UFSP1 Human

What is UFSP1 and what is its role in the UFMylation pathway?

UFSP1 is a Ubiquitin-fold modifier 1 (Ufm1)-specific protease that plays a crucial role in the UFMylation/de-UFMylation pathway. UFMylation is a post-translational modification process where Ufm1, a ubiquitin-like protein, is conjugated to protein substrates, potentially altering their fates. UFSP1 functions as an active protease that mediates both the maturation of Ufm1 precursor (pro-UFM1) and the de-UFMylation of target proteins . The maturation process involves cleaving the C-terminal Ser-Cys dipeptide of pro-UFM1 (approximately 9.1 kDa) to expose its C-terminal conserved Gly residue, yielding mature UFM1 (approximately 8.9 kDa) that can be conjugated to target proteins . Despite previous assumptions about UFSP1 being non-functional in humans, it is now confirmed that both UFSP1 and UFSP2 are active proteases in human cells with distinguishing features in their contributions to the UFMylation/de-UFMylation process.

Why was human UFSP1 previously thought to be inactive?

Human UFSP1 was long considered inactive because when translated from its annotated canonical start codon (445AUG), it appeared to lack a complete protease domain necessary for enzymatic activity . This led to the widespread belief that UFSP2 was the only active Ufm1 protease in human cells responsible for both Ufm1 maturation and de-UFMylation. This misconception persisted because traditional analysis focused only on the canonical open reading frame (ORF) of UFSP1, which would produce a truncated protein of approximately 17 kDa lacking the full catalytic domain . The mystery of why humans would maintain an apparently non-functional UFSP1 gene remained unexplained until recent discoveries about its non-canonical translation.

What is the evidence that human UFSP1 is actually an active protease?

Multiple experimental approaches have confirmed that human UFSP1 is indeed an active protease:

• Overexpression studies: When the full-length cDNA of UFSP1 (including its 5'-UTR) was expressed in 293T and HeLa cells, it produced a ~23 kDa protein (not the predicted ~17 kDa protein from the canonical ORF) that could effectively de-UFMylate Ufm1 conjugates .

• Gene knockout experiments: UFMylation was enhanced when UFSP2 was knocked out and further increased in UFSP1/UFSP2 double knockout cells. Notably, when both proteases were knocked out, no UFMylation was detectable, indicating that UFSP1 must be functional in normal cells .

• Mutational analysis: The decreased UFMylation associated with UFSP1-Long expression was fully abrogated when the potential active site cysteine (349TGC) was mutated to alanine (GCC), confirming that this residue is critical for UFSP1's enzymatic activity .

• Mass spectrometry: Direct evidence from MS peptide sequencing confirmed the translation of human UFSP1 from an upstream near-cognate codon (217CUG) instead of the annotated 445AUG, revealing the presence of a complete catalytic protease domain containing a cysteine active site .

How does the translation of human UFSP1 from a non-canonical start codon occur?

The translation of human UFSP1 initiates at a near-cognate CUG codon (position 217) through eIF2A-mediated translational initiation rather than at the annotated AUG codon (position 445) . This CUG codon normally encodes leucine, but when used as an alternative start site, it can encode methionine instead . The process requires:

• Optimal Kozak consensus sequence: The 217CUG is embedded in an optimal Kozak consensus sequence (ACCGCCCUGG), which plays a crucial role in translation initiation .

• Essential 5' UTR elements: Efficient UFSP1 expression requires approximately 126 nucleotides of 5' UTR sequence upstream of the 217CUG . Within this region, an E-box motif (CAGCTG) has been identified as particularly important for human UFSP1 expression .

• eIF2A-mediated translation: The eukaryotic translation initiation factor eIF2A is critical for translation from the CUG codon. Both chemical inhibition and RNAi approaches targeting eIF2A confirmed its essential role in UFSP1 expression .

This non-traditional translation mechanism explains how human cells produce an active UFSP1 with a complete protease domain despite the apparent truncation when only considering the canonical start codon.

What are the functional differences between UFSP1 and UFSP2 in the UFMylation/de-UFMylation process?

UFSP1 and UFSP2 show distinct functional profiles in the UFMylation/de-UFMylation pathway:

What are the implications of UFSP1's non-canonical translation for other genes potentially using near-cognate start codons?

The discovery of human UFSP1's translation from a near-cognate start codon has broader implications for gene expression regulation:

• Prevalence of non-AUG translation: CUG is the most common near-cognate start codon employed in eukaryotes, but the mechanism behind its use has remained unclear . The UFSP1 example provides insight into how such alternative translation initiations might occur.

• Regulatory elements: The identification of the E-box sequence in UFSP1's 5' UTR raises questions about whether similar elements exist in other genes employing near-cognate start codons as translation initiation sites . This discovery could provide clues to the mechanisms behind near-cognate translation initiation more generally.

• Potential undiscovered protein functions: Other genes currently thought to encode truncated or non-functional proteins based on canonical ORF analysis might actually produce functional proteins through similar non-canonical translation mechanisms. This suggests a need to reevaluate our understanding of the human proteome.

• RNA-binding protein interaction: The findings raise questions about whether specific RNA-binding proteins recognize the E-box sequence to assist CUG start codon usage and how these proteins might coordinate with eIF2A to initiate translation at near-cognate start codons .

How can researchers effectively detect and characterize endogenous UFSP1 expression in human cells?

To detect and characterize endogenous UFSP1 expression, researchers should consider the following approaches:

• CRISPR/Cas9-mediated tagging: Generate homozygous FLAG-tag knock-in cell lines using CRISPR/Cas9, where a FLAG-tag coding sequence is inserted at the C-terminus of the human UFSP1 locus . This allows for specific detection of endogenous UFSP1 without overexpression artifacts.

• Mass spectrometry for protein identification: Use immunoprecipitation with anti-FLAG affinity gel followed by MS peptide sequencing to confirm the translation start site and sequence coverage . In previous studies, this approach revealed eleven peptides covering 75% of the predicted full-length human UFSP1, with six peptides covering more than 90% of the predicted N-terminal region from 217CUG to the canonical 445AUG .

• Western blotting considerations: When designing antibodies or selecting commercial antibodies for UFSP1 detection, ensure they target epitopes present in the full-length protein (~23 kDa) rather than just the canonical ORF region (~17 kDa). Additionally, use appropriate controls including UFSP1 knockout cells to confirm antibody specificity.

• Construct design for expression studies: When studying UFSP1 expression, include the 5' UTR sequence in your constructs, as this is essential for efficient translation from the 217CUG codon. Constructs lacking this region will show extremely low expression efficiency .

What experimental approaches can be used to study the functional relationship between UFSP1 and UFSP2?

To investigate the functional relationship between UFSP1 and UFSP2, researchers can employ these methodological approaches:

• Single and double knockout cell models: Generate UFSP1 knockout, UFSP2 knockout, and UFSP1/UFSP2 double knockout cell lines using CRISPR/Cas9 genome editing . Compare the UFMylation patterns in these cells to determine the unique and overlapping functions of each protease.

• Substrate specificity analysis: Express tagged versions of known Ufm1 substrates (such as ASC1) in the knockout cell lines and analyze their UFMylation status. This can reveal whether UFSP1 and UFSP2 have different substrate preferences .

• Enzymatic activity assays: Perform in vitro de-UFMylation assays using purified recombinant UFSP1 and UFSP2 proteins against various UFMylated substrates to compare their enzymatic activities and specificities .

• Rescue experiments: Reintroduce wild-type or mutant versions of UFSP1 and UFSP2 into the knockout cell lines to determine which functions can be rescued by each protease. This can help define their specific roles in UFMylation and de-UFMylation processes.

• Subcellular localization studies: Use immunofluorescence or cell fractionation approaches to determine the subcellular localization of UFSP1 and UFSP2, which might explain their functional differences. The localization of UFSP2 to the endoplasmic reticulum membrane may be critical for its maximal activity .

How can the non-canonical translation mechanism of UFSP1 be experimentally characterized?

To characterize the non-canonical translation mechanism of UFSP1, researchers can implement these methodological approaches:

• 5' UTR mutation analysis: Create a series of UFSP1 expression constructs with full-length or truncated 5' UTR regions to identify the minimal sequence required for efficient translation from the 217CUG codon .

• E-box motif characterization: Perform site-directed mutagenesis on the identified E-box motif (CAGCTG) to determine its specific contribution to UFSP1 expression. Compare wild-type and mutant constructs for expression efficiency.

• eIF2A dependency experiments:

  • RNAi approach: Use siRNA or shRNA to knock down eIF2A and observe the effects on UFSP1 expression from constructs containing the near-cognate start codon .

  • Chemical inhibition: Employ specific inhibitors of eIF2A to further confirm its role in UFSP1 translation .

  • eIF2A overexpression: Test whether overexpression of eIF2A enhances UFSP1 translation from the CUG codon.

• RNA-binding protein identification: Perform RNA immunoprecipitation followed by mass spectrometry to identify proteins that bind to the UFSP1 5' UTR, particularly around the E-box motif. This could reveal factors that coordinate with eIF2A to facilitate translation initiation at the near-cognate start codon .

• Ribosome profiling: Use this technique to map ribosome occupancy along the UFSP1 mRNA, providing direct evidence of translation initiation at the 217CUG codon under physiological conditions.