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. 2019 Jan 4;38(5):e100276. doi: 10.15252/embj.2018100276

Figure 1. Hel2‐mediated K63‐linked polyubiquitination is crucial for NGD and RQC.

Figure 1

  1. Schematic drawing of the R(CGN) 12 reporter mRNA including the two quality control pathways induced by the R(CGN)12 translation arrest sequence. Ribosome stalling occurs during translation of the R(CGN)12 arrest sequence (shown in orange) and induces RQC and NGD. In the RQC pathway, the stalled ribosome is dissociated into subunits, and peptidyl‐tRNA remaining on the 60S subunit is ubiquitinated by Ltn1 (shown in pink) and degraded by the proteasome. In the NGD pathway, an endonucleolytic cleavage produces two fragments, the 5′ NGD intermediate (5′ NGD‐IM) and 3′ NGD intermediate (3′ NGD‐IM). The green and thin grey lines indicate GFP and HIS3 open reading frames (ORFs), and the black line indicates an untranslated region (UTR).
  2. Schematic drawing of the truncated Hel2 mutant proteins. Activities in RQC or NGD induced by the R(CGN)12 sequence are indicated.
  3. Western blot showing that Hel2(1–315) is defective in RQC but not in NGD. The arrest products derived from the R(CGN)12 reporter in ltn1Δ cells expressing truncated Hel2 mutant protein were detected with an anti‐GFP antibody.
  4. Northern blot showing the 5′ NGD‐IM derived from the R(CGN)12 reporter in ski2Δ cells expressing the indicated Hel2 mutant proteins. 5′ NGD‐IMs were detected with a DIG‐labelled GFP probe.
  5. Primer extension mapping of 5′ ends of 3′ NGD intermediates in Hel2‐WT or Hel2(1–315) mutant cells at nucleotide resolution. The primer extension samples were analysed using 5% TBE‐Urea‐PAGE and detected by fluorescence. Non‐specific reverse transcription (ReTr) products are indicated by asterisks.
  6. Dissection of NGDRQC+ and NGDRQC−: the carboxyl‐terminal region of Hel2 is required for both NGD and RQC which is likely triggered on a disome unit (pale yellow). It contains the primarily stalled, leading ribosome followed the colliding ribosome. For NGDRQC+, cleavages occur on mRNA covered by the disome, whereby the leading ribosome undergoes RQC. In the mutant Hel2 lacking the C‐terminus, an alternative NGD pathway takes place (NGDRQC−). Here, cleavages occur on mRNA covered by the ribosomes following the disome unit (blue) and the leading ribosome is not affected by RQC (grey).