Mechanism for recycling tRNAs on stalled ribosomes

ELAC1 Repairs tRNAs Cleaved during Ribosome-Associated Quality Control

“…As we previously demonstrated (Yip et al, 2019), converting the 2 0 ,3 0 >p to 2 0 -OH and 3 0 -OH by using bacteriophage T4 PNK (polynucleotide kinase) permits CCA addition to generate full-length (FL) tRNA ( Figure 1B, lane 2). In contrast, conversion to 2 0 -p and 3 0 -OH by using CNP (2 0 ,3 0 -cyclic-nucleotide 3 0 Figure 1.…”

“…In the case of tRNAs, recent studies (Kuroha et al, 2018;Yip et al, 2019) demonstrated that RQC complex disassembly does not simply liberate free tRNA as previously thought (Verma et al, 2018;Zurita Rendó n et al, 2018). Instead, ANKZF1/Vms1 is an endonuclease (Kuroha et al, 2018) that cleaves off the universally conserved 3 0 CCA nucleotides (positions 74-76) of peptidyl-tRNA on 60S-RQC complexes (Yip et al, 2019). ANKZF1 cleavage releases nascent proteins for degradation (Verma et al, 2018;Zurita Rendó n et al, 2018) and simultaneously generates a tRNA intermediate containing a 2 0 ,3 0 -cyclic phosphate (2 0 ,3 0 >p) on the ribose of the discriminator base at position 73 (N 73 ) that is incompatible with translation.…”

“…CCA Addition Requires Complete Removal of 2 0 ,3 0 >p from tRNA substrates In earlier work, we established that ANKZF1-cleaved tRNAs contain a 2 0 ,3 0 >p on the discriminator nucleotide at position 73 (N 73 ) that precludes CCA re-addition by TRNT1 (Yip et al, 2019; Figure 1A). To identify the physiologic pathway to CCA addition, we determined which possible processing products of 2 0 ,3 0 >p are competent for CCA addition by TRNT1.…”

“…Supporting this idea are links between ribosome splitting factors and nonfunctional rRNA decay pathways (Cole et al, 2009;Limoncelli et al, 2017;Sugiyama et al, 2019). In the case of tRNAs, recent studies (Kuroha et al, 2018;Yip et al, 2019) demonstrated that RQC complex disassembly does not simply liberate free tRNA as previously thought (Verma et al, 2018;Zurita Rendó n et al, 2018). Instead, ANKZF1/Vms1 is an endonuclease (Kuroha et al, 2018) that cleaves off the universally conserved 3 0 CCA nucleotides (positions 74-76) of peptidyl-tRNA on 60S-RQC complexes (Yip et al, 2019).…”

“…ANKZF1 cleavage releases nascent proteins for degradation (Verma et al, 2018;Zurita Rendó n et al, 2018) and simultaneously generates a tRNA intermediate containing a 2 0 ,3 0 -cyclic phosphate (2 0 ,3 0 >p) on the ribose of the discriminator base at position 73 (N 73 ) that is incompatible with translation. Recycling of ANKZF1-cleaved tRNAs occurs efficiently in the mammalian cytosol through a two-step process (Yip et al, 2019): removal of the 2 0 ,3 0 >p followed by the re-addition of the 3 0 CCA by the CCA-adding enzyme TRNT1. How ANKZF1cleaved tRNAs with 2 0 ,3 0 >p are repaired for CCA addition is not known.…”