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SUMO paralogue-specific functions revealed through systematic analysis of human knockout cell lines and gene expression data.

ABSTRACT: The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogues with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogues and also the best studied. To what extent SUMO1, SUMO2, and the other paralogues impart unique and nonredundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogues during development and studies in cell culture have relied largely on transient paralogue overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralogue gene expression levels in normal human tissues and found unique patterns of SUMO1-3 expression across 30 tissue types, suggesting paralogue-specific functions in adult human tissues. To systematically identify and characterize unique and nonredundant functions of the SUMO paralogues in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, promyelocytic leukemia (PML) nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal nonredundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.

SUBMITTER: Bouchard D

PROVIDER: S-EPMC8684707 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cilia assembly, maintenance and the localization of signal-transduction proteins necessitate a functioning intraflagellar transport (IFT). The IFT machinery moves cargo along a microtubule scaffold from the proximal to the distal end of the cilium. This anterograde transport is facilitated by a large multiprotein complex IFT-B, which consists of 16 proteins in total and recruits motor protein Kinesin-2 for active movement. TTC30A and TTC30B are integral components of this complex and were shown before to have redundant function in context of IFT. One paralogue could compensate the loss of the other, preventing the disruption of IFT-B and thus a severe ciliogenesis defect with no formation of the cilium. Affinity-based protein complex analysis of endogenously tagged cells, generated via CRISPR/Cas9 system as described before, revealed paralogue specific protein interactors proposing the involvement of TTC30A or TTC30B in other ciliary functions, particularly Sonic hedgehog signaling (Shh). Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which intriguingly is also linked to a rare TTC30 variant. In this study we focus on the so far unknown interaction of TTC30A with protein kinase A catalytic subunit α PRKACA, which is a negative regulator of Shh pathway. For an in-depth analysis of this unique interaction and the influence on Shh, we used previously, via the CRISPR/Cas9 system, generated TTC30A or B single- and double-knockout hTERT-RPE1 cells as well as rescue cells harboring the TTC30 mutation. Our systematic approach revealed the paralogue specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA as well as the uptake of Smoothened into the cilium resulting in a downregulation of Sonic hedgehog signaling. Affinity-based protein complex analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in interaction pattern. These interactome alterations combined with Shh downregulation suggest a possible mechanism of how mutant TTC30A and respectively TTC30A KO are linked to synpolydactyly.

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SUMO paralogue-specific functions revealed through systematic analysis of human knockout cell lines and gene expression data.

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