Fig 2: USP36 deubiquitinates Las1L and Nol9 and regulates their levels. A and B, Knockdown of USP36 reduces the levels of Las1L and Nol9. The HEK293 (A) and HeLa (B) cells transfected with scrambled (scr) or USP36 siRNA were assayed by IB. C, The stability of Las1L and Nol9 is dependent on each other. HeLa and HEK293 cells transfected with scr, Las1L, or Nol9 siRNA were assayed by IB. D, USP36 deubiquitinates Las1L. H1299 cells transfected with the indicated plasmids were subjected to Ni2+-NTA PD under denaturing conditions, followed by IB to detect the ubiquitinated species of Las1L. The protein expression is shown in the bottom panels. E, Knockdown of endogenous USP36 increases the ubiquitinated species of Las1L. H1299 cells transfected with the indicated plasmids together with scr or USP36 siRNA were subjected to Ni2+-NTA PD under denaturing conditions, followed by IB to detect the ubiquitinated species of Las1L. The protein expression is shown in the bottom panels. F, USP36 deubiquitinates Nol9. H1299 cells transfected with the indicated plasmids were subjected to Ni2+-NTA PD under denaturing conditions, followed by IB to detect the ubiquitinated species of Nol9. The protein expression is shown in the bottom panels.

Fig 3: The Las1L–Nol9 complex and USP36 play a role in rRNA ITS2 processing. The HEK293 and HeLa cells transfected with scr, Las1L, Nol9 siRNA (A and B), or USP36 siRNA (C and D) were assayed for rRNA processing by Northern blot analysis using probe hybridizing to the ITS2 (A and C). The 47S pre-rRNA, 32S rRNA, 12S rRNA, and the relative ratios of 32S to 28S rRNA normalized to the scr control are shown in the top panels. Ethidium bromide (EB) staining of the RNA gels to indicate 28S and 18S rRNAs is shown in the bottom panels (A and C). The protein expression is shown by IB (B and D).

Fig 4: Las1L SUMOylation at K565 is critical for its function in rRNA ITS2 processing. The HEK293 (A and B) and HeLa (C and D) cells transfected with scr or Las1L siRNA together with siRNA-resistant WT Flag-Las1L or its K565R mutant were assayed for rRNA ITS2 processing by Northern blot analysis using the ITS2 probe. The 32S rRNA precursor and the relative ratios of 32S to 28S rRNA normalized to the scr control are indicated in the top panels. Ethidium bromide (EB) staining of the RNA agarose gel is shown in the bottom panels (A and C). One representative experiment from three independent experiments is shown. The expression of the endogenous Las1L and exogenous Flag-Las1L is shown by IB (B and D).

Fig 5: Mapping the interaction of USP36 with the Las1L–Nol9 complex. A and B, The C-terminal domain of USP36 binds to Las1L and Nol9. H1299 cells transfected with Flag-USP36 or its deletion mutants were assayed by Co-IP with anti-Flag antibody, followed by IB (A). Diagram of USP36 indicating the C-terminal Las1L–Nol9–binding domain is shown in B. USP, ubiquitin-specific protease. C and D, USP36 binds to the CC domain of Las1L. H1299 cells transfected with Flag-Las1L or its deletion mutants together with V5-USP36 were subjected to Co-IP with anti-Flag antibody, followed by IB (C). The diagram of Las1L domains is shown in D. HEPN, higher eukaryotes and prokaryotes nucleotide-binding domain; LCT, Las1L C-terminal tail; CC, coiled-coil. E and F, USP36 binds to the NTD domain of Nol9. H1299 cells transfected with Flag-Nol9 or its deletion mutants together with V5-USP36 were subjected to Co-IP with anti-Flag antibody, followed by IB (E). The diagram of Nol9 domains is shown in F. NTD, N-terminal domain; PNK, polynucleotide kinase; CTD, C-terminal domain. G and H, USP36 binds to Las1L and Nol9 independently. H1299 cells transfected with WT Flag-Las1L or the Nol9-binding defective mutant (G) or Flag-Nol9 or its Las1L-binding defective mutant (H) were subjected to Co-IP with anti-Flag antibody, followed by IB.