Ribosomal protein RPL26 is the principal target of UFMylation

UFM1 is a small, metazoan-specific, ubiquitin-like protein modifier that is essential for embryonic development. Although loss-of-function mutations in UFM1 conjugation are linked to ER stress, neither the biological function nor the relevant cellular targets of this protein modifier are known. Here we show that a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation. RPL26 UFMylation and deUFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. Biochemical analysis and structural modeling establish that UFMylated RPL26 and the UFMylation machinery are in close proximity to the SEC61 translocon, suggesting that this modification plays a direct role in cotranslational protein translocation into the ER. These data suggest that UFMylation is a ribosomal modification specialized to facilitate metazoan-specific protein biogenesis at the ER. ribosome profiling of membrane and cytosolic fractions of UFM1-KO, UFSP2-KO, and wild-type HEK cells in duplicate (12 samples); mRNA-seq of whole cell lysates of UFM1-KO, UFSP2-KO, and wild-type HEK cells in duplicate (6 samples)