Human UFSP1 translated from an upstream near-cognate initiation codon functions as an active UFM1-specific protease

Abstract

Ubiquitin-fold modifier 1 (UFM1) is a recently identified ubiquitin-like posttranslational modification with important biological functions. However, the regulatory mechanisms governing UFM1 modification of target proteins (UFMylation) and the cellular processes controlled by UFMylation remain largely unknown. It has been previously shown that a UFM1-specific protease (UFSP2) mediates the maturation of the UFM1 precursor and drives the de-UFMylation reaction. Furthermore, it has long been thought that UFSP1, an ortholog of UFSP2, is inactive in many organisms, including human, because it lacks an apparent protease domain when translated from the canonical start codon (⁴⁴⁵AUG). Here, we demonstrate using the combination of site-directed mutagenesis, CRISPR/Cas9-mediated genome editing, and mass spectrometry approaches that translation of human UFSP1 initiates from an upstream near-cognate codon, ²¹⁷CUG, via eukaryotic translation initiation factor eIF2A-mediated translational initiation rather than from the annotated ⁴⁴⁵AUG, revealing the presence of a catalytic protease domain containing a Cys active site. Moreover, we show that both UFSP1 and UFSP2 mediate maturation of UFM1 and de-UFMylation of target proteins. This study demonstrates that human UFSP1 functions as an active UFM1-specific protease, thus contributing to our understanding of the UFMylation/de-UFMylation process.

Keywords: UFM1-specific protease; UFMylation; UFSP1; UFSP2; eIF2A-mediated translational initiation.

Figure 1

Human UFSP1 is an active UFM1-specific protease.A, de-UFMylation activity of the human UFSP1 in HEK293T cells (left) and HeLa cells (right). B, mutation of ³⁴⁹TGC in human UFSP1 gene fully abrogates its de-UFMylation function. Human UFSP1-L expression plasmid with ³⁴⁹TGC to GCC mutation, UFSP1-L, UFSP1-S expression plasmid, and the control vector were introduced into HEK293T cells. C, human UFSP1 and/or UFSP2 KO changed the patterns of protein UFMylation and pro-UFM1 maturation. Human UFSP1 can cleave the pro-UFM1-HA (∼11.8 kDa) to produce the mature form UFM1 (∼8.9 kDa). UFSP1 KO (UFSP1^KO), UFSP2 KO (UFSP2^KO), and UFSP1/UFSP2 double KO (UFSP1^KO/UFSP2^KO) cell lines were generated by CRISPR/Cas9–mediated genome editing and monoclonal screening in HEK293T cells. The C-terminal HA-tagged pro-UFM1 plasmid (pSG5-UFM1-HA) or control vector (pSG5-HA) were transfected into the WT or gene KO cells. After 48 h post-transfection, cell lysates were analyzed by Western blot analysis using the indicated antibodies. HA, hemagglutinin; UFM1, ubiquitin-fold modifier 1; UFSP, UFM1-specific protease.

Figure 2

Human UFSP1 is translationally initiated from²¹⁷CUG.A, four CUG near-cognate codon candidates were marked in red and selected for mutation analysis. Generally considered ⁴⁴⁵AUG first start codon was labeled in green. The nucleotide sequences in yellow background is the potential Cys-box upstream of ⁴⁴⁵AUG, the nucleotide sequences in gray background is a potential Kozak sequence. B, mutation of ²¹⁷CTG but not ¹⁹⁰CTG, ²²⁶CTG, or ²⁴⁷CTG to CTA eliminates the expression of human UFSP1. WT or mutant human UFSP1-L, UFSP1-S expression plasmids, and control vector were introduced into HEK293T cells; cell lysates were analyzed by Western blot with the indicated antibodies. C, CRISPR/Cas9 system mediated knock-in of the FLAG-tag at the C-terminal end of the UFSP1 locus in HEK293T cells. The oligodeoxyribonucleotides (ssODNs) functioned as the linear donors and were used to introduce FLAG-tag insertion during homology-dependent repair (HDR) after the Cas9/sgRNA–mediated site-specific double-strand break (DSB). D, verification of expression of FLAG-tagged endogenous UFSP1 in FLAG-KI-6# clone. FLAG-KI-6# or parental HEK293T cells were lysed and immunoprecipitated with anti-FLAG M2 affinity gel for immunoblotting with FLAG or UFSP1 antibodies. Red asterisk indicates nonspecific band. E, identification of the translation initiation codon of human UFSP1 by mass spectrometry (MS). Cell lysates of FLAG-KI-6# or control cells were subjected to immunoprecipitation with anti-FLAG M2 affinity gel. The bound proteins were separated with SDS-PAGE and stained with Coomassie blue. The ∼25 kDa specific band (in red box) was sliced and analyzed using MS, and six peptides in the UFSP1-FLAG knock-in sample that match the region between the potential ²¹⁷CTG start codon and the canonical ⁴⁴⁵ATG codon (in red font) were identified. UFSP, UFM1-specific protease.

Figure 3

The 5′ UTR sequence is essential for human UFSP1 expression.A, schematic diagram of constructs of UFSP1 with different lengths of 5′ UTR upstream of the ²¹⁷CTG codon. B and C, 5′ UTR is essential for human UFSP1 expression. Human UFSP1 expression plasmids with different 5′ UTR lengths (0∼216 nt) upstream of ²¹⁷CTG codon and control vector were transfected into HEK293T cells, followed by Western blotting analysis. D, sequence of nucleotides 97 to 126 plays an important role in human UFSP1 expression. UFSP1-L-Random-1# and 2# plasmids, UFSP1 expression plasmids with different 5′ UTR lengths (96, 126, or 216 nt) upstream of ²¹⁷CTG codon, UFSP1-S, and control vector were transfected into HEK293T cells; cell lysates were analyzed by Western blotting analysis. UFSP, UFM1-specific protease.

Figure 4

eIF2A mediates²¹⁷CUG initiation of human UFSP1 translation.A and B, dose-dependent inhibition of human UFSP1 expression by acriflavine. HEK293T (A) and HeLa (B) cells were treated with different doses of acriflavine for 4 h, followed by Western blotting analysis. C and D, induction of human UFSP1 expression using ATA in a time-dependent manner. HEK293T (C) and HeLa (D) cells were treated with ATA (100 μM) for various periods of time, and the expression of UFSP1 and GAPDH were examined by Western blotting. E and F, eIF2A upregulates human UFSP1 expression. FLAG-tagged human eIF2A was overexpressed in HEK293T (E) and HeLa (F) cells, and UFSP1 expression was evaluated by Western blotting. eIF2A expression was verified by probing the same blot with anti-FLAG and anti-eIF2A antibodies. G–J, reduction of human UFSP1 expression in response to knockdown of eIF2A. HEK293T (G and H) and HeLa (I and J) cells were transfected with eIF2A siRNAs or control siRNAs. Cell lysates were analyzed using Western blotting. Densitometric analysis was performed using image processing software. Data are mean ± SD. Differences in means between two groups were analyzed using two-sided unpaired t test (∗p < 0.05). ATA, aurintricarboxylic acid; UFSP, UFM1-specific protease.

Figure 5

Human UFSP1 functions in UFMylation/de-UFMylation process.A, human UFSP1 can release UFM1 from UFMylated substrate ASC-1. HEK293T cells were transfected with the indicated plasmids, followed by UFMylation assay and immunoblotting with the indicated antibodies. Red asterisks indicate nonspecific band. B, human UFSP1 cleaves pro-UFM1 in vitro. C-terminal HA-tagged UFM1 was purified using anti-HA affinity gel. UFSP1-L-His or control vector were transfected in HEK293T cells. Cells lysates were subjected to immunoprecipitation with Ni-NTA Agarose. Amounts of UFSP1-His or His-tag control was incubated with UFM1-HA for the indicated time at 37 °C. The mixtures were subjected to SDS-PAGE followed by Western blot analysis. C, prokaryotic expressed human UFSP1 has protease activity, which cleaves pro-UFM1 in vitro. The cell lysates of UFSP1^KO/UFSP2^KO HEK293T cells transfected with pSG5-UFM1-HA used as protease substrates. A total of 0.5 μg purified UFSP1-L-His protein or control buffer was incubated with UFM1-HA for the 20 min at 37 °C. The reaction mixtures were subjected to SDS-PAGE followed by Western blot analysis. Red asterisk indicates band of endogenous pro-UFM1 in UFSP1^KO/UFSP2^KO HEK293T cells. D, HEK293T cells were transfected with indicated expression plasmids (UFSP1-L with a C-terminal HA-tag, human UFSP2 with an N-terminal HA-tag, or control vector), followed by Western blotting analysis. E, WT, UFSP1^KO, UFSP2^KO, and UFSP1^KO/UFSP2^KO HEK293T cells were fractionated with sequential detergent extractions before Western blotting analysis with the indicated antibodies. ASC-1, activating signal cointegrator 1; HA, hemagglutinin; UFM1, ubiquitin-fold modifier 1; UFSP, UFM1-specific protease.