the three stop codon variants to all (left) and Rrm4‐bound stop codons (left). Although opal stop codons (UGA) fit best with a UAUG‐containing binding site (UAUGA; present at 32 out of 63 bound UGA stop codons, 51%), they are depleted from Rrm4‐bound stop codons.