The P124A mutation of SRP14 alters its migration on SDS-PAGE without impacting its function

Abstract

SRP14 is a crucial protein subunit of the signal recognition particle (SRP), a ribonucleoprotein complex essential for co-translational translocation to the endoplasmic reticulum. During our investigation of SRP14 expression across diverse cell lines, we observe variations in its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with some cells exhibiting slower migration and others migrating faster. However, the cause of this phenomenon remains elusive. Our research rules out alternative splicing as the cause and, instead, identifies the presence of a P124A mutation in SRP14 (SRP14 ^P124A) among the faster-migrating variants, while the slower-migrating variants lack this mutation. Subsequent ectopic expression of wild-type SRP14 ^P124 or SRP14 ^WT and SRP14 ^P124A in various cell lines confirms that the P124A mutation indeed leads to faster migration of SRP14. Further mutagenesis analysis shows that the P117A and A121P mutations within the alanine-rich domain at the C-terminus of SRP14 are responsible for migration alterations on SDS-PAGE, whereas mutations outside this domain, such as P39A, Y27F, and T45A, have no such effect. Furthermore, the ectopic expression of SRP14 ^WT and SRP14 ^P124A yields similar outcomes in terms of SRP RNA stability, cell morphology, and cell growth, indicating that SRP14 ^P124A represents a natural variant of SRP14 and retains comparable functionality. In conclusion, the substitution of proline for alanine in the alanine-rich tail of SRP14 results in faster migration on SDS-PAGE, but has little effect on its function.

Introduction

The signal recognition particle (SRP) is a ribonucleoprotein complex that plays a critical role in co-translational protein translocation to the endoplasmic reticulum (ER). SRP consists of six proteins (SRP9, SRP14, SRP19, SRP54, SRP68, and SRP72), as well as a single SRP RNA, the 7SL RNA. Notably, the SRP9/SRP14 heterodimer, in conjunction with the 5′ and 3′ termini of 7SL RNA, collaboratively forms what is known as the Alu domain [1].

When the signal sequence emerges from the ribosome, it is captured by SRP, and the elongation of the nascent polypeptide is arrested by the Alu domain. Translation resumes when the ribosome–nascent chain-SRP complex (RNC-SRP) docks with the SRP receptor (SR) on the ER. The function of the Alu domain in arresting translation is believed to extend the time available for RNC-SRP to target the SR, therefore ensuring efficient co-translational translocation [2].

Despite limited primary sequence homology, the crystal structures of SRP9/14 exhibit structural similarity. Both proteins form a three-stranded antiparallel β-sheet stacked against two helices. The six β-sheets of the heterodimer create a highly positively charged concave surface, which serves as the primary Alu RNA-binding site ( Figure 1A) [3]. Notably, despite their structural resemblance, the internal loop between the β1 and β2 sheets of SRP14 is absent in SRP9. Although the affinity between Alu RNA and SRP9/14 lacking the internal loop decreases 10-fold, this does not compromise elongation arrest [4]. In contrast to the indispensable internal loop, the pentapeptide KRDKK following K95 of SRP14 is crucial for elongation arrest both in vitro and in mammalian cells [5]. The insertion of as few as two alanine residues after K95 can completely abrogate the arrest of elongation [4]. The basic patches of SRP14 serve as a positively charged platform for interactions with ribosomal RNA.

Figure 1 .

Two migration rates of SRP14 from different human cell lines can be observed on SDS-PAGE

(A) Sequence and domain analysis of the human SRP14 protein. The amino acid sequence was derived from the UniProt databank (entry: P37108). (B) SRP14 from various cell lines was detected by immunoblotting. (C) The full-length CDS of SRP14 was amplified via PCR using the indicated primers, and the PCR products were separated via agarose gel electrophoresis (D). The band was cut and processed for Sanger sequencing.

The SRP is a universally conserved cellular machinery, with the SRP14 sequence conserved across species, from yeast to mammalian cells, except for its C-terminus. SRP14 in primate species features an additional C-terminal extension, leading to differential migration patterns between rodent and primate cells on SDS-PAGE [6]. In a previous study, we found that ER stress can induce a significant reduction in SRP14, which is mediated by the PERK branch of the unfolded protein response (UPR) and executed through ubiquitin- and proteasome-mediated degradation. The attenuation of translocation, regulated by the PERK-SRP14 axis, can be a novel protective mechanism of the UPR to alleviate ER stress [7]. Furthermore, it is noteworthy that despite the shared human origin of the cells studied, variations in SRP14 migration patterns persist across different cell lines. The underlying reasons for these discrepancies and their potential biological significance require further investigation.

In this study, we demonstrate that the SRP14 ^P124A variant directly leads to faster migration on SDS-PAGE. Additionally, we also find that the SRP14 ^P124A variant retains similar functional properties to SRP14 ^WT.

Materials and Methods

Plasmid constructs

The plasmids pLVX-SRP14-FLAG and pLVX-FLAG-BRM24 were constructed by integrating DNA sequences encoding SRP14 and RNA-binding protein 24 (RBM24) with a FLAG tag into the pLVX-IRES vector (Fenghbio, Wuhan, China). SRP14 (P124A, Y27F, T45A, P39A, P117A, A121P) and RBM24 (A215P) mutants were generated using efficient one-step site-directed plasmid mutagenesis [8], and the mutagenesis primers used for each mutant are listed in Supplementary Table S1.

Cell culture and reagents

The human cell lines HCT116, LoVo, HeLa, and HEK293T were obtained from Dr. Yili Yang at the International Centre for Genetic Engineering and Biotechnology (Taizhou, China). The A549 lung adenocarcinoma cell line and HuH-7 hepatoma cell line were graciously provided by Dr. Xiaofeng Qin at the Suzhou Institute of Systems Medicine (Suzhou, China). The cells were cultured in DMEM supplemented with 10% FBS (FBS; Gibco, Grand Island, USA) and 2 mM L-glutamine and incubated at 37°C in 5% CO ₂. The SH-SY5Y human neuroblastoma cell line was obtained from the National Laboratory Cell Resource (Beijing, China), and maintained in RPMI 1640 medium (Gibco) supplemented with 15% FBS. To package the lentivirus, HEK293T cells were transfected with the recombinant plasmids psPAX2 packing plasmid (Addgene, Watertown, USA) and pMD2.G envelope plasmid (Addgene) was kindly provided by Dr. Xiaodan Hou at the Suzhou Institute of Systems Medicine, using polyethyleneimine (Sigma, St Louis, USA). The lentivirus-containing supernatant was used to infect cells, after which 7.5 μg/mL blasticidin (Gibco) was added for approximately two weeks for selection.

Knockout of SRP14 using CRISPR/Cas9-mediated genome editing

The target sequence for human SRP14 (5′-TATAGACGCTGCCCGACGTC-3′) was incorporated into the PX458 plasmid (Addgene). Subsequently, HeLa cells were transfected with this plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, USA). The resulting clones were isolated and subjected to verification through sequencing and western blot analysis.

Western blot analysis

Whole-cell extracts were prepared by lysing the cells in SDS lysis buffer supplemented with a protease inhibitor cocktail (MCE, Monmouth Junction, USA). Cellular extracts were subjected to 12% SDS-PAGE and transferred to PVDF membranes (Sigma). The blots were preincubated with 5% non-fat milk for 1 h at room temperature (RT) and then incubated with primary antibodies at 4°C overnight. The membranes were incubated with the secondary antibody for 1 h at RT. The bands were analyzed with an enhanced chemiluminescence system (Bio-Rad, Hercules, USA) with GAPDH serving as a loading control. The anti-SRP14 antibody was from NOVUS (Littleton, USA). Anti-FLAG antibody was from MBL (Tokyo, Japan). Anti-GAPDH antibody was from Proteintech (Wuhan, China). The Secondary antibodies HRP-labeled goat anti-rabbit IgG(H+L) and HRP-labeled goat anti-mouse IgG(H+L) were from Beyotime (Shanghai, China).

In vitro translation

The pLVX-SRP14-FLAG plasmid and Q5 High-Fidelity DNA Polymerase (NEB, Beverly, USA) were used to perform PCR to obtain SRP14-FLAG. The relevant primers containing the T7 promoter are listed in Supplementary Table S1. The PCR products were added to a reticulocyte lysate-based in vitro translation system (Promega, Madison, USA), and the procedure was performed according to the manufacturer’s instructions.

Cell counting kit-8 (CCK-8)

To evaluate the impact of SRP14 ^P124A on cell growth, cells (3000 cells/well) were seeded in a 96-well plate with 100 μL of complete medium per well. Following a 24-h incubation, 10 μL of CCK-8 solution (Bimake, Houston, USA) was added to each well, and the cells were incubated at 37°C for 1 h. The absorption values were measured at 450 nm with a microplate reader over five consecutive days.

RNA isolation and qRT-PCR

Total RNA extraction was performed using RNAiso Plus Reagent (TaKaRa, Dalian, China). Subsequently, 1 μg of total RNA was reverse-transcribed into cDNA utilizing the PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa) following the manufacturer’s instructions, using random 6-mers as primers. qRT-PCR was conducted with SYBR Green qPCR Master Mix (TaKaRa) on an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Gaithersburg, USA). To standardize the expression data, the mRNA level of the housekeeping gene GAPDH was used, and calculations were performed using the 2 ^–ΔΔCt method. The sequences of primers used are provided in the Supplementary Table S1. Notably, the primer sequences for 7SL RNA were adopted from a previous study [9].

Statistical analysis

GraphPad Prism 8 was used to generate plots depicting the cell growth curves and for statistical analyses of the resulting data. Data are expressed as the mean±standard deviation, and comparisons among the three experimental groups were analyzed through one-way analysis of variance (ANOVA). P<0.05 was considered statistically significant.

Results

Different migration of SRP14 on SDS-PAGE is not resulted from alternative splicing

Analysis of the expression of SRP14 in various cell lines, such as HCT116, LoVo, A549, HeLa, SH-SY5Y, HEK293T and HuH-7, revealed that the migration of SRP14 was faster in HCT116, A549, SH-SY5Y, HEK293T and HuH-7 cells than in LoVo and HeLa cells ( Figure 1B). The human SRP14 gene comprises five exons, and it is of interest to investigate whether the alternative splicing of these exons contributes to the observed size differences. To explore this possibility, we designed primers targeting the 5′ and 3′ untranslated regions (UTRs) and performed PCR on the complete coding sequence (CDS) ( Figure 1C). Interestingly, we found that the length of the PCR products was consistent across different cell lines, suggesting that alternative splicing is unlikely to be the underlying cause of the distinct SRP14 migration patterns observed ( Figure 1D).

The P124A mutation in SRP14 alters migration, as determined by SDS-PAGE

Sanger sequencing analysis of the PCR products shown in Figure 1D revealed intriguing results. In addition to a few synonymous mutations ( Supplementary Figure S1), the faster-migrating SRP14 had a missense mutation at position 370 within the CDS ( Figure 2A,B). Specifically, this point mutation involved the conversion of G to C, resulting in an amino acid substitution from proline (P) to alanine (A) at residue 124 (SRP14 ^P124A). Conversely, the slower-migrating SRP14 proteins showed no mutations at this site ( Figure 2C,D).

Figure 2 .

Sanger sequencing analysis of SRP14 from various cell lines

(A) A diagrammatic representation of the SRP14 mRNA (NM_003134.6). (B) The Sanger sequencing results for the indicated regions from various cell lines were aligned. The base’s point mutation is highlighted with black background. (C) The amino acid sequences from different cell lines were derived from Sanger sequencing and aligned as shown. The amino acid point mutations are highlighted in black, and the predicted molecular masses of SRP14WT and SRP14P124A are presented on the right. (D) Chemical structural formulas for the reference and mutated amino acid sequences of the indicated regions.

Modifications of proteins, such as glycosylation, phosphorylation, or sumoylation, can result in altered migration on SDS-PAGE. Prolyl residues of proteins can be hydroxylated, and this modification may be involved in many important cellular processes, including oxygen sensing [10], epigenetic regulation [11], cell proliferation [12], and viral biogenesis [13]. To investigate whether the altered migration is due to point mutations in different cell types, we introduced both SRP14 ^WT and SRP14 ^P124A into various cell lines. FLAG tags were added to the C-terminus of the proteins to distinguish them from the endogenous proteins. These proteins were then ectopically expressed in HeLa and HEK293T cells, whose native SRP14 proteins migrated more slowly and quickly, respectively on SDS-PAGE. Surprisingly, regardless of whether SRP14 ^P124A was expressed in HeLa or HEK293T cells, it consistently migrated faster than SRP14 ^WT ( Figure 3A). To address whether the altered migration is due to different modifications, we used an in vitro translation system to express SRP14 ^P124A and SRP14 ^WT. The results showed that the migration of SRP14 ^P124A was faster than that of SRP14 ^WT ( Figure 3B). These results suggest that the altered migration is primarily attributed to the substitution of alanine for proline at residue 124 rather than being influenced by the specific cell lines in which the proteins are expressed.

Figure 3 .

Western blot analysis of the expressions of WT, P124A, Y27F, and T45A SRP14 translated in vivo or in vitro

(A) SRP14WT and SRP14P124A, each with a FLAG tag at the C-terminus, were expressed in HeLa and HEK293T cells. The whole-cell extracts were subjected to western blot analysis using anti-FLAG and anti-SRP14 antibodies. (B) Both SRP14WT and SRP14P124A were translated in vitro, and the resulting products were processed for western blot analysis. HeLa cell extracts expressing SRP14WT was utilized as a control. (C) SRP14WT and its Y27F and T45A mutations, each with a FLAG tag at the C-terminus, were expressed in HeLa cells. Protein expression was detected via western blot analysis using an anti-SRP14 antibody. (D) The WT, P124A, P39A, P117A, and A121P SRP14 were translated in vitro or in HEK293T cells and subjected to western blot analysis using anti-FLAG and anti-SRP14 antibodies. (E) The WT and A215P RBM24 were translated in vitro or in HEK293T cells and subjected to western blot analysis using an anti-FLAG antibody.

Typically, the migration of proteins on SDS-PAGE gels is determined mainly by their molecular weight. However, even when proline (0.097 kDa) is replaced by alanine (0.071 kDa), the molecular weight of SRP14 is only slightly reduced by approximately 0.026 kDa. This raises the question of whether SDS-PAGE is sensitive enough to detect such small changes in molecular weight and whether other types of point mutations may also affect protein migration. To explore this further, we constructed two additional SRP14 mutants, Y27F and T45A, which target two potential phosphorylation sites. These substitutions resulted in molecular weight reductions of approximately 0.016 kDa and 0.030 kDa, respectively. However, Figure 3C shows no significant difference in migration between the mutated SRP14 and SRP14 ^WT strains. These results suggest that the altered migration observed in SRP14 ^P124A is not solely due to small changes in molecular weight.

To further elucidate the mechanism of action of SRP14, additional point mutations were generated. Interestingly, the introduction of the proline-to-alanine mutation at site 39 did not result in any significant changes in migration. However, it became evident that only point mutations in the alanine-rich domain located at the C-terminus of SRP14 affected its migration pattern. Specifically, the point mutation from proline to alanine (P124A, P117A) can lead to faster migration, whereas the inverse mutation from alanine to proline (A121P) slows this process. Interestingly, despite all of these mutations being located within the alanine-rich domain of SRP14, the extent of the alteration in migration varies depending on the specific mutation site ( Figure 3D). To investigate whether the migration on SDS-PAGE is consistently affected by point mutations in alanine-rich regions, we examined RBM24 (24.8 kDa), a protein that contains a sequence of 13 consecutive alanines at its C-terminus. Surprisingly, mutation of the alanine to proline in its poly-alanine region (A215P) did not result in significant alteration in migration ( Figure 3E). Therefore, the migration observed in SRP14 due to point mutations in its alanine-rich tail seems to be unique.

SRP14 ^P124A and SRP14 ^WT exhibit similar functions in terms of SRP RNA stability, cell morphology, and cell growth

Previous studies have shown that mutations involving the substitution of proline (P) for alanine (A) can impact protein stability and modification, potentially affecting cell viability [14]. Therefore, it remains important to determine the physiological significance of the SRP14 ^P124A variant. As shown in Figure 4A,B, ectopic expression of both SRP14 ^WT and SRP14 ^P124A in HeLa and HEK293T cells did not result in significant changes in cell growth compared to that in vector-expressing cells. To further assess their functions, we introduced SRP14 ^WT and SRP14 ^P124A into HeLa cells, in which endogenous SRP14 was knocked out. Confirmation of the knockout and ectopic SRP14 expression was achieved by western blot analysis ( Figure 4C). Depletion of SRP14 resulted in a change of HeLa cell morphology from spindle-shaped to round, with reduced adherence to the cell substrate. These alterations in cell morphology were partially rescued by the ectopic expression of either SRP14 ^WT or SRP14 ^P124A ( Figure 4D).

Figure 4 .

SRP14 ^WT and SRP14 ^P124A exhibit similar functions

SRP14WT and SRP14P124A were ectopically expressed in HeLa (A) and HEK293T (B) cells. The cells were seeded into a 96-well plate and subjected to CCK-8 assay. (C) Western blot analysis of four cell lines: HeLa cells, HeLa cells with endogenous SRP14 knocked out (SRP14–/–), and HeLa cells with endogenous SRP14 knocked out and ectopically expressing SRP14WT-FLAG, and HeLa cells with endogenous SRP14 knocked out and ectopically expressing SRP14P124A-FLAG. (D) Bright-field images of the four cell lines. (E) Relative expression levels of SRP RNA in four cell lines. (F) Growth curve analysis of the four cell lines. ns, not significant; *P<0.05, ***P<0.001 by Student’s two-tailed t test.

Since SRP14 plays a pivotal role in maintaining the stability of SRP RNA, the 7SL RNA [9], we examined 7SL RNA expression and found that SRP14 knockout reduced 7SL RNA level by approximately 70%. Ectopic expression of SRP14 ^WT or SRP14 ^P124A effectively restored 7SL RNA to similar levels ( Figure 4E). Given that 7SL RNA serves as the structural backbone of SRP, a decrease in 7SL RNA level signifies SRP dysfunction. Consequently, cells exhibited significantly slower growth when SRP14 was knocked out, a phenomenon that was rescued by the ectopic expression of either SRP14 ^WT or SRP14 ^P124A ( Figure 4F).

Based on these results, we conclude that SRP14 ^WT and SRP14 ^P124A perform similarly in terms of SRP RNA stability, cell morphology, and cell growth. As mentioned above, the additional amino acids at the C-terminus of SRP14 are unique to primates, are rich in alanine, and are resulted from the insertion of repetitive sequences (CAX) _n into the coding region [15]. Previous studies have shown that SRP14, which lacks the C-terminus, is fully active in both translational arrest and translocation assays [4]. It is therefore not surprising that the SRP14 ^P124A variant exhibits a similar functionality to that of SRP14 ^WT.

Discussion

In our study, we observed that, on SDS-PAGE, SRP14 in various human cell lines exhibited two different migration positions. Coincidentally, a previous study [6] showed that SRP14 in various human cell lines, including AMA (transformed human amnion cell line), U-937, MRC-5, and Hep G2, had an apparent molecular mass of approximately 18 kDa. However, the molecular mass of SRP14 was different at approximately 20 kDa in HeLa cells. To determine the underlying cause, Bovia et al. [6] introduced AMA-derived SRP14 cDNA into HeLa cells. Surprisingly, when expressing SRP14 cDNA from AMA cells in HeLa cells, a protein of the same size as SRP14 from AMA cells can be detected, in addition to the endogenous SRP14 protein in HeLa cells. This intriguing finding suggested that posttranslational modification may not be responsible for the decreased migration of SRP14 in HeLa cells. However, further experiments were not performed to determine the exact cause of the altered band migration.

In this study, we obtained full-length SRP14 coding sequences from several human cell lines by PCR amplification and verified them by Sanger sequencing. Although sequencing of these PCR products revealed identical lengths, we observed a correlation between the presence of the P124A point mutation and faster-migrating SRP14 variants, whereas slower-migrating SRP14 lacked this mutation. When SRP14 ^WT and SRP14 ^P124A were expressed in cell lines or translated in vitro, altered migration was consistently observed and reproduced. Based on these results, we believe that the P124A mutation in SRP14 is responsible for the faster migration observed on SDS-PAGE.

Intriguingly, a single point mutation can result in significant migration on SDS-PAGE. Although previous studies have suggested that a high proline content in proteins can cause slow migration on SDS-PAGE [ 16, 17], the faster migration observed in SRP14 ^P124A remains a special case. Several proline-to-alanine mutations have been introduced into several proteins, including HIF-1α (93 kDa) with the P564A mutation [10], UCP1 (33 kDa) with P33A, P133A, and P232A [18], histone H3 (15 kDa) with the P16A mutation [11], and DYRK1B (69 kDa), in which a number of prolines were mutated to alanine [19]. However, these mutations did not result in significant differences in migration. In addition, Figure 3C,D shows that point mutations such as Y27F, P39A, and T45A in SRP14 do not cause any change in migration. The observed migration in the P124A, P117A, and A121P point mutants may be due to their location within the alanine-rich domain at the C-terminus of SRP14 ( Figure 3D). However, the A215P mutation in the alanine-rich region of RBM24 also did not result in significant differences in migration ( Figure 3E). This discrepancy could be attributed to the larger size of RBM24, which decreases the extent of the changes in migration. Another possibility is the longer alanine-rich region present in SRP14. These factors may contribute to the distinctive nature of SRP14 migration.

Not surprisingly, the P124A mutation in this domain had no significant effect on cell viability. This domain is unique to primates, and previous studies have shown that SRP14 with this domain removed still has full activity in translation arrest and translocation assays. Furthermore, although the reference allele of the 370th base of the SRP14 CDS is G, the frequency of the P124A variant of the SRP14 gene or its CDS 370th base substitution from G to C is approximately 92% in the global population, as reported in the dsSNP (rs7535).

In summary, this study demonstrated that the P124A mutation in the alanine-rich domain at the C-terminus of SRP14 can lead to faster migration on SDS-PAGE, while leaving its essential functions intact. It is anticipated that these unique characteristics of the alanine-rich tail may have broader applications beyond SRP14 detection.

Supplementary Data

Supplementary data is available at Acta Biochimica et Biophysica Sinica online.

COMPETING INTERESTS

The authors declare that they have no conflict of interest.

Funding Statement

This work was supported by the grants from the National Key Research and Development Program of China (No. 2019YFA0109902) and the National Natural Science Foundation of China (No. 32200419).